1.
Antibody Response Of Buffalo To Inactivated Foot And Mouth Disease (Aphtho) Virus Vaccine
by Nadeem Murtaza | Prof. Dr. Khushi Muhammad | Prof. Dr. Masood Rabbani | Faculty of Veterinary Sciences.
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Publisher: 2005Dissertation note: Buffaloes when vaccinated against Foot and Mouth Disease (FMD) vaccine containing serotype "O" of the virus, showed detectable level of complement fixing (CF) antibodies. Buffaloes vaccinated with Aftovaxpur vaccine showed undetectable level of CF-antibodies when tested through complement fixation test using local vaccinal serotype "O" of FMD virus. However, buffaloes irrespective of age and sex when vaccinated with aluminum hydroxide adsorbed FMD vaccine containing serotype "O" of the FMD virus, showed detectable level of CF-antibodies, when tested through CFT using the local serotype "O" Of FMD virus. These antibodies disappeared fourth month post boosting. Buffalo calves immunized with oil based FMD vaccine showed high-level GMT titer (17.6) of the CF-antibodies. Rabbits immunized with the oil based FMD vaccine showed high level GMT (31.2) of the CF-antibodies. Low level of CF-antibodies might be sufficient to induce resistant to field exposure of the FMD virus serotype in the presence of blood complement. Sera of buffalo, cattle, sheep, goat and guinea pigs contained complement titer 35.2, 32.6, 19.2, 20.8, 614.4, respectively. Moreover, it was observed that complement activity remained stable when stored at -200C for 24 hours. The complement activity decreased from 1:512 to 192, 70.4, and 13.6 when stored at 40C, 250C and 370C, respectively. The complement activity was detectable when diluents containing Ca++and Mg++ ions were used.
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2.
Development Of Standard Protocols For Preparation And Evaluation Of Liver Homogenate Vaccines Against
by Sahidullah | prof. Dr. Masood Rabbani | Prof. Dr. Khushi Muhammad | Faculty of Veterinary Sciences.
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Publisher: 2005Dissertation note: Twelve vaccines were prepared from HPS infected liver homogenate by using two different virus concentrations (1x102 &1x103 LD50) and two virus inactivants (1%formalin and 0.001%Binary ethyleneimine) with and with out different adjuvants. These vaccines were evaluated in 13 groups of day old broilers (105 chicks) for their comparative immunogenicity and protection. At day 14 of age, groups A1, B1, C1 & D1 were vaccinated with 4 oil base vaccines (OB-HPSV) with different virus concentration and different inactivants. Similarly groups A2, B2, C2 & D2 were vaccinated with aluminized vaccines (AH-HPSV) and groups A3, B3, C3 & D3 with non adjuvant vaccines (NA-HPSV). Groups E was kept as unvaccinated control group. All the vaccinated birds were found sero-positive 7 days post vaccination (PV). IHA GMT results indicated no difference for different virus concentrations and different virus inactivants but same adjuvant. The IHA GMT recorded weekly during 0-28 days post vaccination was the highest and more consistent (52-181) for OB-HPSV followed by AH-HPSV (52-147) and then NA-HPSV (73.3-104). All the birds vaccinated with OB-HPSV resisted the virus challenge 21 days PV (100% protection). While protection percentage recorded for AH-HPSV and NA-HPSV was 87.5 % & 62.5% respectively. It was concluded that 1x102 LD50 oil base vaccines inactivated with either formalin or binary amine may be recommended for commercial use being the best in experimental trails.
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3.
Standardization Of Indirect Enzyme Linked Immunosorbant Assay For Detection Of Antibodies Against Foot and Mouth Disease Virus Sdrotype "O"
by Yasmeen Siddique | Prof. Dr. Khushi Muhammad | Dr. Mansur-ud-Din Ahmad | Faculty of Veterinary Sciences.
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Publisher: 2005Dissertation note: An indirect ELISA was standardized for titration of anti-FMD-serotype-specific antibodies. In this test polystyrene plates were coated with known FMD serotype "O" virus using carbonate/bicarbonate buffer. The blank spaces were blocked with horse serum. The immunoplate was coated with anti-FMD "O" virus specific serum from vaccinated calves. After washing, the plate was coated with rabbit anti-bovine-Ig-specific-antibodies-horse radish peroxidase conjugate. After washing, the plate was coated with HRP specific substrate. Development of color was recorded in form of OD value using ELISA reader. During the standardization of ELISA, flat bottom ELISA plates among all types of plates, 1:10 diluted virus among different dilutions of FMD "O" type virus, 1:10 diluted serum from buffalo calves vaccinated with FMD "O" type specific vaccine, 1:4000 dilution of conjugate and incubation of 4º C for coating the virus showed good results. In each experiment, plateau region, test back ground and plate back ground was recorded. Results of the study will help in establishment of an economical, sensitive, reliable indirect ELISA that subsequently be helpful to understand the percent prevalence of FMD serotypes in Pakistan and efficacy of FMD vaccines.
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4.
Passive Immunization Against Canine Distermper Virus In Dogs
by Ali Ahmed Malik | Prof. Dr. Masood Babbani | Prof. Dr. Khushi Muhammad | Faculty of Veterinary Sciences.
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Publisher: 2005Dissertation note: Canine distemper is an important, highly contagious disease of dogs, caused by morbillivirus of family paramyxoviridae. The disease occurs worldwide in variety of hosts. In the present study, data relative to breed, sex and age susceptibility in clinically suspected cases of canine distemper was collected and analyzed. The disease is mostly seen in young nonvaccinated dogs of 4 to 6 months of age when maternal anti-CDV antibodies are decreased. Immune serum was raised in experimental dogs with commercially available measles live virus vaccine. The level of antibodies in the immune serum was determined by agar gel precipitation test (AGPT) and an ELISA based assay. Immune serum containing 128 AGPT units of anti-CDV antibodies was effective to control the disease in infected dogs after natural exposure to canine distemper virus. Finally the effective time for passive immunization against canine distemper was determined in experimental dogs. It was noted that immune serum offered protection to canine distemper immediately after infection, during the incubation period of the disease , 48 hours after infection and early phase of the disease(at the appearance of clinical signs). Passive immunization is not rewarding in the terminal phase of the disease (when infected dogs show nervous signs of the disease).Thus it is very useful for the prevention of disease in dogs kept with infected dogs in kennels and pet shops.
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5.
Standardization Of Indirect Sandwich Enzyme Linked Immunosorbent Assay For Detection Of Foot And Mouth Disease
by Muhammad Mujahid Amjed | Prof. Dr. Khushi Muhammad | Professor Dr. Masood Rabbani | Faculty of Veterinary Sciences.
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Publisher: 2005Dissertation note: Foot and Mouth Disease (FMD) is one of the most troublesome and infectious diseases of livestock, caused by the FMD virus. In this study Indirect Sandwich Enzyme Linked Immunosorbent Assay (IS-ELISA) was standardized to characterize the FMD serotype "0" virus. Oil based FMD serotype "0" vaccine was prepared and injected at the neck region of guinea pigs and rabbits. The vaccine induced anti-FMD serotype "0" virus antibodies in the vaccinated animals after 21 days post boosting. The serum thus separated was purified through ammonium sulfate salt (NH4)2S04 and ion exchange column chromatography. Total protein content in the guinea pig serum (whole serum), Ammonium Sulfate Precipitated Guinea Pig Serum (ASPGPS) protein and Ion Exchange based purified Guinea Pig Serum (IEGPS) protein when analyzed through spectrophotometer at 280 nm and 260 nm was found to be 52 ug/mi, 24 ug/ml and 10 ug/mi respectively. Virus Neutralization (VN) test was performed to monitor the neutralizing antibody titer. The whole serum of guinea pigs and rabbits showed the 1:32 and 1:64 anti-FMD serotype "0" virus neutralizing antibody titers. While anti-FMD serotype "0" virus neutralizing antibody titer was 1:128 in the IEGPS proteins. IEGPS protein with 1:128 neutralizing antibody titer were used as capture/trapping antibody in the standardization of the assay. The IEGPS protein 1:1000 diluted with 10 ug/ml of protein content was found to be optimum as capture/trapping antibody. To cover residual blank spaces, different available blocking buffers were evaluated and Skimmed Milk Solution 5 % in Phosphate Buffered Saline (PBSSKIVI-5%) proved best amongst blocking buffers. Coating of 1:1000 diluted IEGPS at 37 °C for 1 hour followed by storage at 4 °C for overnight was best incubation time in the study. FMD serotype "0" virus 1:100 diluted was optimum in IS-ELISA. Similarly rabbit anti-FMD serotype "0" virus specific immune serum 1:10,000 diluted and goat anti-rabbit IgG horseradish peroxidase conjugate 1:4000 diluted were found to be optimum during the standardization of the assay. Lastly ELISA plates were proved to be best amongst the available plates for assay. In each experiment, plateau region, test background and plate background were recorded. Results of the study helped for establishment of an economical, sensitive, reliable, robust IS-ELISA technique in research and diagnostic laboratories in the country.
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6.
Studies On In Vitro Culture Characteristics Of Adherent Baby Hamster Kidney-21 (Bhk-21) Cell Line
by Saddeq-ru-Rahman | Prof. Dr. Masood Rabbani | Prof. Dr. Khushi Muhammad | Faculty of Veterinary Sciences.
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Publisher: 2005Dissertation note: Baby Hamster Kidney-2 1 (BHK-2 1) cell line growth pattern, growth requirements, growth effectors, cryopreservation and its susceptibility to different animal viruses were studied to optimize the in vitro culture requirements and conditions for maintenance and long time cryopreservation in liquid nitrogen of this cell line. It was observed that BHK-2l cells multiplied fast during first 48 hours and made a complete monolayer after getting confluency with in 72 hours post incubation which was followed by a decline phase. Fetal calf serum has growth stimulating effect and 5 - 10% serum level was satisfactory for the maintenance of cell line. While harvesting the cells from a flask, Trypsin (0.25%) with neutralization by fetal calf serum (5-10%) was found better. For cell storage 10% Dimethylsulfoxide (DMSO) through gradual cooling maintained maximum recovery of viable cells during cryopreservation.
Footh and mouth disease virus (FMDV; serotype "0" and "A") were adapted to cell this cell line, while canine parvo virus (CPV), Newcastle disease virus (NDV LaSota strain), canine distemper virus (CDV), and hydropericardium syndrome virus (HPSV) could not adapted to this cell line through five blind passages in this study.
Availability: Items available for loan: UVAS Library [Call number: 0916,T] (1).
7.
Preparation And Evaluation Of Cell Culture Vaccines Against Hydropericadium Syndrome Virus In Poultry
by Jamshid Akhter | Masood Rabbani | Prof. Dr. Khushi Muhammad | Faculty of Veterinary Sciences.
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Publisher: 2005Dissertation note: In this study a total of 9 vaccines were prepared, 6 vaccines were prepared from cell culture passaged HPS virus having TCID50 i' and inactivated with formalin and binary ethyleneimine (BET ) with and with out different adjuvant combinations. While other 2 vaccines were prepared from more diluted virus suspension with PBS (10 and 100 times) and were inactivated by binary amine and adjuvant was oil base. The 9th vaccine was prepared from infected liver homogenate (aqua base) and inactivated with formalin. These vaccines were evaluated in 10 groups of day old broilers (100 chicks) for their comparative immunogenicity and protection. At day 14 of age, groups Al, B 1, Cl, and C were vaccinated with four oil base vaccines with different virus concentration and different inactivants. Similarly groups Dl & D were vaccinated with aluminized vaccines and groups A, B, and El with non adjuvant vaccines. Groups E was kept as unvaccinated control group. Serum samples were collected from all groups on 0, 14, 28 and 42 day of age and subjected to AGPT for seroconversion. AGPT GMT results indicated difference for different virus concentrations and no difference for different virus inactivants but same adjuvant. The AGPT GMT recorded 0 & 14 day of age pre vaccination indicated the maternal antibodies against HPS in chicks were not protective level. The chicks became protective against the disease in most susceptible age. The AGPT GMT recorded 14 and 28 days post vaccination indicated the highest and more consistent (149.2 and 182) for oil base vaccines with virus concentration having TCID50 104.1 but for vaccines of diluted virus suspension then GMT was variable. Similarly aluminized vaccines showed (149 and 94.4) and non adjuvant cell culture vaccines showed (116 and 2.7) while non adjuvant liver homogenate showed (80.5 and 2.3). On day 42 of age, all birds were challenged with virulent HPS virus and percentage mortality and percentage protection for each group were recorded. Lowest mortality (0%) and highest protection (100%) were recorded for groups vaccinated with oil base vaccine. There was zero mortality and 100 percent protection were recorded for group Dl (BET inactivated) while in group D (formalinized) there was 10 percent mortality and 88 percent protection in groups vaccinated with aluminized vaccines. While mortality and protection in groups vaccinated with non adjuvant cell culture vaccines were 25% and 71.5% while in group vaccinated with non adjuvant liver homogenate vaccine was 50% and 44%, respectively. Cell culture oil base vaccine against HPS virus, having io' TCID50 inactivated with BET was concluded the best in experimental trails and has been recommended for commercial production after field evaluation.
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8.
Standardization Of An Indirecto Enzyme Linked Immuno Sorbent Assay Elisa For Measuring Antibodies Of Infectious Bursal Disease Virus
by Faisal Amin | Dr. Mansur-ud-Din Ahmad | Prof. Dr. Khushi Muhammad | Faculty of Veterinary Sciences.
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Publisher: 2005Dissertation note: This project was conducted to make an attempt to develop an in-house ELISA to measure antibodies against IBDV. ELISA is the most commonly used serological test for evaluation of IBDV antibodies, but the cost of imported ELISA kit is usually very high and not affordable by the average farmer. The present study was designed with an aim to develop a cost effective ELISA kit under local conditions.
Various steps involved in the development of ELISA were standardized. Two types of antigens i.e. "A" and "B" were used. Antigen "A" was prepared by propagating the live IBD virus (D-78, Intervet) in CEF cells and further concentrated by dialysis against PEG-6000. Antigen "B" was the reconstituted live IBDV vaccine. Both the antigens produced acceptable and comparable results but antigen "B" is conventional due to ease of preparation and to avoid a time consuming and costly procedure of cell culture. The optimum dilution for antigens "A" and "B" were found to be 1:300 and 1:600 respectively. The optimum dilution of conjugate was selected as 1:2000 and incubation time was standardized as 30 minutes at room temperature (25-30°C) after addition of ABTS as substrate. Standard curve (two fold dilution of the positive sera up to 1 1th dilution) was constructed and 3 standards were selected to cover the range of strong reactor with non-reactor sera.
The in-house developed ELISA was evaluated with field chicken sera samples of different age groups. The sera samples of different groups (A, B, C and D of 1-day-old broiler breeder chicks, 1-day-old broiler chicks, 13 weeks old vaccinated layer breeder bird and 30 weeks old vaccinated broiler breeder birds respectively) were evaluated with in house ELISA and its efficiency was compared with commercially available ELISA kit. The samples that were either positive (strong or weak) or negative with commercial ELISA kit also had similar pattern with in-house ELISA. Groups A, C and D were strongly positive while group B was found negative for IBDV antibodies.
It is concluded that in-house developed ELISA is comparable with the commercially available kits.
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9.
In Process Quality Control Factors Affecting Efficacy Of Hydropericardium Syndrome Virus Vaccine
by Muhammad Danish Mehmood | Prof. Dr. Khushi Muhammad | Dr. Irshad Hussain | Prof. Dr. Zafar | Faculty of Veterinary Sciences.
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Publisher: 2007Dissertation note: The objective of this project was to study in process quality' control factors affecting the efficacy of Hydropericardium Syndrome virus vaccine in broilers. The parameters studied were mortality and protection percentage, seroconversion and affect of HPS infected liver homogenate vaccine on body weight gain of broiler birds. In this different vaccines were prepared from HPS infected liver homogenate having different biological titer (105.6,104.6 and 103.6 units of infectivity Animal Lethal Dose 50-ALD50) inactivated with 0.15% formalin. The other type of Hydropericardium Syndrome vaccine was prepared from chicken embryo hepatocytes having biological titre 1036 tissue culture infective dose-TCID50. At day 14th of age, groups Al, A2 and A3 were vaccinated with HPS infected liver homogenate aqueous based vaccines having different biological titre. While groups Bl, B2, B3, B4 and B5 were vaccinated against 20, 25, 30, 35 and 40 doses per gram of HPS infected liver homogenate vaccine and groups Cl, C2, C3 were vaccinated with lanolin based HPS vaccine, gel based HPS vaccine, montanide based HPS vaccine respectively. Similarly group Dl, D2 were vaccinated with HPS virus chicken embryo hepatocyte vaccine and HPS liver homogenate vaccine respectively. The group El, E2 and E3 were vaccinated with HPS virus infected liver homogenate vaccine containing preservative (thiomersal sodium) stored for 30, 60 and 90 days respectively. The birds in group F served as uninnoculated controls. The HPS infected liver stored for 0-45 days at -20 C and processed for determination of its biological activity at fortnightly interval. It was observed that HPS vaccine containing more than 104.6 and 105.6 units of the immunogen provided protection to 100% in vaccinated birds. The 20 doses and 25 doses of the gel based HPS vaccine per gram of the liver developed 90% protection in vaccinated birds. Montanide based HPS vaccine provided 100% protection while HPS virus infected homogenate vaccine containing thiomersal sodium provided 80% protection up to 90 days and HPS virus chicken embryo hepatocyte vaccine provided 40%protection in the vaccinated birds. Se3rum samples were collected form all groups on 14 and 28 day post vaccination and subjected to AGPT for seroconversion. Each serum sample when monitored for anti-HPSV antibodies through agar gel precipitation test, showed undetectable titre.
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10.
Effects Of Different Disinfectatnts On Pathogens In Poultry
by Asif Abbas Malik | Prof. Dr. Masood Rabbani | Prof. Dr. Khushi Muhammad | Faculty of Veterinary Sciences.
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Publisher: 2006Dissertation note: Poultry sector is the second largest industry after textile in Pakistan. It is threatened by various diseases i.e; Newcastle disease (ND), Avian Influenza (Al), Colibacillosis, Hydro-pericardium syndrome (HPS) and Infectious bursal disease (IBD, Gumboro). The efficacy of various available disinfectants (Hygen 275 — 2000 H, Virkon S and Aldekol) was tested at 2x, lx and Y2 x dilution against Staphylococcus aureus, Escheria Coil, Newcastle Disease virus and Avian Influenza virus. Each dilution of all the disinfectant was divided into 4 aliquots i.e; a, b, c and d (each of the
aliquot, for each pathogen). Each aliquot were mixed with equal volume of either of the pathogen. The mixture of the disinfectant and the pathogen was incubated at 37°C for a period of 15, 30 and 45 minutes of interaction. The contents were collected aseptically and processed to evaluate the effectiveness of the disinfectants. Disinfectant A (Hygen 275-2000H) showed good bactericidal as well as virucidal activity at 1% dilution. Disinfectant B (Virkon S) was able to kill all the bacteria and viruses even at 0.25 % dilution. While, disinfectant C (Aldekol) effectively killed the bacteria and viruses at 0.5 % and I % dilutions. Results of the study will help the farmers to adopt effective biosecurity measures to minimize the challenges at farm level.
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11.
Dvelpoment And Optimization Of Multiplex Pcr For The Detection Of Avian Influenza Strains In Pakistan
by Mirza Salman Saleem | Asso. Prof. Dr. Muhammad Hanif | Prof. Dr. Khushi Muhammad | Faculty of Veterinary Sciences.
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Publisher: 2009Dissertation note: The pathogenic Influenza A viruses (subtype H5N1, H7N2 and H9N3), are emerging avian influenza (AI) viruses that have been causing global concern as a potential pandemic threat. Some forms having zoonotic importance (H5N1 and H7N7). So it is a matter of priority to develop quick and efficient methods for detection of Influenza viruses.
For the detection of avian influenza, HA (haemagglutination) test and HI (haemagglutination inhibition) tests are being used for long time. But studies have shown that Influenza virus shows variability and diversity and a high rate of mutation, which makes diagnosis difficult. For this reason the reverse transcriptase PCR (RT-PCR) assays are considered to be a helpful tool.
In this study design, a multiplex RT-PCR strategy was optimized and developed for the detection of AI virus (subtypes H5, H7 and H9). Primers were designed from sequence available Influenza Database (IVDB) for Pakistan and neighboring regions. The primers were annealed at different temperatures so as to optimize a temperature at which all three primers can amplify their respective subtypes. The results clearly indicated that a multiplex RT-PCR is a quick and efficient method for the detection and it is also economical as fewer reagents are utilized. The PCR products of the reaction can potentially be used to provide additional information about strain variation, either by restriction analysis or PCR product sequencing.
The core objectives achieved are the development of an efficient and economical method for detection of avian influenza viruses by designing indigenous primers and optimization of a multiplex RT-PCR for the avian influenza virus.
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12.
Identification And Genotyping Of Vp1 Genses Of Fmd Viruses
by Atia Bukhari | Prof. Dr. Irshad Hussain | Prof. Dr. Khushi Muhammad.
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Publisher: 2009Dissertation note: Within two decades after its first report in 1954 from Pakistan, Foot and mouth disease has become endemic in the country and poses a serious threat to large as well as small ruminant population. Foot and Mouth Disease (FMD) is prevailing in cattle and buffaloes and is caused by either 0, A, Asia-i serotype of the FMD virus in Pakistan. The present study was undertaken to study the mutation rate of FMD virus and also molecular typing of the strains prevalent in Pakistan was done.
A total of 60 samples from buffalo and cattle were collected from five districts of Punjab including Lahore, Faisalabad, Sialkot, Okara and Sheikhupura. Soon after extraction of their RNA, all of them were reverse transcribed and then subjected to amplification by using different sets of the primers including universal as well as serotype specific primers. Then their VPI portions were amplified by using VP1 specific primers. Among 60 samples, 48 were positive with universal primers. Other 12 samples were not amplified with these primers hence not processed.
Among 48 FMD positive samples, 24 were positive with serotype 0 specific primers, 16 with serotype Asia-i and remaining 8 were positive with serotype A specific primers. After their amplification, the amplicons were run on the gel. These amplicons were extracted by using DNA extraction kit. After their purification, they were sent to Macrogen® (Seopl, Korea) and Centre of Excellence for Molecplar Biology, Pakistan (CEMB) for sequencing. Each amplicon was sequenced thrice and the consensus sequence was established eliminating sequencing errors.
Sequence identity and multiple sequence alignment of molecular sequences (nucleotide and amino acids) were performed with Clustal W algorithm (Thompson et al., 1994). Neighbour joining trees were constructed by using MEGA version 4.0 (Kumar et al., 2004). Nucleotide distance matrices were computed by Kimura two parameter algorithm based on the total nucleotide substitutions and evolutionary trees for VP1 genes were constructed.
For FMDV serotype '0' phylogenetic analysis, 14 VPI sequences from various field isolates were compared with some previously published Pakistani FMD 0 type VP1 specific sequences available with GeneBank and some recently published VP1 sequences reported by countries bordering with Pakistan including India, Iran and Afghanistan Similarly, 12 VP 1 sequences of FMDV serotype Asia-I isolates of this study were compared with previously published sequences and their phylogenetic relationship was established. However, the sequencing results of serotype A were inconclusive and were not included for phylogenetic analysis. Three sequences of three locally available FMD vaccines were also studied and compared with the outbreak strains.
Polymerase chain reaction was optimized with respect to MgCI2, buffer pH, annealing temperature, primer concentration, template concentration, and Taq polymerase. A concentration of 2.5 mM of MgCl2 resulted in the best amplification of the target sequences (Figure 1). The buffer with pH 8.8 yielded the best results (Figure 2) Although, the suggested annealing temperatures for various primers (of various serotypes) ranged from 48 °C to 63 °C, however, a temperature of 56 °C was found to be the best with all sets of primers (Figure 3). The best intensity DNA bands were observed with 0.3 pM concentration of the primers (Figure 4). Moreover, the best cDNA template concentration giving optimum amplification was found to be 3.0 p1 per reaction (Figure 5). Lastly, a concentration of 0.5 U of Taq polymerase was not sufficient for amplification of cDNAs, however, 1.0 U of enzyme was found to yield better amplification (Figure 6).
VP 1 DNA sequences of six previously published Pakistani FMD serotype 0 strains were analyzed phylogenetically with VP 1 DNA sequences of 14 isolates of the study. Serotype 0 isolates of this study distributed themselves into two distinct clusters (Figure 19). First cluster comprised of Sheikhupura 1 and 2, Muridkey 1, Raiwind 1, Nankana 1, Gujranwala 1 and Gujrat I isolates (Figures 19 and 20), whereas the second cluster included Depalpur 1, Sahiwal 1, Okara I, Multan 1, Toba 1, Faisalabad I and Pattoki 1 isolates (Figures 19 and 21). The first cluster was found to be associated with previously published Pakistani isolates of 2006 mostly. However, it also showed association with Afghanistan's isolates of 2004 (Figure 20). The second cluster seemed to be mostly related to previously published Pakistani isolates of 2003 (Figure 21). The overall grouping of the 14 sequences, when compared with each other, depicted a three clustered phylogram (Figure 22). Serotype 0 isolates from Depalpur, Sahiwal, Okara, Multan, Pattoki, Toba Tek Singh and Faisalabad grouped together into a clan and had more than 85% sequence similarity with each other. The second cluster consisted of isolates of Sheikhupura, Nankana, Raiwind and Muridkey. These sequences had more than 86% similarity with each other. The third cluster consisted of only two isolates which were 100 % similar to each other. However the third cluster had only 74 % sequence similarity to cluster I and 73 % sequence similarity when compared with cluster 2.
When the phylogenetic relationships with previously reported isolates of Asia 1 was evaluated, FMD Asia I isolates of this study were found to be scattered into two distinct groups (Figure 16). Group one consisted of isolates of Lodhran, Toba and Hafizabad that were more closely related to Indian isolates sharing more than 98% identity with each other and more than 94 % sequence identity with isolates of Indian 2001 to 2004 (Table 5 and Figures 16 and 17). However, they shared more than 86% sequence similarity with Pakistani isolates of 2002-2005 (Table 5). Group two comprised of isolates of kasur, Lahore, Pakpattan, Okara, Faisalabad, Jhang, Rahim Yar Khan, Bahawalpur and multan alongwith vaccine A and B (Figure 16). The isolates of group 2 were found to be closely associated with previously published isolates of Pakistani and Afghani origin of year 2003 and 2004 (Figures 16 and 18). Collectively, they shared an overall 70% sequence identity with each other. However, isolates of Bahawalpur, Rahim Yar Khan and Multan shared more than 98% similarity with each other, a measurement of close relationship denoting a likely common origin as one clan or dade. Similarly, isolates of Pakpatan, Faisalabad, Okara, Kasur, and Lahore shared 88% sequence identity with each other and qualified as one clade.
Although, overall amino acid sequence similarity of our isolates was not strikingly different from that of the published isolates, however, amino acid substitutions with dissimilar properties were found with a scattered pattern of distribution. For example, 15th amino acid residue which is hydrophilic in the previously published isolates had a substitution with a hydrophobic amino acid residue in our three isolates namely Sheikhupura 2, Muridkey I and Raiwind I (Figure 25). Similarly, 14th amino acid residue which is hydrophobic in nature was found to be replaced with a hydrophilic one in our last five isolates. Amino acid residue number 13 (Figure 25) had a substitution with a hydrophobic residue in some of our isolates etc. etc. It is interesting to note that such substitutions with amino acids having dissimilar properties have also been found, albeit at lower rate, in previously published sequences by many researchers (Figure 25).
A comparison of the deduced amino acid sequences in the critical VP I region of FMD serotype Asia I revealed that most of this study isolates shared very high homology with sequences of Vaccine A. However, the sequences of isolates of Lodhran, Hafizabad and Toba did not match much with that of either vaccines, A or B (Figure 23). Sequences of Vaccine A had a "K" which seemed to be replaced by a "T" in the sequences of most of the isolates. Considering the properties of various amino acids, this change does not signify a major shift in the three dimensional picture of the protein as K is a lysine, a positively charged amino acid, whereas a T is threonine, a hydrophilic amino acid in nature. Next substitution in most of the isolates is a "P" for "A" in comparison to the vaccines. Again, it is not a significant change as both P and A share the same property, hydorphobicity. Similarly a K with an R can be substituted without much change in the overall shape of the protein molecule. Next amino acid substitution is a leucine instead of methionine. Again both are hydrophobic in nature; hence their impact on the overall picture is minute, if at all. However, glycine and arginine are two very different amino acids; the former is a hydrophobic amino acid whereas the latter is positively charged one. Such amino acid substitutions may have the potential to make a major impact in terms of the epitopic differences in the capsids of vaccinal and field viruses. A comparison of the deduced amino acids of FMD serotype 0 isolates also exhibited such changes with the vaccinal virus (Figure 24).
Of the three hyper immune sera raised against three different vaccines in rabbits, only one vaccine induced a measureable immune response yielding good precipitation line against various FMD virus antigens.
In summary, RT-PCR for diagnosis of serotypes A, 0 and Asia 1 of FMDV was optimized and could be used for prompt and precise diagnosis of FMD in the country. Although, RT-PCR data pertains to bovines in the current project, but PCR optimization parameters are equally applicable to FMDV infections in other FMD susceptible animal species such as sheep and goat. The combination of PCR and sequencing of the VP1 gene to detect and analyze FMDV in disease outbreaks is fast (less than 6 hours for PCR and about 24 hours for sequencing), and it can give an accurate immunologic characterization of the virus, thus providing a rational basis for choice of vaccine. In fact, the molecular epidemiology of field isolates is a powerful tool to monitor the circulation of viruses (Saiz et al., 1993).
Secondly, various isolates of serotypes 0 and Asia 1 were sequenced along with some vaccinal strains. Sequence similarity tree analysis indicated that most of our isolates were closely related to previously reported Pakistani isolates and to those of neighboring countries such as India, Afghanistan and Iran. Additionally, amino acid sequence similarity data of major immunogenic site that forms 13G-13H loop in FMDV serotypes revealed that serotype Asia 1 vaccinal strain and Asia 1 isolates of this study possessed high degree of similarity suggesting a likely host immune response against the vaccine that may afford some protection against most field isolates of serotype Asia 1 type. Lastly, of three vaccines tested, only one was found to afford protection against field isolates of FMDV suggesting more work on vaccine issue in the country.
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13.
Effect Of Differnet Physico Chemical Substances On The Production Peotential Of Phycocyanin From Spirulina and its Characterization
by Firasat Hussain | Dr. Imran Najeeb | Prof. Dr. Khushi Muhammad.
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Publisher: 2010Dissertation note: Spirulina is a multi-cellular, filamentous Cyanobacterium, belonging to a blue-green alga of Cyanophyta. Spirulina is recently proven in animal experiments to exhibit various biological activities such as lowering plasma cholesterol levels and blood pressure.
The principal phycobiliproteins present in spirulina are phycocyanin and allophycocyanin which are made up of dissimilar ? and ? polypeptide sub units. The fresh biomass was found suitable for phycocyanin extraction. Freezing and thawing of cells was proved the best method for extraction of phycocyanin (0.4mg/ml), as compared to homogenization, hydrochloric acid and sonication. Nitrogen effects phycocyanin production from spirulina.
Different concentrations of nitrogen spirulina medium were provided. Among which 1.875g/L spirulina produced phycocyanin (0.412mg/ml). Phosphate effects phycocyanin production from spirulina. Different concentrations of phosphate spirulina medium were provided.Among which 1.5g/L spirulina produced phycocyanin (0.354mg/ml). There is also effect of temperature on phycocyanin production. Spirulina medium 0.192mg/ml at 25oC, 0.390mg/ml at 30oC, 0.184mg/ml at 35oC. There is also effect of light on phycocyanin production. 0.361mg/ml were produce at 1500 Lux.
Molecular weight (66kDa) of phycocyanin was confirmed by SDS-PAGE and explored potential production of phycocyanin from indigenous spirulina.
Availability: Items available for loan: UVAS Library [Call number: 1204,T] (1).
14.
Characterization Of Indigenious Species Of Mycotoxins Producing Aspergilli
by Gull Naz | Dr. Aftab Ahmad Anjum | Prof. Dr. Khushi Muhammad.
Material type: ; Format:
print
Publisher: 2010Dissertation note: Pakistan's economy is based on agriculture. Agriculture crops are harvested and stored in feed mills for production of thousands ton of feed for livestock as well as poultry through out the year. In Pakistan, July and August are hot and humid months during which moulds grow abundantly on the heaves of wheat! rice/maize straw and feed ingredients and produce variety of toxins.
Present study has been designed to explore different groups of moulds prevailing in and around Lahore city in each month of the year. Samples of soil and air were collected from ten different places of Lahore city.
A total of 240 samples were cultured on a common Saboraud's Dextrose Agar to get single colonies of each mould. These single colonies were identified by colony characters, slide cultures and biochemical tests. Mycotoxin producing Aspergilli were isolated by culturing on specified media and placing the cultures under Wood's lamp. Mycotoxin productions potential were assessed by extracting mycotoxins of these Aspergilli. Mycotoxins produced by the Aspergilli were identified and purified through Thin Layer Chromatography. These mycotoxins were then quantified through High Performance Liquid Chromatography.
The identified and purified mycotoxins can be used as standards. Reference standards are important and critical for qualitative and quantitative detection of mycotoxins in field samples screening. Presently mycotoxin is a ban item. The occurrence of toxinogenic Aspergilli have economic impact directly on livestock and poultry products export.
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15.
Effectof Mentofin (Herbal Product)On Antibody Response Of Broilers To Newcastle Disease Vaccine
by Saif- Ur- Rehman | Prof. Dr. Khushi Muhammad | Dr. Tahir Yaqub | Prof. Dr. Muhammad.
Material type: ; Format:
print
Publisher: 2011Dissertation note: Immunostimulants such as Mentofin® are commonly used to enhance the immune response of birds to vaccines. In the present study immunomodulatory effect of Mentofin® on antibody response of broilers to Newcastle Disease virus vaccine was evaluated. For this purpose one hundred one day old broilers were divided in to four groups (A, B, C and D) each containing 25 birds. Each bird of group A and B was vaccinated against ND and each bird of group A and C was treated with Mentofin. Anti-ND-HI antibody titer of each bird of each group was monitored on 14, 21, 28 and 35 days of age. Mentofin® treated broilers showed higher consistent antibody (anti-NDV HI antibody titer) response as compared to untreated broilers. These birds when given challenge infection of velogenic ND virus on 35 days of age showed same protection as that of untreated vaccinated birds. However non vaccinated broilers treated with Mentofin showed higher protection as compared to that of in non treated unvaccinated birds. Weight gain in Mentofin® treated broilers was same as that of non-treated birds. Similarly, there was no effect of the Mentofin on FCR. Droppings from Mentofin treated birds showed no urease producing bacteria while 100% droppings of the herbal untreated birds showed urease producing bacteria. Mentofin at 1% concentration in nutrient broth inactivated the proteus species while its 0.0001 % concentration inactivated the bacteria in urea broth. In in vitro studies, 0.5 % concentration of Mentofin inactivated the lentogenic strain of ND Virus within 15 minutes at interaction temperature of 37 0C.
The results can be used to formulate the vaccination policy along with herbal based immunostimulatant such as Mentofin®.
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16.
Passive Immunization Of Infectious Bursal Disease Virus Infected Birds Using Chemically Purified Immune Yolk
by Ammara Akram | Prof. Dr. Masood Rabbani | Prof. Dr. Khushi Muhammad.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2011Dissertation note: Infectious bursal disease (IBD) is a major killer disease of poultry. It is also known as
Gumboro disease where the disease was reported first time. It is double stranded RNA
virus belongs to the family birna viridae. This disease is quite endemic in Pakistan
which has huge impact on poultry industry. Besides vaccination if immune yolk is
properly harvested and purified it can be used for treating of IBDV infected birds.
Therefore this work has been outlined to study the effectiveness of immune yolk in
experimentally produced IBDV infected birds. Refinement of yolk IgY from egg yolk
of immunized hens. Suitability in using hyperimmune egg yolk in IBD infected bird
in field conditions. In order to get hyper immunized egg yolk 20 commercial layers
were raised in poultry shed of the university. They were supplied with fresh water and
feed ad libitum with proper hygienic condition. The birds were vaccinated with oil
based killed Gumboro vaccine twice at 15 days interval at the age of 26 weeks to get
immune yolk. Eggs were collected at two weeks interval till two and half months after
boosting. Immune yolk was purified by chemical means. Antibody against IBD in egg
yolk and semi purified egg yolk IgY was measured by indirect ELISA kit method.
The eggs which were collected 15 days interval after boosting had the highest
antibody titre which decline with the passage oftime and lowest was recorded 75 days
after boosting. Similar pattern of results were also observed in semi purified egg yolk.
However significant antibody titre was lost during purification process. 50
commercial chicks of 15 days old were purchased and they were reared in poultry
shed in the university up to 36 days. They were splitted in eight groups and two
experiments were carried out side by side. In experimental chicks the birds were
challenged with the Gumboro infected bursal homogenates which were confirmed by agar gel diffusion tests. In first experiment the birds were challenged at the day of 30 days and they were provided with passive therapy of immune yolk and semi purified
IgY after 3 days of challenge. In the second experiment the birds were challenged and
passive immuno therapy was provided 24 hours interval of challenge and concurrently. The birds which received semi purified immune yolk and antibody titre having more or less 4000 they showed 20% mortality in the each group.
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17.
In Process Quality Control Factors Affecting Sensitivity Of Rapid Serum Agglutination Antigen Of Mycoplasma
by Rana Khurram Khalid | Prof. Dro. Masood Rabbani | Prof. Dr, Khushi Muhammad.
Material type: ; Format:
print
Publisher: 2012Dissertation note: Mycoplasma gallisepticum (MG) is one of the smallest self replicating infectious agent, it lacks cell wall so cell membrane is the outer most boundary. Its cell membrane is made up of sterols which not only gave it rigidity but also make it fastidious to grow. MG is the causative agent for chronic respiratory disease (CRD). Isolation identification and serodiagnosis are routinely used in laboratories. However among the serodiagnosis rapid serum agglutination (RSA) test is mainly used for early detection for its sensitivity, rapidity and cost effectiveness.
Keeping in VIew the importance of RSA antigen test a study was conducted to prepare
standardize RSA antigen from local isolate and compare its sensitivity with a commercial RSA antigen. Molecular characterized local isolate of MG procured from University Diagnostic Lab University of Veterinary And Animal Sciences, Lahore, was grown in 11 different media formulations to identify a media with better antigenic yield. Affects of different in process quality control parameters; bacterial concentration (0.75%, 1 %,
1.25% PCV), diluents (normal saline, PBS, HBSS) inactivants (heat, formalin) and preservatives (thiomersal sodium, sodium azide, phenol) were studied in terms of their influence on the sensitivity of prepared RSA antigens. These prepared antigens from local isolate were further compared with a commercial RSA antigen. The results of antigenic yield were statistically analyzed through One Way A OVA test. All the
media formulations support the growth of MG isolate except Frey's media with 7.5% fetal
bovine serum and 7.5% chicken serum that revealed no growth or packed cell volume. The
maximum bacterial growth in terms of packed cell volume was obtained from frey's media with 12% horse serum. So this media was further use in the production of antigens. Sensitivity of RSA antigens with different bacterial concentrations in terms of packed cell volume, preservatives, inactivants was more using HBSS followed by PBS and normal saline.
However difference in increasing the sensitivity of RSA antigen in terms of agglutination with
known serum was statistically found significant. Formalin inactivated RSA antigens were
somewhat more sensitive as compared to the heat inactivated using different diluents and
bacterial concentrations. Sensitivity of RSA antigens in different diluents, inactivants and
preservatives was a little bit more with 1.25% pev followed by 1.00% pev however it was least with 0.75% Thiomersal sodium added RSA antigens were more sensitive followed by phenol and sodium azide. PBS and HBSS based, formalin inactivated and preservative (thiomersal sodium, phenol, sodium azide) added antigens having bacterial concentration of 1.25% and 1 % (peV) given agglutination at 1 :30 dilution of known positive MG serum. The sensitivity of all these antigens was equal to the commercial RSA antigen. All the prepared RSA antigens and commercial anitigen showed no agglutination with known negative serum. Sensitivity of most of the prepared and a commercial antigen was comparable. The findings of this study will be helpful for further recommendations of local RSA MG antigen used in the diagnosis of chronic respiratory disease.
Availability: Items available for loan: UVAS Library [Call number: 1406,T] (1).
18.
Comparison Of Diagnostic Approaches For The Detection Of Bovine Viral Diarrhea Persistency In Dairy Herds
by Arfan Ahmad | Prof. Dr. Masood Rabbani | Prof. Dr. Khushi Muhammad.
Material type: ; Format:
print
Publisher: 2012Dissertation note: Bovine viral diarrhea is one of the most important diseases of cattle which are causing
continuous economic losses to the cattle industry primarily due to decreased reproductive
I performance. Without doubt, direct contact between BVDV persistently infected, and susceptible animals is the most important transmission route of virus. All control programs which are in use in many countries of the world, mainly depend upon the detection of PI animals, eliminating them and preventing their return into the herds. Therefore, in this study diagnostic suitability of ear notch biopsies and serum samples were compared for the detection of PI animals, as well as proficiency of various diagnostic approaches like VI, AC-ELISA, IHC and real time RT-PCR were evaluated using ear notch biopsies. A total of 468 samples were collected from 12 participating dairy cattle farms located at Prince Edward Island, Canada. The samples were divided into two groups on the basis of age, A " 6 months), and B (> 6 months).
PI calves remain immunotolerant to the infecting strain but if exposed to a heterogonous strain postnatally, they may develop low level of antibody. Accordingly, serum neutralization was applied for initial screening of samples for further testing. The samples of animals of group B, having SNT (:S 1 :64) were selected, while all samples of younger aged group A were processed without considering the serum neutralizing titres, because unlike older animals, P.1. animals below 6 months of age can have passive colostral antibodies in the course of persistency. Diagnostic suitability of ear notch biopsy and serum sample for confirmation of BVDV
A significant discrepancy was observed between ear notch biopsies (51198 positive) and
serum samples (71198 positive) during first round of testing by real time RT-PCR. However, on
follow up testing, 30 days post first round of testing, a complete agreement between ear notch
biopsies and serum samples was observed. On second round of testing, a total of 4 animals out of
197 (one positive animals died before re-sampling) were confirmed with PI, using both ear notch
biopsies and serum samples. The decrease in the positivity using RT-PCR on serum samples in
the second round of testing reflected the presence of 2 transiently infected animals. Ear notch
biopsy (EN) testing did not detect any transiently infected animal indicating the lack of
delectability of the virus in EN during transient infection under conditions of this study. After
follow up testing, 2 animals in each of group A and B were identified as PI. These findings have
led us to conclude, that either serum or ear notch biopsy can be used for the detection of
persistent infection. Of 468 collected and 197 tested samples, an overall 0.85% and 2.03%
prevalence of PI animals with BVDV was observed respectively. A complete agreement (P value=l) was observed when three diagnostic approaches (Real time RT- PCR, AC-ELISA, and IHC) were compared with standard of VI. A total of 197 ear notch biopsies (145 of group A and 52 of group B) were tested by the four diagnostic tests, four animals (2 from group A and 2 from group B) were found positive by all the tests applied. A complete agreement was observed between the first and the second round of testing. All four assays were found specific but real time RT-PCR was found to be more sensitive. Both, VI and IHC were found labour intensive, as diagnosis may take more than one week to be made. Further PI calves remain immunotolerant tothe infecting strain but if exposed to a heterogonous strain postnatally, they may develop low leved
ofantibody. Accordingly, serum neutralization was applied for initial screening of samples for further testing. The samples of animals of group B, having SNT (:S 1 :64) were selected, while all samples of younger aged group A were processed without considering the serum neutralizing titres, because
unlike older animals, P.1. animals below 6 months of age can have passive colostral antibodies in the course of persistency. Diagnostic suitability of ear notch biopsy and serum sample for confirmation of BVDV persistent animals were evaluated by real time RT-PCR. TaqMan probes and primers specific for BVDVI and BVDV2 were used. They were found specific and able to detect 10·s and 10-4 TCID50 units ofBVDVI and BVDV2, respectively.
Availability: Items available for loan: UVAS Library [Call number: 1407,T] (1).
19.
Monitoring Of Mixed Vegetable Salads For Microbial Quality
by Sana Hameed | Dr. Aftab Ahmad Anjum | Prof. Dr. Khushi Muhammad.
Material type: ; Format:
print
; Nature of contents: ; Literary form: Publisher: 2012Dissertation note: The present research was planned to investigate the microbial load and
identification of bacteria in mixed vegetable salads from three different locations such
as road side vendors, fast food outlet and family restaurants. Total 90 samples were
collected 30 from each location. Samples collected carefully in sterilized plastic bags
and processed in microbiological lab of UV AS Lahore. The results were compared
with different food standards given by developed countries like UK.
In present study results showed that for aerobic plate count 30 samples from
road side vendors was in range of 105_ 1014, fast food outlets range of 105_ 1012 and
family restaurants range of 105_ 1013. Coliform count from road side venders was in
range of 105_ 1013, fast food outlets range of 105_1012 and family restaurants range of
105 - 1013. Fungal count from road side vendors was in range of 103_106, in fast food
out lets range of 103 - 106 and in family restaurants range of 103 - 106.
In our study from road side vendors 12 Salmonella, 24 Aeromonas, 11
Bacillus, 29 Entarobacter spp, 29 E.coli, 30 Staphylococcus aurues 29 Klebsealla spp
5 Aspergillus fumigates, 10 Aspergillus niger and 3 Aspergillus flavus have been
identified. In fast food outlets 26 Salmonella, 21 Aeromonas, 9 Bacillus, 30
Entarobacter spp, 30 E.coli, 30 Staphylococcus aurues 30 Klebsealla spp and 9
Aspergillus fumigates and 2 Aspergillus flavus have been identified. In family
restaurants 21 Salmonella, 15 Aeromonas, 9 Bacillus, 30 Entarobacter spp, 30 E. coli,
30 Staphylococcus aurues 30 Klebsealla spp 4 Aspergillus fumigates, 3 Aspergillus niger and 4 Aspergillus flavus have been identified.
Availability: Items available for loan: UVAS Library [Call number: 1413,T] (1).
20.
Microbiological Quality Of Commercial Fruit Juices Sold In Lahore City
by Muhammad Naeem Iqbal | Dr. Aftab Ahmad Anjum | Prof. Dr. Khushi Muhammad.
Material type: ; Format:
print
Publisher: 2012Dissertation note: Fruit juices are used for their nutritional value, thirst quenching properties and stimulating effect or for their medicinal values. Due to poor hygienic conditions during processing and packaging, fruit juices are becoming a health hazard. Many outbreaks are caused by consuming poor quality juices. Food borne infections are caused by eating food or drinking beverages contaminated with bacteria and parasites. Most of the food borne infections remains undiagnosed and unreported due to poor documentation system and failure in the implementation of law regarding food borne diseases in Pakistan. The present study was conducted to compare the quality of commercial fruit juices so as to provide data for local authorities to deal food security issue. A total of ninety packed fruit juice samples were obtained from retail shops in Lahore city. The fruit juice samples included, apple, mango and orange juices of five various brands. The pH value of the fruit juices measured using pH meter was found between 2.0 to 4.0. Bacterial load of fruit juices was assessed using Total viable count, Staphylococcal count and Coliform count to compare the quality of fruit juices.All the samples were positive for total viable count, 60 samples were positive for staphylococcus count and 30 samples ere positive for coliform count.The mean total viable count in fruit juice samples was3.70xIQ5CFU/ml (log 5.56±1.47CFU/ml)with the range from log 2.69 to log 7.67CFU/ml.Mean staphylococcal count in fruit juice samples was 1.34x 102CFU/ml (log 2.11±1.97CFU/ml) with the range from log 0.00 to log 5.62CFU/ml. Sixty out of 90 fruit juice samples showed staphylococcal counts.Mean coliform counts of 1.80xlOI CFU/ml (log 1.25±1.57CFU/ml) with the range from log 0.00 to log 5.50 CFU/ml. Thirty out of 90 fruit juice samples were positive for coliforms. Identification of bacteria was done on the basis of culture characters, microscopic characters and biochemical tests as per standard protocols described in Manual of Food Microbiology. Among the 226 bacterial isolates, Bacillus spp. were (150),Staphylococcusaureus (49)and E. coli (27), and no Salmonella were detected from the collected samples. Although fruit juices have low pH, still higher viable counts and prevalence of bacterial isolates suggest poor hygienic conditions during manufacturing procedures. Antimicrobial sensitivity profile of the isolates was studied by standard Disk diffusion method (Kirby Bauer method) for commonly used antibiotics. Among various antibiotics used, highest97.78% resistance toAzlocillinand lowest 25.22% resistance against Sulphafurazole. These findings suggest that the antibiotic resistance is transferred through fruit juices. After microbiological examination, it was cleared that fruit juices were as contaminated as compare to our country standards and hygienic conditions.
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21.
Physico-Chemical Factors Affection Survival Of Mycoplasma Gallisepticum
by Javed Muhammad | Dr. Aftab Ahmad Anjum | Prof. Dr. Khushi Muhammad.
Material type: ; Format:
print
Publisher: 2012Dissertation note: Poultry industry is second largest industry in Pakistan. Mycoplasma gallisepticum causes chronic respiratory disease in poultry and has a great impact on economy of country. The present study was conducted to check the effect of physical factors including pH, temperature, ultra violet light (UV), atmospheric condition and sodium chloride, chemical factors including formalin and sodium hypochlorite and extracts of herbal plants including Garlic, glycyrhiza and Neem on survival of Mycoplasma gallisepticum (MG). Referenced isolate of MG received from University Diagnostic Lab (UDL), University of Veterinary of animal Sciences, Lahore was used in Bacterial count 0.1 at optical density 450 equal to approximate 108 cfu/ml was used in the entire experiment.
Survival of MG at pH level 4.8 and 10.8 is significantly lower (p?0.05) as compared to pH level 7.8. Optimum pH was found 7.8 showing best growth while pH 10.8 indicated more lethal effect on survival of MG as compared to 10.8. Temperature study showed that MG exposed to 43°C more lethal effect on survival as compared to 31°C while showed growth occurred at 37°C. Ultra violet light (254nm) showed significant effect (P?0.05) on viability at a distance of 2, 4 and 6 centimeter which indicated that MG at 2 cm from UV light leading to death with increase in exposure time. Survival of MG was best in presence of 5 to 10% CO2 or candle jar as compared to incubate in closed container or without closed container and candle jar (open air). Sodium chloride 3 and 5 percent occupied a drastic effect on MG viability but resistance was existed up to some extant to 1 percent.
Culture of MG exposed to formalin 0.1 and 0.2 percent for 15 minutes resulted in high lethal effect significantly (p?0.05) as compared to 0.05 percent. Non significance difference (P?0.05) was present between 4 and 6 percent sodium hypochlorite in terms of effect on survival of MG and has lethal effect when exposed for 5 minute which differ significantly from 2 percent which resulted in death after exposing for 10 minutes.
Glycyrhiza and Neem indicated minimum inhibitory effect against MG with similar concentration of 6.25 mg/mL while garlic stop growth at concentration of 3.125 mg/mL.
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22.
Molecular And Serological Characterization Of Avian Influenza Viruses In Domestic And Wild Birds
by Mobeen Sarwar | Prof. Dr. Khushi Muhammad | Prof. Dr. Masood Rabbani.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2012Dissertation note: Avian Influenza virus (AIV) has been recognized one of the most important pathogen in poultry industry. AIVs play an important role in the pandemic spread that could cause high morbidity and mortality in human beings. A total of 1500 tracheal and cloacael swabs were collected from the seven live bird poultry markets of Lahore Pakistan for surveillance of AIV. The samples were processed for virus isolation in chicken embryos. The isolates were processed for HA test and AIV Antigen Rapid Test Kit to differentiate Newcastle disease virus and Avian influenza virus. Only four samples were positive for Avian influenza H9N2 subtype and 17 were positive for Newcastle disease virus. Four HA virus suspension of AIV showed high titers of anti AIV H9N2-HI antibody titer in chicken as well as rabbit serum, raised using Ottoman Pharma AIV-H9N2 (Oil based) vaccine.
The isolates of AIV H9N2 were confirmed using laboratory optimized RT-PCR, mRT-PCR and LAMP tests. All isolates were sequenced and analyzed to develop phylogenetic relationship. Two isolates A/Chicken/Pakistan/Micro-1/2009 and A/ chicken/ Pakistan/ Micro-2/ 2009 showed 99.1% nucleotide homology with each other and 95- 99% homology with the other Pakistani isolates, 95.1% homology with A/Chicken/Iran/B102/2005. The nucleotide sequence of "A/Duck/Pakistan/Micro-3/2009" showed 98.8% homology with "A/chicken/Pakistan/micro-4/2009", 98-98.7% homology with other Pakistani Isolates and 95.8% homology with "A/Chicken/Iran/B102/2005". The nucleotide sequence of "A/Chicken/Pakistan/Micro-4/2009" showed 99.6% homology with "A/duck/Pakistan/micro-3/2009", 95-96% homology with other Pakistani isolates and 94.3% homology with "A/Chicken/Iran/TH lBM863/2007".
Three out of four isolates had PARSSRGL cleavage sites and one isolate A/chicken/Pakistan/micro-1/2009 had PAKSSRGL cleavage site. The four isolates of the study contained a 226-Leu and 228-Gly at receptor binding sites. Substitution of Glutamine (Q) into Leucine (L) at 226 receptor binding site in HA glycoprotein increased the binding specificity for Sialic acid ? 2, 6 Galactose linkage of human receptor. The HA gene of the live poultry market isolates had 7 predicted glycosylation sites at 29-32, 105-108, 141-144, 298-301, 305-308, 492-495 and 551-554, positions. The NA gene of the live poultry market isolates had 8 predicted glycosylation sites at 44-46, 61-63, 69-71, 146-148, 200-203, 234-237 and 402-403, positions. Predicted glycosylation sites affected the structure and stability of NA protein.
It is concluded that continuous epidemiological and virological surveillance of live bird poultry markets may help scientists to develop an effective control and preventive measures for AI viruses.
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23.
Epidemmiology Of Foot And Mouth Disease In Buffaloes Of Punjab Province
by Farhat Nazir Awan | Prof. Dr. Khushi Muhammad | Prof. Dr. Muhammad Akram Muneer.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2009Dissertation note: This study indicates that the ranking order of buffalo diseases, with respect to their incidence in descending order in Punjab province is Foot and Mouth Disease, Mastitis, Diarrhea, Haemorrhagic Septicemia, Sudden Death Syndrome and haemoglobinuria. Similarly the disease ranking order in cattle in descending order is FMD, Mastitis, Diarrhea, Hemorrhagic Septicemia, Haemoglobinuria and Sudden Death Syndrome. FMD is top most economic important disease both in buffaloes and in cattle in the province.
Morbidity rate in the adult cattle and buffalo was higher as compared to the younger stock. However, the mortality rate was higher in young stock as compared to the adult animals of both the species. Moreover, adult and young males of both the species were more susceptible to the disease as compared to females.
Cross-sectional survey revealed the economic loss of Rs. 41.32 million due to loss of milk, cost of dead animals and treatment cost of sick and complicated cases of FMD. The loss due to milk reduction was 57.3% of the total losses followed by mortality loss (26.4%), morbidity effect expenses (15.2%) and treatment charges in FMD complicated cases (1.0%). The findings of present study clearly indicate the association of age, feeding pattern, vaccination status and season as risk factors in the incidence of FMD in Punjab.
Data obtained from the EPI-Unit Lahore showed that 719 FMD outbreaks occurred in the district of Punjab during 2007-2008. The highest number of outbreaks (212) was recorded in Rahim-Yar-Khan followed by Bhakkar (118), T.T. Singh (81) and Faisalabad (72). Of the total 309 disease outbreaks in buffalo, 174 (56.3%) were recorded in adults, whereas this number in cattle was 169 (61%). The incidences of the outbreaks increased gradually following the post-monsoon period. The greatest number of outbreaks was observed during the winter season, from December to February. Data from FMD Research Center, Lahore revealed the involvement of only FMDV serotype "O" in all the outbreaks during 2007-2008.
Studies of the factors (age, feeding pattern, stage of pregnancy and species) on the immune response of local trivalent FMD vaccine revealed that buffaloes of all age groups responded well to vaccination against disease. It was also observed that 7-9 months pregnant buffaloes elicited significantly lower antibody response to vaccine as compared to the control groups. Similarly, buffaloes on grazing have shown lower anti-FMD-CF GM titer as compared to buffaloes on manger feeding. Sheep and goat were found to be late and poor responder to vaccine as compared to cattle and buffalo.
Analysis of 300 serum samples from FMD affected buffaloes of 12 districts of the Punjab indicated the highest incidence of serotype "O" (62.3%) followed by Asia-1 (32.4%) and "A" (3.30%) in the population tested.
FMD virus was inactivated at 61 ºC within 15 minutes and at pH 4, 8, and 10 within 24 hours. However, ultraviolet radiation was unable to inactivate the virus even after 45 minutes. The disinfectants/chemicals evaluated in this study including sodium hydroxide, sodium carbonate, citric acid, acetic acid, formalin, sodium hypochlorite, virkon-s, aldekol and Gas-G were effective in inactivating the FMDV at recommended concentration levels of 2%, 4%, 0.20%, 4%, 0.15%, 3.0%, 1.0%, 0.50% and 0.1% after 60, 30, 60, 60, 30, 30, 30, 60 and 30 minutes, respectively, at 300C. Sodium hypochlorite and Gas-G were equally good in inactivating the virus at half (1.5% and 0.05%) of the recommended concentration.
Efficacy trial of local and imported oil based trivalent FMD vaccine in six villages, of the Faisalabad district clearly showed that 81.8% of FMD cases were prevented by the local inactivated vaccine in vaccinated animals whereas; this percentage was 70.6 in case where imported vaccines were employed. Moreover, efficacy of the local vaccine was higher than the imported vaccines.
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24.
Factors Affecting Biomass Producation Of Baby Hamster Kidney Cell Line In Roller Bottle Culture System For The Production of Foot and Mouth Diseas Virus
by Qaiser Akram | Prof. Dr. Khushi Muhammad | Prof. Dr. Masood Rabbani.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2012Dissertation note: Foot and Mouth Disease (FMD) is endemic in Pakistan and killed virus vaccines against prevalent serotypes are used for the control of disease. Baby Hamster Kidney (BHK-21) cells as monolayer culture are routinely used for the propagation of FMD viruses. Various nutritional factors: amount of growth medium and serum concentration as well as physical conditions: seeding density, rolling speed, growth time, and incubation temperature for the propagation of BHK-21 cells on roller culture bottles were optimized. The roller culture bottles having surface area of 480 cm2 were used for the propagation of cells. Feeding of cells with 100 ml of growth medium per bottle and supplementation of 5% serum supported the growth of the cells in optimum way. Seeding of ten million cells per bottle resulted into the development of complete monolayer with maximum cell density within 48 hours. The cultured cells remained confluent up to 60 hours while a rapid decline in the number of harvested cells was recorded after 72 hours of incubation. Growth rate of the cells was slower at 33° C that increases at 35° C, reaching to its maximum at 37° C while cells could not tolerate the temperature of 39° C. The bottles kept at rolling speed of 3 rpm yielded maximum amount of cells while higher or lower rotation speed negatively affected the cell proliferation.
Antibody response of buffalo calves to different levels of FMD virus immunogen in trivalent vaccine was investigated. The vaccine containing 106.2 units of immunogen/TCID50 of each of the three serotypes of FMD virus induced log2(1.3± 0.4) units of anti-FMD "O" Complement Fixing Cumulative Geometric Mean antibody (FMD"O" CFT-CGM) titer, log2(1.4±0.3) units of anti-FMD"A" CFT-CGM titer and log2(2.0±0.7) units of anti-FMD"Asia-1" CFT-CGM titer. The vaccine containing 2x106.2 units of immunogen of each of the three serotypes of FMD virus induced log2(2.2±0.2) units of anti- FMD"O" CFT-CGM titer, log2(2.1±0.25) units of anti- FMD"A" CFT-CGM titer and log2(3.4±0.8) units of anti-FMD"Asia-1" CFT-CGM titer. The vaccine containing 3x106.2 units of TCID50 of each of the three serotypes of FMD virus induced log2 (5.3 ± 2.0) units of anti-FMD"O" CFT-CGM titer, log2 (4.6±1.9) units of anti-FMD"A" CFT-CGM titer and log2 (5.0±2.2) units of anti- FMD"Asia-1" CFT-CGM titer. The vaccine containing 4 x 106.2 units of TCID50 of each of the three serotypes of FMD virus induced log2(5.5±1.5) units of anti-FMD"O" CFT-CGM titer, log2(4.4±1.9) units of anti-FMD"A" CFT-CGM titer and log2(5.2±1.9) units of anti-FMD"Asia-1" CFT-CGM titer. Moreover, buffalo calves (n=3) which were primed and boosted with 60 days interval using vaccine containing 2x106.2units of immunogen of each serotype of FMD virus, showed log25.0 and log26.3 units of anti FMD"O"-CFT-GMT antibody titer, log24.6 and log2 6.0 units of anti FMD"A"-CFT GMT antibody titer, log25.6 and log26.0 units of anti FMD"Asia-1"-CFT GMT antibody titer, on 30 and 120 days post boosting.
Each serotype of the virus grew well on BHK-21 cell line. The virus showed poor TCID50 (log 103.2±0.2) in BHK-21 cells having Glasgow Minimal Essential Medium (GMEM) without Fetal Calf Serum (FCS). Addition of FCS in the medium at the rate of one percent increased log 107.1±0.2 units of virus TCID50. Incubation temperature of 350 C and 370 C supported the multiplication and maintenance of BHK-21 cells and yielded log106.6±0.1 and log107.0±0.2units of virus TCID50, respectively. Each serotype of FMD virus showed log106.29±0.07 units of virus TCID50 in the stationary monolayer of BHK-21 cells in Roux flask (75cm2), log107.66± 0.02 units of virus TCID50 in roller bottles (490 cm2) and log108.34 ± 0.07 units of virus TCID50 on 0.2 g of micro-carriers suspending in 200 ml of the growth medium in stirring bottle. The infectivity titer/TCID50 of each of the virus serotypes was significantly higher in roller bottles than that achieved in Roux flasks (p<0.05) and was significantly higher in stirring bottle containing micro-carriers suspending in the growth medium than that of harvested in roller bottle (p<0.05). It is concluded that the infectivity titer of the virus is directly proportional to number of BHK-21 cells in the culture system.
Availability: Items available for loan: UVAS Library [Call number: 1554,T] (1).
25.
A Metagenomic Analysis Of The Respiratory Microbiota Of Birds
by Muhammad Zubair Shabbir | Prof.Dr. Masood Rabbani | Prof. Dr. Khushi Muhammad.
Material type: ; Format:
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not fiction
Publisher: 2013Dissertation note: The respiratory systems of birds are susceptible to and are a reservoir for numerous bacterial species, including those of significance to public health. A number of bacteria, either as primary or secondary infectious agents, have been associated with respiratory outbreaks in poultry and subsequent losses worldwide. A key component of a poultry development policy is the proper diagnosis and control of infectious diseases, which requires substantial knowledge of the microbiome in diseased and healthy birds. Because only a small proportion (< 1%) of organisms are culturable, limited as well as highly variable and time-consuming conventional microbiological procedures have typically excluded the normal flora present in the respiratory tract or have restricted the analysis to potential pulmonary pathogens. This limitation provides only a partial representation of the airway microbiota of birds and has little potential for determining or discovering novel organisms/pathogens and their association with clinical outcomes. Using the hypervariable region of the 16S rRNA gene, culture-independent techniques such as 454-pyrosequencing, can provide species-specific sequences of any bacteria in a given clinical sample. This approach has identified a number of novel bacterial species in recent years.
Based on the quality and quantity of the double-stranded gDNA, a total of 30 T-BAL samples including houbara bustard and ostrich, were collected from equal numbers of clinically diseased and healthy birds originating from flocks within different management systems, including free range, open house, and controlled house. Using 454 bar-coded pyrosequencing, the hypervariable regions of the 16S rRNA gene corresponding to V1 - V5 (~ 1,000 bp) were sequenced. Of the high-quality reads obtained (296,811) using the MOTHUR platform, the sequences were processed for sequence alignment with the 16S RDP database via BLASTn, and subsequent taxonomic analysis through MEGAN programs using a homology-based method to bin sequence reads.
Almost all of the read were classified to the bacterial domain and its subsequent descendants. The birds were shown to be susceptible to a diverse microbial community belonging to a variety of phyla, families, genera, and well-characterized bacterial species. The bacterial communities were relatively conserved at the phylum level; however, at lower taxonomic levels, differences were observed in the phylotypes and abundance between the clinically diseased and healthy birds as well as between different management systems. The biodiversity and richness in the taxonomic content was higher in the clinically healthy birds compared with the diseased birds, as indicated by the rarefaction plot and the Shannon-Wiener and Simpson-Reciprocal diversity indices. Regardless of the management type, bird species, and health status, a number of new bacterial species were identified. Although the clinical importance of these bacteria as part of the respiratory microbiome of birds has not been established, a number of these bacterial species have been found to be associated with infectious diseases in humans and other species.
The interactions of bacterial species with one another and, potentially, with the birds themselves provide a fascinating avenue for continued research. Further clinico-pathological studies will be needed to establish the links between causes versus effects. This information may help us gain insight into the ecological roles of these bacterial species and their potential co-evolution with birds.
Availability: Items available for loan: UVAS Library [Call number: 1560,T] (1).
26.
Sources Of Salmonella Contamination In Poultry Meat During Processing And Its Resistance To Antibioties
by Atif Masood Ahmad Khan | Prof. Dr. Tahir Yaqub | Prof. Dr. Khushi Muhammad | Veterinary and Animal.
Material type: ; Format:
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drama
Publisher: 2013Dissertation note: The unhealthy birds which are slaughtered at poultry retail shops may be transferring the pathogen to the healthy meat via butcher's block, clothe (used for cleaning carcass), weighing scale, table, knife and drum in which they are bled after slaughter. The tools which are used in butchers' chicken sale point ; different objects in their shop; the feed they give to birds and bird droppings ; all were analyzed and not surprising they were found heavily contaminated with Salmonellae. Salmonellae is an enteric organism and at chicken sale points contamination to objects through birds' intestine is not much surprising. Therefore a strong need to push the pressure on government to devise laws and set standards for clean premises at chicken sale points. Extensive and irrational use of antimicrobials in human and veterinary sector in the treatment, prophylaxis and as feed additive have made this organism resistant to many of the commonly used antimicrobials.These resistant organisms are being transferred to human body due to the consumption of contaminated poultry meat. Therefore the Salmonellae in humans show resistance to many antibiotics. It is assumed that Salmonellae can transfer resistant genes via bacterial conjugation, transformation and transduction.As a result it is becoming resistant to many antimicrobials. Therefore a strong check on irrational use of antibiotics is needed.
The purpose of current study was to estimate the prevalence of antimicrobial resistant Salmonellae at chicken sale points in Lahore city. In current study 250 samples of 8 different types were collected from different poultry meat sale points in Lahore city.The selection of sale points was random. The samples included50 samples of each poultry feed and bird droppings. 150swab samples of butcher wooden blocks, table, drum( in which the birds are slaughterd), butcher's balance, knife , cloth (used to clean block, knife, table and chicken meat) were also collected from different chicken meat sale pointsin Lahore city.The Samples were analyzed for the presence of Salmonellae by culturing and biochemical tests.The percentage of Salmonellae positive samples inwooden block, weighing balance, cloth, birds' droppings, drum, bird feed, knife and table surface was 44, 24, 36 ,16, 32, 8, 28, 20 respectively.Overall prevalence of Salmonellae was 23.2 %. The isolated Salmonellae were then checked for antimicrobial resistance against 18 antimicrobials by using disk diffusion method. All the Salmonellae isolates were resistant to atleast four antimicrobials. 49 different antimicrobial resistance patterns were found.
Availability: Items available for loan: UVAS Library [Call number: 1563,T] (1).
27.
Biomass Production Of Pasteurella Multocida By Using Biofermentor For Preparation Of Montanoid Based Vaccine
by Noreen Sarwar | Prof. Dr. Khushi Muhammad | Dr. Atif Hanif | Prof. Dr. Muhammad.
Material type: ; Format:
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drama
Publisher: 2012Dissertation note: Hemorrhagic septicemia is a contagious bacterial disease of large ruminants principally in cattle and buffalo with high morbidity and mortality. The disease is endemic in nature and outbreaks are common during hot, humid and wet season. The acute and fatal nature and brief duration of the disease limit the antimicrobial therapy. In Pakistan, the disease causes heavy economic losses to dairy industry. Vaccination therefore, is an option for controlling the disease. For a quality vaccine, biomass production of P. multocida along with well developed capsule (immunogen) is necessary. The problem associated with the production of a quality vaccine is poor biomass production of P. multocida when grown in ordinary or routine media.
Present study was designed to isolate P. multocida from sick animals and its molecular characterization in the laboratory and study factors (temperature, media composition, pH incubation time and agitation or shaking) affecting its immunogen production and "in process quality control" factors (biological titer, dry mass, adjuvant and storage time) that affect antibody response. Finally, biomass production of the organism using biofermentor and monitoring of the antibody response of buffaloes to inactivated Montanide ISA-70 based P. multocida vaccine.
Each of the field isolates showed grey, viscous, mucoid, translucent and non hemolytic colonies on blood agar. There was no growth on MacConkey's agar. It was Gram negative coccobacilli or thin rods and bipolar when stained with Leishman's stain. The isolates were positive for Catalase, Oxidase, Hydrogen sulphide and Indole production along with nitrate reduction while it was negative for urease production, citrate utilization and gelatin liquefaction. The bacteria fermented glucose, sucrose, mannitol, mannose, but failed to ferment arabinose, maltose, salicin, lactose, dulcito and inositol.
Polymerase chain reaction (PCR) was performed on isolated colonies by using P. multocida specific and HS causing serotype B specific primers. P. multocida specific PCR gave product of 465 bp while HS causing serotype B specific primers amplified a product of approximately 590 bp.
Growth of the bacteria in casein yeast sucrose broth was optimized under different conditions. CSY broth showed dense growth of P. multocida during incubation for 18 hours. A temperature in between 35°C and 40°C showed its optimum growth. Poor growth was observed below 30°C and no growth was detected at 50°C and above. No growth occurred at pH 0.5 and 10.0 but best growth was obtained at pH 7.0 and 8.0. There was positive correlation between shaking in terms of rpm and growth. There was optimum growth at 500 rpm for 24 hours.
Inactivated HS Vaccine was prepared from dense growth in biofermentor on the basis of dry mass and bacterial count. The effect of biomass, adjuvant, storage of the vaccine, priming alone or with boosting on its potency was also studied along with boosting effect of montanoid ISA 70 oil based vaccine. Dry mass 1.7 mg/dose produced protective antibody titer while bacterial count 10-14/ml was sufficient to produce the protective antibody titer. Montanoid ISA 70 based vaccine provided immunity to buffalo calves better than aluminium hydroxide gel and bacterins. Boosting with oil based vaccine can help to keep the animal immunized for whole year. For better results of vaccine, it can be stored at 4oC for six months.
It is concluded that the proposed study improved quality of the vaccine and reduced volume of the vaccine dose, cost of its production and frequency of vaccination.
Availability: Items available for loan: UVAS Library [Call number: 1581,T] (1).
28.
Transovarian Transmission And Molecular Characterization Of Hydropericardium Syndroe Virus In Experimentally Infected Poultry Birds
by Rabia Tahir | Prof. Dr. Khushi Muhammad | Prof. Dr. Masood Rabbani.
Material type: ; Format:
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drama
Publisher: 2013Dissertation note: Poultry is one of the important and growing industries in Pakistan. It has now become the tremendously growing sub-sector of livestock. But the major constraint in growth of this industry is infectious diseases of poultry which leads to high mortality and morbidity rate resulting in heavy economic losses. The present study was conducted on one of the infectious poultry diseases named Hydropericardium syndrome which commonly affects broilers between 3-5 weeks of age. The causative agent is highly infectious virus belonging to Avian Adenovirus serotype 4. These are non-enveloped DNA viruses. It is an acute, infectious disease characterized by high mortality and excess pericardial fluid and multifocal hepatic necrosis. The incubation period ranges from 2-5 days followed by inoculation with liver homogenate or purified virus. This disease is characterized by the accumulation of straw coloured jelly like fluid in the pericardial sac, discoloured and inflamed liver with basophilic intranuclear inclusion bodies, congested kidneys and mortality up to 70%.
The present research was planned to prove that hydropericardium syndrome virus besides horizontal transmission is also transmitted by vertical transmission through transovarian route. For this purpose Liver sample was used as source of this virus. These liver samples were processed for further propagation of virus in live birds. Moreover, Virus neutralization test was also conducted for the confirmation of virus. To prove the transovarian transmission of this disease liver homogenate of infected birds was injected in 22 wk old breeder. Eggs were collected at 7-14, 15-21 and 22-28 days post infection. The day old hatched chicks were slaughtered to obtain liver and spleen sample for confirmation of virus through PCR. For confirmation, DNA was extracted using KIT method followed by polymerase chain reaction and amplified genomic material was visualized through gel doc system.
After validation of PCR 90 samples of liver and spleen were processed for DNA extraction followed by PCR. None of the samples of extracted DNA processed for each tissue from chicks produced a visible band of DNA in agarose gel after ethidium bromide staining. The possible reason for these negative results may be that viral load was below detectable limit or the presence of high titre of neutralizing antibodies. The implications of these findings are that vertical transmission via transovarian route does occur in poultry birds but the exact mechanism and establishment of latent infections further need to be investigated.
Availability: Items available for loan: UVAS Library [Call number: 1585,T] (1).
29.
In Process Quality Control Factors Affecting Potency Of Inactivated Black Quarter Vaccine
by Kashif Hanif | Prof. Dr. Khushi Muhammad | Dr. Aftab Ahmad Anjum | Prof. Dr.
Material type: ; Format:
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drama
Publisher: 2013Dissertation note: Black quarter (BQ) is a majorbacterial disease of cattle and buffalocharacterized by loss of appetite, lameness, depression, fever, swelling of the skeletal muscles, crepitating sounds followed by sudden death without any clear signs of disease. It results in irrecoverable economic losses. The disease prevails in all provinces of Pakistan especially in District Dera Ismail Khan,Cholistan and Chakwal etc. Morbidity losses comprise of losses due to reduced milk production, work hindrance, treatment charges etc. The survey revealed that 15.91% losses were due to morbidity and 84.09% losses occurred due to mortality caused by black quarter in cattle and buffalo.
Reliable diagnosis, mass scale vaccination and clamping strict bio-security measures are the only ways to control the disease.The present study was aimed to optimize the PCR for prompt and reliable diagnosis of BQ and to evaluate the inactivated whole culture vaccine with variable biological titer to induce protective immune response in calves. The comparative antibody response of animals to adjuvanted (Aluminium hydroxide gel & Montanide ISA 70) and non adjuvanted vaccine and duration of immunity was also studied. Effect of boosting on the humoral immune response of animals as well as shelf life of various vaccines was also evaluated. All of the vaccines were inoculated in a group of four animals. Serum samples were collected at specified time intervals and antibody levels were detected through antihemolytic units.
The PCR was optimized for diagnosis of C. chauvoei in clinical specimens from infected carcasses. Study of factors (temperature, media composition, pH, incubation time and anaerobic agents)for biomass production of bacteria and its hemolytic toxins revealed that certain growth parameters can be improved to enhance the bacterial growth and its hemolytic toxins. Like use of RCM medium for vaccine production enhances the growth of C. chauvoei and its toxins under in vitro conditions. Supplementation of nitrogen gas in culture medium can enhance the bacterial growth and hemolysin. Proper incubation time, temperature and pH can be very helpful factors for the growth and biomass production of C. chauvoei under in vitro conditions. Finally, biomass production of the organism using manual biofermentor is a very cheap and cost effective method for concentrated vaccine production in our country where commercial biofermentor cannot be afforded. So by using these techniques we can make more no. of vaccine doses from less quantity of bacterial culture. It will also help us in developing bivalent, trivalent or multivalent vaccine.
Study of in process quality control factors (bacterial biomass and toxins) production and "in process quality control" factors (biological titer, bacterial count, hemolytic units, adjuvants and storage time) that affect antibody response of vaccinatesrevealed that vaccine with 250 HU / dose showed relatively similar antibody titer in calves as the vaccine with 500 HU or 750 HU per dose. So this can be helpful to produce more doses of vaccine with same culture. The Montanide ISA 70 gave best result for development of good and prolonged immunity but gel based vaccine also produce satisfactory results.So in future oil based vaccine may be used to attain long term and effective immunity. Effect of priming and boosting revealed that boosting give better results as compared to primed group by producing prolonged immunity. The results were very encouraging. Effect of storage showed that the quality of immunogen was not affectedwith the passage of time if the vaccine is properly stored at 4 °C upto three months.
Availability: Items available for loan: UVAS Library [Call number: 1586,T] (1).
30.
Physicochemical Factors Affecting Infectivity Of Pesti Des Petits Ruminants Virus
by Kinza Khan | Prof. Dr. Khushi Muhammad | Dr. Jawad Nazir | Dr. Mutti-ur-Rehma.
Material type: ; Format:
print
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drama
Publisher: 2013Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1636,T] (1).
31.
Comparative Efficacy Of Hand Sanitizers And Liquid Soaps Against Commonly Encountered Microbes On The Experimentally Contaminated Palm Surfaces
by Taiba Tahir | Dr.Jawad Nazir | Prof Dr | Prof Dr. Khushi Muhammad.
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drama
Publisher: 2013Dissertation note: Hand hygiene plays a key role in the prevention, control and reduction of many communicable infections as contaminated hands are the major source of transmission for microbes. Three categories of hand hygiene products; hand sanitizers (Safeguard, Dettol, and Cool & Cool hand sanitizers), antibacterial soaps (Safeguard, Dettol, and Lifebuoy liquid soaps) and plain soaps (Lux, Capri, and Pears liquid soaps) were evaluated against five bacterial cultures (E. coli, K. pneumonia, Salmonella. spp, S. aureus, P. aeruginosa) for their antibacterial activity through in vivo and in vitro techniques. In vivo testing was performed through palmar surface contamination techniques. Palm surfaces of volunteers' hands were artificially contaminated followed by recovery of the bacteria through glove juice method both before and after the application of product for 30 seconds. Each of the experiment repeated thrice and means log reduction (MLR) in the bacterial count after the application of each product was calculated. In vitro efficacy of hand hygiene products was carried out through calculation of minimum inhibitory concentration (MIC) and phenol coefficient values.
MLR values of the sanitizers were ranged from 2.0 - 5.5 log10 CFU/ml, while that of antibacterial and plain soaps were 3.0 - 4.1 and 3.0 - 4.6 log10 CFU/ml. MIC values for the sanitizers, antibacterial, and plain soaps were ranged from 1:10 - 1:40, 1:6 - 1:20, and 1:2 - 1:8 against all of the 5 bacteria. Hand sanitizers were proved to be superior to medicated and plain soaps during in vivo and in vitro testing. Both of the antibacterial and plain soaps were equally effective in reducing bacterial load on the contaminated hands because during hand washing procedure mechanical removal of contaminants through surfactant activity of soaps is mostly responsible for the removal of bacteria. While a relatively higher MIC values of the antibacterial soaps were attributed to the presence of certain antibacterial agents in them. It was not possible to calculate the phenol coefficient values for any of the hand hygiene product because even least dilutions (1:2) of the products did not stop the bacterial growth. Present study emphasizes the suitability of using hand sanitizers in health care centers as well as in routine life. Because of comparable efficacy of medicated and plain soaps, excessive use of antibacterial soaps should be avoided due to risk of developing antibiotic resistance.
Availability: Items available for loan: UVAS Library [Call number: 1662,T] (1).
32.
Production And Evaluation Of Peste Des Petits Ruminants Virus Vaccine
by Muhammad Aness | Prof. Dr. Khushi Muhammad | Prof. Dr. Masood Rabbani.
Material type: ; Format:
print
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not fiction
Publisher: 2013Dissertation note: PPR is an acute highly contagious viral disease of small ruminants which is endemic in Pakistan. Present study was aimed to evaluate the freeze dried PPRV vaccine with variable biological titer to induce protective immune response in beetal goats. The comparative immune response of animals to adjuvant and non-adjuvant vaccine and duration of immunity was also studied. Effect of boosting on the humoral immune response of animals as well as shelf life of various vaccines was also evaluated. Each of the vaccines was inoculated in a group of five animals. Serum samples were collected at specified time intervals and antibody levels were detected through cELISA as PI values and neutralization test as MNA titer.
The virus was propagated on the Vero cells. It was estimated that infecting 2 x 107cells with 104.00 TCID50 virus concentrations added to a T-175 cell culture flask at the time of subculture yielded maximum virus titer in the cell culture harvest following three freeze thaw cycles of the contents. The freeze dried vaccine with a biological titer of 105.00 TCID50 per dose provoked maximum antibody titer followed by the ones with a titer of 104.00 or 103.00TCID50 which provoked nearly equivalent protective immune response while the animals inoculated with a vaccine having 102.00 TCID50virus concentrations developed minimum antibody titer. The oil adjuvant PPRV vaccines elicited significantly higher antibody titer in comparison to gel based vaccines but however minimum antibody titers were detectable in response to freeze dried vaccines. Although protective antibody level (? 10 neutralizing antibody units) was detectable in the animals vaccinated with either oil based, gel based or freeze dried vaccine containing biological titer of 104.00 TCID50 but however the extent and duration of immunity was found to be most superior in response to oil based vaccines. It can be concluded that a single shot of either gel or oil based vaccine can provide protection in the vaccinated animals for a minimum of one year duration. Goats receiving a booster dose of the vaccines had a significantly higher antibody tier in comparison to the ones who received single dose of the vaccines. The freeze dried and wet vaccine kept at 4 °C did not show any significant drop in the biological activity of the virus even after 12 months of storage. Immunogenicity of the both adjuvant and non-adjuvant vaccines, as measured through the immune response in the vaccinated animals, also remained unaffected after 12 months of storage at 4 °C.
Availability: Items available for loan: UVAS Library [Call number: 1731,T] (1).
33.
Antibody Response Of Buffalo Calves To Oil Based Multivalent (Pasteurella Multocida, Clostridium Chauvoei And FMD Virus "O' "A" and "Asia1") Vaccine
by Muhammad Farooq | Prof. Dr. Khushi Muhammad | Dr. Aftab Ahmad Anjum | Prof. Dr.
Material type: ; Format:
print
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not fiction
Publisher: 2013Dissertation note: Microbial diseases are one of the constraints for further development of dairy industry as a profitable enterprise. The diseases are causing heavy economic losses to the industry. The diseases such as Foot and Mouth Disease (FMD), Hemorrhagic Septicemia (HS), Black Quarter (BQ),etc., are endemic in Pakistan and perpetuate among the dairy animals. These diseases can be effectively controlled by vaccination.
FMD virus "O", "A" and "Asia 1" were grown on BHK-21 and were inactivated with BEI. Culture of P. multocida and Cl. chauvoeiwere grown on CSY and RCM media, respectively and inactivated with formalin. The vaccine containing 0.2 x 107 units of TCID50of each serotype of FMD virus ("O", "A" and "Asia1"), 2 mg of Pasteurella multocida and 250 Hemolytic units of Clostridium chauvoei per dose were prepared. Oil adjuvanted vaccines of HS, HS + BQ, HS + FMD ("O", "A" and "Asia 1"), BQ, BQ+ FMD ("O", "A" and "Asia 1"), FMD ("O", "A" and "Asia 1") and HS + BQ + FMD ("O", "A" and "Asia 1") were prepared and injected into the buffalo calves in 7 group of 3(n=3) animals each separately at Living Dairies, Chunian. 8th group of three animals was kept as negative control. Antibody response against FMD virus, Cl. chauvoei and P. multocida were measured by CFT, Anti hemolytic Assay and IHA, respectively at day 0, 30, 60 and 90 post vaccinations. Two groups (n=3) of calves vaccinated with whole culture FMD vaccine and NSP free FMD vaccine. Data was analyzed by one way ANOVA procedure and significance was determined by Duncan Multiple Range Test through SPSS version 13.
The vaccine when injected in buffalo calves induced Log22.00±1.00units of anti FMD "O" CFT antibody titer, Log22.22±1.00 units of anti FMD "A" CFT antibody titer, Log22.22±0.84 units of anti FMD "Asia 1" CFT antibody titer; Log22.99±0.58 units of Indirect Haemagglutinating (IHA) units of antibody against Pasteurella multocida and Log25.44±1.02, Anti Hemolytic Units (AHU) of the antibodies against hemolytic toxins of Clostridium chauvoei. There was no significant difference among the titers of FMDV "O", "A" and "Asia 1"; Pasteurella multocida and Clostridium chauvoei whether used in monovalent or in multivalent.In present study anti-NSP-FMD virus ELISA antibodies in the animals vaccinated with FMD (whole culture) vaccine were undetectable on 15 days post priming while detectable on 30 and 45 days post priming. However anti-NSP-FMD virus ELISA antibodies in the animals vaccinated with FMD (NSP free) vaccine were undetectable on 15, 30 and 45 days post priming. Moreover these antibodies were detectable in FMD carrier animals on 15 days post recovery.Cellular pellet of Pasteurella multocida, Clostridium chauvoei can be used to further minimizing the volume of culture required and further Brucella abortis vaccine can be added in it in conjunction with FMD. This will revolutionize the field of vaccination in Pakistan.
Availability: Items available for loan: UVAS Library [Call number: 1732,T] (1).
34.
Comparative Analysis Of Respiratiory Microbiota From Clinically Healthy And Deseased Broiler Breeders
by Husnain Ahmed | Prof. Dr. Masood Rabbani | Prof. Dr. Khushi Muhammad.
Material type: ; Format:
print
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drama
Publisher: 2014Dissertation note: The pulmonary system of birds is a considered a reservoir of innumerable bacterial pathogens including those which are subject to public health significance. This scenario makes respiratory tract of birds prone to many bacterial infections as well. There are many respiratory outbreaks in poultry that causes huge economic losses and a number of bacterial pathogens either acting as primary or secondary pathogen can be held responsible for these losses. A very small fraction of (<1%) of bacterial species are culturable, limited as well as highly variable and time consuming conventional microbiological procedures have typically excluded the normal flora present in the respiratory tract or have restricted the analysis to potential pulmonary pathogens. Due to unculturable nature of many bacterial species there is a very little room left for discovering or determining novel organisms or pathogens and their association with clinical outcomes through conventional microbiological procedures. With the advancement of technology metagenomic analysis of a given sample has emerged as a major culture independent technique for identification of many pathogens, by reading the hypervariable region of the 16S rRNA gene, culture-independent technique such as 454-pyrosequencing, can provide species specific sequence of any bacteria in a given sample.
A total of 12 T-BAL samples from breeder birds were selected based upon the quality and quantity of the double-stranded gDNA. Using 454 bar-coded pyrosequencing, the hypervariable regions of the 16S rRNA gene corresponding to V1 – V5 (~ 1,000 bp) were sequenced. Of the high-quality reads obtained (296,811) using the MOTHUR platform, the sequences were processed for sequence alignment with the 16S RDP database via BLASTn, and subsequent taxonomic analysis through MEGAN programs using a homology-based method to bin sequence reads. The results of study indicate that birds harbor a diverse microbial community including number of phyla, families, genera and characterized bacterial species. The bacterial communities were relatively conserved at the phylum level; however, at lower taxonomic levels, differences were observed in the phylotypes and abundance between the clinically diseased and healthy birds. As indicated by the rarefaction plot and the Shannon-Wiener and Simpson-Reciprocal diversity indices, the biodiversity and richness in the taxonomic content was higher in the clinically healthy birds compared with the diseased birds. Regardless of the bird health status a number of new species were identified. A number of these bacterial species have been found to be associated with infectious diseases in humans and other species, although the clinical importance of these bacteria as part of the respiratory microbiome of birds has not been established.
As the nature of bacterial species is to constantly act with one another and, potentially, with the birds themselves provides an interesting avenue for continued research. There is a need to conduct further clinico-pathological studies to establish the link between causes versus effects.
Availability: Items available for loan: UVAS Library [Call number: 1826,T] (1).
35.
Newcastle Disease Virus Act As An Adjuvant For Antibody Response Of Broilers To Inactivated Mycoplasama Gallisepticum Vaccine
by Rabia Riaz | Prof. Dr. Khushi Muhammad | Dr. Muhammad | Dr.Ali ahmad sheikh.
Material type: ; Format:
print
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not fiction
Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1932,T] (1).
36.
Microbiological Quality Assessment And Antibiotic Resistance Of Microbes Present In Fresh Indigenous Juices And Milk
by Hafza Raahat Farooq | Prof. Dr.Aftab ahmad Anjum | Prof. Dr. Khushi Muhammad.
Material type: ; Format:
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not fiction
Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1934,T] (1).
37.
Antibody Resoinse Of Broilers To Oil Based Combined Mycoplasma Gallisepticum And Avian Influenza H9
by Sadi Sarfaraz | Prof. Dr. Khushi Muhammad | Prof.Dr. Asim | Prof.Dr.Tahir yaqub.
Material type: ; Format:
print
Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1976,T] (1).
38.
Screening And Characterization Of Phytase And Bile Salt Hydrolases Producing Probiotic Lactobacilli Isolated
by Madiha arif | Dr. Muhammad Nawaz | Prof. Dr. Khushi muhammad.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 2001,T] (1).
39.
Effect Of Temperature And Relative Humidity On The Survival Of Newcastle Disease Virus Isolates Using Germ Carrier Techniques
by Tayyeba Sohail (2009-VA-209) | Dr. Jawad Nazir | Prof. Dr. Khushi Muhammad) | Prof. Dr. Asim Aslam.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Newcastle Disease (ND) is a highly contagious viral disease that affects almost all avian species including poultry, cage and wild birds around the globe (Terregino et al. 2003; Vidanovic et al. 2011). ND is economically important disease and included as list-A disease of Office des International Epizootics (OIE) (Anonymous). Mortality of infected birds ranges from negligible to as high as 100 % depending on the pathotype of the virus involved and health status of the birds (Alexander and Manvell 2004). NDV is an enveloped virus with single stranded, non-segmented, negative sense RNA genome (Makoui et al. 2013). The virus belongs to Avulavirus genus of Paramyxoviridae. There exist only one serotype of NDV designated as avian paramyxovirus-1 (Kapczynski et al. 2013) however, different virus strains do vary in their pathogenicity. There are 3 pathotypes of NDV; velogenic (highly virulent), mesogenic (moderate virulent), and lentogenic (mild virulent) based upon diseases producing potential and severity of signs in the infected birds (de Leeuw and Peeters 1999).
NDV is primarily transmitted to the susceptible birds through aerosol and fecal oral route (Martin 1992). Infected birds secrete high amount of the virus in their feces, saliva, mucous and nasal secretions which might contaminate the premises. Inanimate objects or fomites are a potential reservoir of viruses outside the host and might play an important role in the transmission of pathogens (Nicas and Sun 2006). Several factors can influence the survival of viruses outside the host (Sobsey and Meschke 2003; Weber and Stilianakis 2008; Stallknecht and Brown 2009). A number of studies show that respiratory pathogens can survive from hours to months on fomites (Abad et al. 2001; Kramer et al. 2006). Certain physical factors like temperature, humidity, pH, salinity, exposure to ultraviolet (UV) rays etc drastically affects the
Introduction
2
virus persistence in the environment. Effect of such physical insults is more pronounced on enveloped viruses than non-enveloped ones (Mbithi et al. 1991; Schaap et al. 2012; Tuladhar et al. 2012). High humidity and temperatures not only reduces the survival of influenza viruses on contaminated surfaces but also modulates their transmission to the susceptible birds (Shaman and Kohn 2009; McDevitt et al. 2010; Paynter 2014). Similarly lower temperature and less humidity promote the survival of NDV in the environment (Dat and Chuc 1985; Kournikakis et al. 1988).
ND is endemic in Pakistan but since last few years several new virus strains are circulating in commercial and rural poultry of the country (Munir et al. 2012; Shabbir et al. 2013). Central Punjab region is densely populated with commercial poultry and serve as disease epicenter every year. It has been observed that the disease outbreaks usually start in December, attain peak in the late winter and spring season, start decline in June and disappear in the rainy season. Apart from several other contributing factors, environmental survival of the viruses might contribute to the disease outbreaks. Availability: Items available for loan: UVAS Library [Call number: 2227-T] (1).
40.
Prevelance Of Brucellosis In Aborted Women Visiting Tertiary Care Hospitals Of Lahore City
by Saba Yasmin (2009-VA-211) | Prof. Dr. Aftab Ahmad Anjum | Dr. Tayyaba Ijaz (Co Supervisor) | Prof. Dr. Khushi Muhammad | Dr. Muhammad Tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Pakistan is an agriculture based country whose rural population depends upon livestock for livelihood. Contribution of livestock to agriculture sector is 55.9 percent while 11.8 percent to the national GDP during 2013-14 (GOP 2013-2014). A number of infectious diseases hamper the growth of livestock sector. Some of the livestock diseases are zoonotic in nature and threat to human health. Brucellosis is considered among major zoonotic diseases throughout the world. The Mediterranean Basin, south and Central America, Eastern Europe, Asia, Africa, the Caribbean and the Middle East are considered as high-risk countries (Memish 2001).
Brucellosis in human beings is a major concern of community health. It causes acute and chronic illness, physical incapacity and loss of health. Bacterial species involved include Brucella abortus, Brucella melitensis or Brucella suis. Brucellosis is acquired by human beings from infected animals by close contact with vaginal secretions, urine, feces, blood, aborted fetus, or consumption of unpasteurized milk or other raw milk products. Shepherds, milkmen, butchers, knackers, veterinary assistants and abattoir workers are at high risk (Agasthya et al. 2007). Prevalence of brucellosis recorded by Mukhtar and Kokab (2008) in abattoir workers of Lahore Pakistan was 21.7 percent. Higher prevalence of brucellosis was observed in females (37.06%) than males (24.2%) in patients admitted at Peshawar, Pakistan (Shahid et al. 2014).
Symptoms of disease vary among human patients, ranging from non–specific, flu-like symptoms (acute form) to undulant fever (chronic form). Some of the serious complications of skeletal system, cardiovascular and central nervous systems may develop. Other important signs observed include arthritis, orchitis, epididymitis, abortion, retained placenta and stillbirth (Baba et al. 2001; Grilló et al. 2006). In animals, brucellosis in most of the cases results in abortion, birth of weak calves, death of young stock, infertility in males and reduced milk yield in females (Maadi et al. 2011; Abubakar et al. 2012).
There is actual need for teamwork between public health officials and veterinary officers to reduce communication of brucellosis between animals and human in endemic areas (Jelastopulu et al. 2008; Makis et al. 2008). Clinical picture of brucellosis is nonspecific and may vary from patient to patient. Therefore, laboratory diagnosis by isolation and culture or recognition of specific anti–Brucella antibodies is essential for confirmation of brucellosis (Al-Attas et al. 2000).
Diagnosis of brucellosis by culture and phenotypic description is time-consuming. Furthermore, risk of infection to worker is always there. Serological tests are commonly preferred for brucellosis in cattle and small ruminants, especially at farm level screening. Chance of cross-reactions with other gram negative bacteria is a major problem. Rose Bengal Plate Agglutination Test (RBPT) and Slow Agglutination Test (SAT) are extensively used for detection of anti-Brucella antibodies (Halling et al. 2005). Enzyme Linked Immunosorbent Assays (ELISA) have been developed to resolve suspected samples by RBPT. ELISA is more sensitive, so it can detect Brucella carriers which are negative by RBT, SAT and CFT (Aert et al. 1984). Molecular techniques are more reliable and specific than serological tests. Final confirmation of brucellosis is carried out using polymerase chain reaction (PCR), a molecular technique. Real-time PCR offers enhanced sensitivity, specificity and rapidity of performance when compared to conventional PCR (Gwida et al. 2012). Availability: Items available for loan: UVAS Library [Call number: 2225-T] (1).
41.
Modulation Of Antibiotic Resistance In Salmonella Enterica By Different Plant Extracts
by Haleema Adil (2009-VA-233) | Dr.Muhammad Nawaz | Prof. Dr. Khushi Muhammad | Dr.Sanaullha Iqbal.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2015Dissertation note: Poultry, being the 2nd largest industry in Pakistan provide jobs for more than 1.5 million people.
Salmonella enterica infections continue to pose a significant risk for poultry industry.Salmonella
infections have been controlled by antibiotics but in recent times antibiotic resistance in
microorganisms especially in Salmonellais a global health issue. Antibiotic resistant Salmonella
has further compounded the problem. It is dire need of time to search for alternative therapies or
to improve existing ones. Plant extract not only have antibacterial activity but also can modulate
the antibiotic resistance in Salmonella. Poultry isolate of Salmonella enterica (n=5) were
procured from Department of microbiology UVAS Lahore and identified by genus specific PCR.
Antibiotic Susceptibility was checked by disc diffusion method against amikacin (30μg),
amoxicillin (30μg), ampicillin (10μg), cefixime (5μg), cefotaxime (30μg), ceftazidime (30μg),
ceftriaxone (30μg), cefuroxime (30μg), ciprofloxacin (5μg), gentamicin (10μg), and tetracycline
(30μg) and resistant pattern was 100% in ampicillin and tetracycline and 20% and 40% in
gentamicin and ciprofloxacin respectively while Cephalosporin antibiotic show 0% resistance.
Leaves and stems of six medicinal plants including Zingiber officinalis,Gymnema
sylvestre,Astragalus,Opuntia dellinii, Nigella sativa and Calotropis procera were processed for
extraction using ethanol, hexane and chloroform solvents.The antibacterial activity of these plant
extractswere determined against the Salmonella enterica isolates by agar well diffusion method.
Only ethanolic extract of all plant show zone of inhibition.Minimum inhibitory concentration of
plant extracts and antibiotics alone and in combination against Salmonella enterica were
determined by broth dilution method.
Summary
73
MICs of plant extract alone show undesirable effect up to 16mg concentration but in
combination with Ciprofloxacin plant extract were able to reduce the MICs of antibiotics while
in combination of plant extracts and ampicillin MICs were increase which show negative
modulation of antibiotic resistance in Salmonella enterica by using plant extracts. Availability: Items available for loan: UVAS Library [Call number: 2295-T] (1).
42.
Isolation And Molecular Characterization Of Causative Agent Of Equine Strangles
by Tayyaba Naz (2008-VA-235) | Prof. Dr. Khushi Muhammad | Dr. Aamir Ghafoor | Prof. Dr. Aneela Zamir Durrani.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Strangles is an important infectious and contagious disease of horses that affects upper respiratory tract. This disease is very much prevalent in Pakistan. Disease can be controlled by early diagnosis, strict quarantine measures and proper immunization. Disease is characterized by nasal discharge, submandibular lymph node swelling, raised temperature and anorexia. The disease can terminate into encephalitis, purpura hemorrhagica and bastard strangles. 20 nasal swabs as samples were collected from horses showing signs of nasal discharge or swollen lymph nodes from Remount depot Sargodha, 10 samples from clinically sick horses and 10 from apparently healthy horses with the history of disease. Samples were swabbed on to blood agar with 5% defibrinated blood of sheep. Isolated hemolytic colonies were undergone biochemical testing with the help of API strep 20 kit. Samples which appeared as streptococcus equi through biochemical testing were subjected to molecular amplification by targeting two genes. Two different PCR were performed PCR 1 targeted the Sod A gene this gene is present in Streptococcus equi and PCR-2 targetted SeM gene this gene is specific for Streptococcus equi subsp. Equi only. 5 samples were confirmed positive for Streptococcus equi through biochemical and molecular testing. SDSPAGE on the isolated bacterial samples were performed and it appeared as no protein diversity was observed among different isolates. However the protein pattern varied with number of passages as less number of bands appeared from older cultures. Availability: Items available for loan: UVAS Library [Call number: 2445-T] (1).
43.
Antibody Response Of Goats To Bivalent Pprv And Goat Pox Virus Vaccine
by Muhammad Farooq (2009-VA-146) | Prof. Dr. Khushi Muhammad | Dr. Muhammad Anees | Professor Dr. Aftab Ahmad Anjum | Dr. Muhammad Avais.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: PPR is one of the most acute viral disease of sheep, goats, deer and other similar animals. This disease is caused by Morbillivirus which belongs to familyParamyxoviridae. In this disease oculo-nasal discharge, stomatitis, diarrhea, high temperature, and pneumonia is common sign.along with foul breath.Goat pox virus (GPV) disease is extremely transmissible described by temperature, falling down and different stages of pox lesion development such as, vesicles, scars, pustules, erythema and papules, all over the body.
This study was aimed to evaluate the monovalent lyophilized PPRV and GPV vaccine with bivalent PPRV and GPV vaccines. Moreover effect of amount of immunogen of the vaccines, and nature of adjuvant used in the vaccine on antibody response of goats was also evaluated.Seven types of vaccines PPR (FD), GPV (FD),PPR+GPV (FD), PPR+GPV(gel 102.5), PPR+GPV(gel 103.5), PPR+GPV(gel 104.5) and PPR+GPV(oil) were prepared. All vaccines other than gel based contained 103.5 immunogen level. Each vaccine was inoculated to each of the six goats of the respective group. Blood was collected at 21, 42 and 63 dayspost vaccination. The antibody response of goats was measured with CFT.
There was non-significant difference between the anti-PPR antibodies induced by either monovalent or bivalent vaccines. Similarly goat Pox vaccines also produced non-significant difference in both monovalent and bivalent form. Antibody response was directly proportional to the amount of specific immunogen in the vaccine. There was non-significance effect of gel or oil in the vaccine as an adjuvant on the antibody response of goats to the vaccine. Availability: Items available for loan: UVAS Library [Call number: 2496-T] (1).
44.
Antibiotic Resistance Pattern Of Staphlococcus Aureus And Its Resistance Modulation Using Medicinal Plant Extracts
by Iqra Asif (2010-VA-279) | Prof. Dr. Aftab Ahmed Anjum | Prof. Dr. Khushi Muhammad | Ms. Tehreem Hussain.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: This project was designed to evaluate the antimicrobial efficacy of Chloroform and
ethanol extracts of Calotropis procera (C .procera) and Eucalyptus globulus (E. globulus)
against Multiple Drug Resistant (MDR) Staphylococcus aureus isolated from human origin. This
study was conducted to evaluate the antimicrobial potential of C. procera and E. globulus alone
and in combination with antibiotics to check synergism between medicinal plants and resistant
antibiotics.
S.aureus is a major pathogen which causes various infections. Infectious diseases affect
millions of people around the world and in the history these diseases are major cause of mortality
and morbidity across the globe. In past few decades rate of mortalities are continuously
increasing because of acquired resistance of S. aureus against multiple drugs, thus it is utter need
of time to discover some alternatives to antibiotics so that we can resolve this dilemma of
antibiotic resistance. Plant extracts are hope for this purpose as they have many compounds
which have potential to lower down the number of micro-organisms. Plants have benefits over
other as they are non toxic, non-reactive and have least side effects.
Total 20 samples of human origins were procured from Department of Microbiology,
UVAS Lahore and were subjected to check their antibiotic resistance profile against
Erythromycin, Amoxicillin and Ciprofloxacin by Kirby Bauer disc diffusion assay. Out of 20,
nine resistant isolates were separated. Among them three were resistant to Erythromycin, three to
Amoxicillin and three to Ciprofloxacin.
Summary
72
Leaves of C. procera and E. globulus were processed in Chloroform and ethanol
Solvents. Antimicrobial activity was evaluated by agar gel well diffusion assay in which zone of
inhibitions were measured. Minimum inhibitory concentration (MIC) of plant extracts was
evaluated by micro broth dilution method. Best antimicrobial activity was observed by ethanolic
extract of E. globulus.
Then combine effect of sub-inhibitory concentrations (SICs) of plant extracts and
minimum inhibitory concentration of antibiotics were determined by Well Diffusion assay. Four
different sub inhibitory concentrations of plant extracts i.e.10μg/ml, 20μg/ml, 40μg/ml and
80μg/ml were used in combination with fixed concentration of antibiotics i.e.100μg/ml to check
combinational effect of both. At selected sub-inhibitory concentration plant extract alone did not
show any antibacterial activity. Two of the isolates had shown modulation when amoxicillin and
plant extracts combination was used against them. The isolate labeled as S.aureus 4 showed
modulation with the use of Ethanolic extract of Calotropis procera and S.aureus 5 had shown
modulation with the use of chloroform extract of Calotropis procera. For further confirmation
two more concentrations of 160μg/ml and 320μg/ml were used along with100μg/ml Amoxicillin
against same isolates S.aureus 4 and 5. Zone of inhibition was observed with increased diameter
indicating modulation of two isolates. While erythromycin and ciprofloxacin resistant isolates
didn’t show any modulation.
Data of antibiotic resistance and resistance modulation using plant extracts was analyzed
by one way analysis of variance (ANOVA) followed by Duncan’s multiple range(DMR)
posthoc test using Statistical package for social sciences (SPSS) 17.0 Statistical software at α <
0.05. Availability: Items available for loan: UVAS Library [Call number: 2501-T] (1).
45.
Effect Of Stabilizers On Biological Titre Of Freeze Dried Ppr Virus Vaccine
by Muhammad Zubair Latif (2009-VA-382) | Prof. Dr. Khushi Muhammad | Dr. Muhammad Anees | Dr. Imran Altaf | Dr. Muhammad Imran.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: PPR is an acute and highly contagious viral disease of small ruminants caused by Morbillivirus. It causes high morbidity and mortality in small ruminants and heavy economic loses to farmers. Live attenuated vaccines are commonly used to control the disease. During freeze drying and after dilution of freeze dried vaccine, there is lose of virus titre so vaccine efficacy is reduced. Stabilizers are therefore added to protect from freeze drying stress and heat shock during storage and transportation. Each stabilizer has different protective effect. The present project was therefore designed to evaluate different stabilizers to act as the best one for maximum stability of the virus titre.
Fifty vials of PPR vaccine with each of six stabilizers (Weybridge medium-WBM, Lactalbumin hydrolysate sucrose-LS, lactalbumin hydrolysate sorbitol-LSbG, Tris sucrose-TS, Tris Trehalose-TT and Goat skimmed milk-GSM) was formulated, freeze dried and three vials from each formulation was selected and evaluated by biological titration just after freeze drying and dilution in PBS (7 pH). Each of the vaccine diluted with the PBS and stored at 40C, 250C, 370C for 36 hours. Biological titration of each of the vaccines stored at different temperature and time was determined on Vero cell lines. The virus infectivity was calculated as mean TCID50 by MTT assay.
It is concluded from the study that stabilizers having carbohydrates (sucrose, sorbitol, trehalose), salts (sodium) and hydrolyzed proteins (lactalbumin hydrolysates) are effective to make compact mass (freeze drying) of PPR virus vaccine. WBM, LS, and LSbG maintain infectivity of the PPR virus vaccine (if reconstitution with PBS and stored at 4°C) for 12 hours. However, TT is able to protect infectivity titre of the PPR virus vaccine during freeze drying and even during its storage after hydration with PBS at 4°C for 24 hours. Availability: Items available for loan: UVAS Library [Call number: 2546-T] (1).
46.
Study On The Status And Risk Factors Of Brucellosis In Small Ruminants Of District Bagh Azad Jammu And Kashmir
by Hafiz Muhammad Atique Ghafoor (2008-VA-160) | Dr. Aamir Ghafoor | Prof Dr. Khushi Muhammad | Dr. Aqeel Javeed.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Brucellosis is one of the most important zoonotic diseases of large and small ruminants with worldwide distribution. Brucellosis is declared as serious problem in 86 countries as it is widely distributed throughout the developing world (WHO, 1996). It mainly affects sexually mature animals and causes significant reproductive losses (Wadood et al. 2009). In Azad Jammu & Kashmir (AJK), research work on seroprevalence of brucellosis in small ruminants is very limited. In Bhimber Azad Jammu and Kashmir, 13.33% prevalence of brucellosis in goats has been reported (Din et al. 2013). In another study in Kashmir valley, overall 6.5% small ruminants were found positive by RBPT (STAT 2013). The data was based only on screening test. No confirmatory test like ELISA or PCR was performed to know the true picture of disease problem among RBPT positive ones. To find out seroprevalence of brucellosis in small ruminants, 400 animals ( n= 173 sheep, n=227 goat) were randomly selected and were screened for brucellosis in District Bagh Azad Jammu and Kashmir. A questionnaire was filled for each animal which carried entries including breed, specie, sex, lactation status, abortion history, housing, feeding, age and management etc. Sera samples from these animals were collected and examined by RBPT. Sera samples declared positive by RBPT were subjected to further analysis through ELISA. All lab analyses were done at University diagnostic Lab UVAS, Lahore. The data originated from this study was tested by using Chi square by using “SPSS version 20” and probability level <0.05 was considered significantly different. The results showed overall prevalence 30.75% through RBPT while 2.25 % by ELISA in sheep and goats of District Bagh Azad Jammu and Kashmir. The results showed that the prevalence through RBPT was 14.7 % in
Summary
33
sheep and 16 % in goats while through ELISA it was 1 % and 1.25 % in sheep and goats respectively.
The results revealed that overall prevalence was higher in goats than in sheep. Moreover the prevalence was higher in female then in male. The prevalence of brucellosis was higher in age group (1.6 Y to 3 Y) in both sheep and goats. The results showed that prevalence was higher in non-pregnant as compare to pregnant animals while prevalence was higher in dry as compare to lactating sheep and goats. Area wise results revealed that prevalence was higher in Bagh followed by Harighel, Arja and Mongbajri. Availability: Items available for loan: UVAS Library [Call number: 2616-T] (1).
47.
Molecular And Serological Characterization Of Soilborne Francisella Tularensis
by Javed Muhammad (2010-VA-65) | Prof. Dr. Masood Rabbani | Prof. Dr. Khushi Muhammad | Dr. Muhammad Wasim.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Tularemia, caused by Francisella tularensis (F. tularensis ), is a zoonotic disease transmitted through contact with infected animals and contaminated environment. The disease has been reported from many countries of the world but no study has been done in Pakistan. In the current project, a total of 2280 soil samples representing 456 villages of eight districts of Punjab province were collected from way-points having human-animal interaction, processed for genomic extraction and tested through real time PCR for presence or absence of F. tularensis. Association of risk factors was determined from data such as gender and age of animals, plough method, irrigation system, fertilizer type used, availability of veterinary services, level of farmer education, physical and chemical composition of the soil. Moreover, sero-prevalence against F. tularensis in cattle, buffaloes, sheep and goats was determined using ELISA. Seventy four soil samples (3.24 percent) were found positive for F. tularensis. Phylogenetic analysis showed 100 percent similarity index with F. tularensis sub specie holarctica reported from other regions like USA, Sweden, Spain, Turkey and Germany. Presence of F. tularensis in soil showed negative association with increase in number of human density (0.7159; 0.3834-0.2054). Prevalence of anti- F. tularensis ELISA antibodies were significantly higher (p<0.05) in large ruminants (cattle and buffalo) as compared to small ruminants (goat and sheep). Age and gender-wise analyses showed non-significant differences (p>0.05) between small and large ruminants. Whereas, rain-irrigation system (2.96: 1.35- 6.48), lack of veterinary services (4.77:1.26-18.03) and use of organic fertilizer (5.3: 11.38- 20.39) have positive association with prevalence of anti- F. tularensis ELISA antibodies in the serum. Sero-prevalence of Ft in the animals has significant association with quantity of clay in soil (p<0.05). A conventional PCR based test has also been optimized for
Summary
103
detection of F. tularensis using tul4 gene specific primers. Specificity of primer showed F. tularensis detection in soil DNA in the presence of other cross-reactive organism. Sensitivity was determined in two fold dilutions with detection limit of up to 320 pg/μL. Utilizing pET28a vector, a construct was prepared containing transformed tul4 gene (450bp) showing 100 percent sequence homology to query gene sequence. For manufacturing diagnostic assays especially in developing countries where availability of BSL-3 facilities and positive control reagents is an issue, provision of tul4 gene based constructs in vector can act as positive control and biosafe to use.
It is recommended that similar studies may be done in other parts of Pakistan to have spatial distribution of F. tularensis all over Pakistan. In future studies, other sources of transmission like water, ticks and rodents may be considered with soil for complete analysis. Transportation of whole genome of F. tularensis has been prohibited by Russian government, ATCC and CDC, WHO is working on designing a complete protocol for transportation of this bacteria or genome to other countries. Under such situation, conventional PCR optimization can be done for diagnosis of F. tularensis and pET28+tul4 constructs, developed in this study, can be used as a PCR positive control reagents. Availability: Items available for loan: UVAS Library [Call number: 2640-T] (1).
48.
Epitope Mapping Of Fusion And Hemagglutinin Genes Of Ndv Isolates From Pigeon And Peacock, And Efficacy Of Lasota Vaccine In Broiler Against The Isolates
by Sameera Akhtar (89-ag-433) | Prof. Dr. Mohammad Akram Muneer | Prof. Dr. Khushi Muhammad.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Poultry industry in Pakistan has experienced huge economic losses in the recent past by NDV. Simultaneous appearance of disease outbreaks and an increased genetic dissimilarity between the isolates recovered from commercial, backyard poultry and captive wild-birds raised the concerns whether the commonly used vaccine strains and vaccination schedules were good enough to provide protection against the circulating diverged strains reported by various researchers (Munir et al., 2012a; Shabbir et al., 2012a; Shabbir et al., 2013; Siddique et al., 2013; Rehmani et al., 2015). The fact that wild birds are known to act as reservoirs of viruses of low virulence that may emerge as vNDVs with the mutation in F0 cleavage site especially for chickens (Alexander et al., 2012) and that phylogenetically related vNDVs of class II causing outbreaks in chickens have previously been isolated from wild birds (Miller et al., 2010, Dortmans et al., 2011), viruses (in this study) were isolated from disease outbreaks in pigeons and peacock and the evolutionary trends of the isolates were studied. Deduced amino acid profiling of F and HN genes of the isolates along with their pathogenic potential and virulence for the broilers vaccinated with currently used live LaSota and killed ND vaccines were also studied.
Both the isolates had the genome size of 15,192 nt similar to NDV genotypes reported previously. The complete genome and residue analysis of F and HN genes of the isolates revealed a low divergence to APMVs which was classified as genotype VI/lineage 4 and VII/lineage 5, respectively for pigeon and peacock isolates. The deduced amino acid residue analysis of cleavage site of the study isolate suggested that both the isolates carried a motif characteristic for velogenic/mesogenic ND viruses. The presence of polybasic F-protein
Discussion
74
cleavage site, mean death time (48 – 61 hrs), severe clinical, macroscopic and microscopic lesions, all highly suggestive of the virulent nature of under-study isolates.
The sites for glycosylation and cysteine residues are thought to be conserved for F and HN protein. However, in comparison to each other and to representing genotype particularly the vaccine strain, we found differences in residues composition for a given glycosylation site and variations in both number and site of cysteine residues. Further, comparison of functional domains of F and HN protein to other genotypes and vaccine strain revealed several substitutions that were more often in F protein than HN protein. The substitutions particularly in fusion peptide, hydrophobic regions and transmembrane region of F protein and neutralizing epitopes of HN protein could results in altered proteins that may result in increased or decreased capacity of the these protein to bind to the host cells. However, it may be noted that percent divergence for fusion protein in both isolates when compared with the vaccine strain was found higher for nucleotides than the deduced amino acids indicating synonymous nucleotide substitutions (homologous recombination) for amino acid residues. Though there exists low frequency of such recombination in negative sense RNA viruses especially in non-segmented, such homologous recombination in all the coding and some non-coding region of NDV particularly the vaccine and circulating lineages is supposed activity and neutralizing escape variants (Hu et al., 2010; Wang et al., 2015). They play an important role in generating genetic diversity and evolution to NDV (Chare et al., 2003; Wang et al., 2015). Taken together, variation in nucleotide and subsequent substitutions/alternations in amino acid profile such as observed in this study is consistent with previously described theories of evolution of RNA viruses particularly the NDVs (Yu et al., 2001; Umali et al., 2013).
Discussion
75
F and HN are glycoproteins. Two oligomers of HN are connected through disulphide bonds to form a dimer and two such dimers get associated noncovalently to form a tetramer which is a functional protein. F protein, on the other hand, is a class I viral membrane fusion protein which undergoes a significant refolding transition during virus entry. The role of cysteine residues in forming intermolecular as well as intramolecular bonds is important for proper functioning of HN protein. Similarly other amino acid residues also play important role in the proper folding of HN and F proteins to maintain their integrities and biological activities. Generally speaking, amino acid substitutions with residues having similar properties do not result in major changes in the overall three dimensional shape of a protein molecule. For example if a hydrophobic amino acid is substituted with another hydrophobic amino acid in a primary sequence of a protein, the overall three dimensional shape of the protein remains more or less conserved. However, if a hydrophobic amino acid residue is replaced with a hydrophilic amino acid residue, this kind of substitution results in a major change in the overall three dimensional structures or shape of a protein molecule impacting the epitopes. Although, overall amino acid sequence similarity of our isolates was not strikingly different from that of the published isolates, however, amino acid substitutions with dissimilar properties were found with a scattered pattern of distribution. For example, 36th amino acid residue (isoleucine) of HN gene of peacock isolate (Figure 4.13) in our study is hydrophobic but it is polar (threonine) in the previously published NDV isolates. On the other hand, 50th aa in our isolate (peacock) is polar but it is hydrophobic in previously reported isolates. Similar substitutions could be appreciated at various regions of the genes. It is interesting to note that such substitutions with amino acids having dissimilar properties have also been found, albeit at lower rate, in previously published sequences (Figure 4.13). The
Discussion
76
implications of these changes in the critical regions of F and HN genes could be serious as these changes may mean escape of the virus from the established immunity. Since the peacock flock had the history of vaccination with lentogenic strain (LaSota), identification of cleavage motif similar to velogenic strain raises concerns for type of vaccine used to protect the flock and need for post-vaccine evaluation. Substitution and subsequent mutations at fusion peptide, HR regions and transmembrane domain could affect the fusion activity of NDV (Umali et al., 2014) and alteration in antigenic epitopes particularly those that are involved in virus attachment could result in escape variants and subsequent vaccine failure (Cho et al., 2007; Wang et al., 2015).
Phylogenetic analysis of complete genome and hypervariable region (F gene, 374 bp) of pigeon-originated APMVs revealed evolutionary relationship to lineage 4/genotype VI (sub-genotype VIbii), which is predominantly represented by the pigeon isolates (PPMV-1). The NDVs belonging to same genotype have been reported previously in Sindh province (genotype VI, Khan et al., 2010) and Khyber PakhtunKhwa province (genotype VIc, Shabbir et al., 2013) indicating both are in circulation in the environment. However, this is the first report detailing complete genetic and clinicopathological characterization of pigeon-originated NDVs (PPMV-1, VIbii) from Pakistan. The isolate represents currently circulating PPMV-1 since it had cleavage motif (112RRQKR↓F117) different than the viruses isolated prior to 1980s (112GRQKR↓F117). Given the significant genetic variability in PPMV-1 belonging to VIb, the viruses were divided into two major sub-groups (VIbi and VIbii). The isolates belonging to VIbii have been observed as predominant strains in the latter period of pigeon-originated panzootic while strains of VIbi are known to be diminished in the late 1980s (Aldous et al., 2003; Awu et al., 2015) imitating selective pressure from vaccine usage (Aldous et al., 2004). Though it is difficult to speculate
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about the origin of the study virus of genotype VIbii, the clustering and genetic relatedness to isolate from Russia than recently reported PPMVs-1 from China [(VIa and VIb), (Awu et al., 2015; Wang et al, 2015)] indicates possible introduction through migratory birds from North to South Pole.
The peacock isolate clustered to lineage 5/genotype VII (sub-genotype VIIi) and was found to be closely related to isolate previously reported from chickens in Pakistan. Lineage 5/genotype VII is thought to originate from the Far East with the first isolation from Taiwan in 80s (Yang et al., 1999). Since then, the presence of this genotype has been indicated from various parts of the globe (Aldous et al., 2003). Varying at sub-lineage level, a number of vNDVs of class II have been reported from many Asian countries including those that shared borders with Pakistan. Genetically related NDVs to sub-genotype VIIa have been previously reported from wild birds while sub-genotype VIc, VIIb and novel VIIi were reported from backyard and commercial poultry. Interestingly, novel sub-genotype VIIi was reported previously from backyard and commercial poultry (Munir et al., 2012a; Siddique et al., 2013; Rehmani et al., 2015). Nevertheless, it is the first time that this genotype has been identified from peacock, a wild bird, indicating that this genotype has the potential for its transmission between the two species (peacocks to poultry).
We observed sudden deaths in challenged birds and in one of the vaccinate group. This was not unexpected since death with no apparent clinical indications are considered the most noteworthy evidence of velogenic NDVs. Similar observations have been reported by Samuel et al (2013) in immunogically naive birds challenged with virulent isolates of African origin. Though severity
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of observed clinical symptoms was relatively less for pigeon isolate than that of the peacock, it resulted in almost comparable morbidity, mortality and shedding even in vaccinates. All the birds in Ch group died on day 7 p.i. The nervous symptoms were observed in non-vaccinates and vaccinates however it varied in duration (days p.i): symptoms were evident on day 6 p.i in non-vaccinates while it were observed on day 9 p.i in vaccinates. In general, even with the characteristic F protein cleavage site for velogenic strains (112GRQKRF117 or 112RRKKRF117 or 112RRQKRF117), most of the pigeon-originated NDVs do not result in significant disease in poultry and differences in pathogenicity index vary from moderate to no virulence for chicken (Collins et al., 1994; Dortmans et al., 2010). In an effort to characterize pigeon-originated PPMVs, Awu et al. (2015) suggested a low rate of viral replication and an increased antibody level to the low pathogenicity of pigeon upon challenge to chicken. However, differences in pathogenicity and efficiency of viral RNA replication have been described previously demonstrating varying level of proneness to different host species (Dortmans et al., 2010). Some PPMV-1 have been reported to be highly pathogenic for chicken after passage either in chicken or chicken embryo indicating their potential to cause ND outbreaks (Dortmans et al., 2011) similar to what has been observed in this study. Furthermore, variants of genotype VIb originating from pigeon have been shown to produce neurological symptoms (Ujvari et al., 2003).
While comparing pre- and post-challenge antibody titers, we found varying but an increased immune response indicating that challenge virus have replicated. The immune response generated by pigeon isolate was found greater than peacock isolate indicating its efficient replication than peacock’s isolate (Fig.15). Furthermore, nervous symptoms were evident one day earlier in birds challenged with pigeon isolate than that of the peacock. The potential reason
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could be the fact that both group of vaccinates received killed vaccine containing genotype VII that may have hindered replication of challenged virus of genotype VII to some extent than genotype VI. Viral shedding together with increase in antibody titer suggests that the commonly practiced vaccine types and schedule (LaSota and killed vaccine of genotype VII) are not able to provide protection or provide only partial protection (if at all) from ND. Genetic and antigenic differences as observed in the functional domains and neutralization epitopes of F and HN protein of study isolates and vaccine strain could be attributed for increased virulence and escape from vaccine. The deduced amino acid residues (F and HN protein) for pigeon isolates were 11.8% and 12.1% while it were 11.6 and 13.5 for peacock isolate, respectively. It is believed that genome-heterologous vaccines may prevent the disease but are unable to prevent infection and subsequent shedding of challenged viruses compared to genome-homologous vaccination (Yu et al., 2001; Hu et al., 2009; Miller et al., 2009). For example, in an immunization and subsequent challenge experiment, Samuel et al. (2013) have attributed variations in pre- and post-challenge immune response of immunized birds, viral replication and shedding to the genetic distance between vaccine and challenge strains. Although, they did not find disease upon challenge but infection, shedding and increased titer were noted for strains that were much divergent to vaccine strains and had subsequent break-through replication. Contrary to this, Susta et al., (2014) revealed that genomic variation in vaccine and challenge strains did not affect virus shedding in the presence of protective immune response. Comparing classic vaccine (LaSota) and adopted vaccine containing F and HN protein of challenged virus (genotype VII virus NL/93), Dortmans et al. (2012) revealed that classic vaccines are well able to protect from disease and results in reduced shedding even with suboptimal vaccine dose of classic vaccine against virulent strains of NDV. Further, they reported that it may be poor flock immunity due to inadequate vaccine
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practices than genetic and subsequent antigenic evolution for potential outbreaks and spread of vNDVs. Beside potential compromise in procedures used in vaccine storage and administration, the expected genetic distance between vaccine strains and study isolate seems to be well-explained by Wang et al. (2015) through cross-HI assay. While evaluating the antigenic diversity of different strains through cross-HI assay, they reported a lower R-value (0.13-0.18) for interaction of PPMVs to LaSota than between PPMVs (VIa and VIb, 0.7) indicating an obvious antigenic difference with vaccine strain. This is consistent with our results where we observed an increased antibody response in vaccinated birds challenged with genotype VI than birds challenged with genotype VII indicating lack of or partial cross-reactivity of genotype II and VII to genotype VI.
Given the antigenic similarity (serotyping) among all APMVs, lentogenic strains (LaSota, B1) are being used as live vaccine to protect birds from virulent NDVs (vNDVs). These strains cluster phylogenetically closely to the viruses isolated approximately 60-70 years ago, and have a high genetic gap in relation to viruses isolated in the fields (Miller et al., 2007; Munir et al., 2012a). These classic vaccines are known to prevent disease but not infection, replication and shedding of the virus even in vaccinates that may be reduced compared to immunologically naive or non-vaccinates. This reduced shedding depends but not limited to species affected and its immune status, concentration and virulence of challenged isolate, vaccine type and its dose as well as the time between vaccine and challenge (Miller et al., 2009). Relating to differences in virus shedding upon challenge to vaccinates, differentiation in antigenicity have been reported at the level of classes and genotypes by cross HI assays (Miller et al., 2009; Li et al., 2010; Gu et al., 2011). Since the genetic distance or dissimilarities exist between classic and prevailing
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strains of vNDVs, spread as well as control of disease is always a matter of concern. The situation becomes further worse particularly in settings (e.g., Pakistan) where there lacks routine vaccination to backyard poultry and wild-birds, lack of or infrequent serological monitoring of the poultry flocks, uneven vaccination schedules and breach in bio-security practices.
Currently, lentogenic (LaSota) strains are being used to vaccinate commercial poultry flocks. Killed vaccine has also been added in the schedule following frequent ND outbreaks in the recent years. However, vaccination schedule widely varies particularly in broiler flocks. Together, in a life-time of approximately 40 days, 3 time administration of LaSota and one time intramuscular injection of indigenous prepared killed vaccine (type and genome characteristic are unknown but most probably genotype VII) is being practised in broilers. The backyard poultry remains unvaccinated in Pakistan; however, occasionally LaSota or Muketesawr strains are being applied depending upon the owner awareness and access of government institutes to birds. The backyard poultry, characterized by small flock with an absolute lack of biosecurity measures and poor management, often intermingles with wild birds including pigeons and could serve as potential hot-spot for virus spread and disease transmission. Beside concerns that whether these vaccine strains are able to elicit a protective immune response against the diverged circulating variants, the excessive use of vaccination could be problematic than benefits. It is interesting to note that classic vaccines are supposed to give better protection against the vNDVs isolated in 1930-70s than the variants isolated in the recent years (Czegledi et al., 2006; Munir et al., 2012a).
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CONCLUSIONS
Two isolates originated from pigeon and peacock were genotypically and pathobiologically characterized. Both the isolates had the potential to cause disease and subsequent shedding even in vaccinates following the commonly practiced vaccine schedule. The result presented may be useful in revising the vaccine schedule being practiced currently in Pakistan. Furthermore, it ascertains the need to establish and maintain the active surveillance for appropriate diagnostics and control of NDVs that could be spilled out in the environment by wild birds. Availability: Items available for loan: UVAS Library [Call number: 2740-T] (1).
49.
Spatial Ecology And Distribution Of Soil Borne Burkholderia Mallei In Punjab, Pakistan
by Muhammad Asad Ali (2002-VA-73) | Prof. Dr. Khushi Muhammad | Prof. Dr. Aftab Ahmad Anjum | Prof. Dr. Mansur-Ud-Din Ahmad.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Burkholderia mallei is a causative agent of glanders, the disease of equines. The disease is characterized by pulmonary, nasal and cutaneous forms. B. mallei is excreted through nasal discharge, lacerated skin/wounds and expiration. Diseased animals shed bacteria through the discharges contaminating soil, water, fodder and other susceptible animals in its vicinity. The present study was designed to map and investigate the association of different physical factors and soil chemistry analytes with persistence of B. mallei genome in soil of 10% percent villages (n=456) from eight selected districts of Punjab province, Pakistan.
Eleven (0.48%) out of 2, 280 soil samples were positive for B. mallei genome in varied locations of Punjab. Higher prevalence (2.37%) for genome was detected in Sheikhupura district followed by Chakwal district (2.10%). None of the samples from Gujranwala, Sahiwal, DG Khan, Attock, Faisalabad and Sargodha districts were found positive for B. mallei genome. The genome of B. mallei was distributed in 25% study districts of Punjab, Pakistan. In Chakwal district, the genome of B. mallei was strongly associated with moisture (p=0.008) in all positive samples ranging from 0.80 to 39.20%, Phosphorous (p=0.050) ranging from 1.74 to 21.75 mg/Kg. While, this association in Sheikhupura district soil samples was with Sodium (p=0.018) and moisture (0.026) ranging from 1.90 to 133.59 mg/Kg and 0.80 to 39.20%, respectively. The odds of detecting DNA of B. mallei were recorded higher (1.4, 6.8, 5.0, 2.8 and 10.6 ) when soil sample sites were < 500 meters away from vehicular traffic roads, < one kilometer from animal markets, < 100 meters from canal, animal density < 1,000 animals and human population < 300 houses/village. While the odds of detecting DNA of B. mallei were 0.1, 0.3, 0.4, 0.2 and 0.5 when soil sample sites were > 500 meters from vehicular traffic roads, > one kilometer from animal markets, > 100 meters from canal, animal density > 1000 animals and human population > 300 houses/village, respectively. Soil-borne B. mallei DNA is more likely to be detected in areas closer to roads with vehicular traffic along the interstate routes in Punjab and soil containing low level of moisture.
It was concluded that soil of two districts out of eight selected was positive for B. mallei genome in Punjab province. Odds of less distance from main road to animal farm and high animal density at farm were positively associated with B. mallei DNA persistence in soil. Moisture, sodium and phosphorus were positively associated with persistence of B. mallei DNA in soil.
Availability: Items available for loan: UVAS Library [Call number: 2900-T] (1).
