1.
Physicochemical Factors Effecting The Survival Of Newcastle Disease Virus
by Rizwan Qayyum | Dr. Muhammad Naeem | Dr. asif Rabbani | Prof. Dr. S.A.R. Rizvi | Faculty of Veterinary Sciences.
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Publisher: 1997Dissertation note: For this research project, about 305 fresh fertile hen eggs were obtained from Veterinary Research Institute, Lahore. These eggs after cleaning were incubated at 37°C in automatic incubator for 11 days. At the 11th day, candling was done to confirm the fertility of eggs, either they are embryonated or not,. Eggs found dead at the time of candling were discarded. Fertile eggs 305 in number were inoculated with physically and chemically treated mesogenic strain of Newcastle disease virus which had already been treated and stored in plastic vials at -20°C. Each egg was inoculated with about 0.lnil of the treated viral sample. Four eggs were set for each of the factor for each time period. Four eggs were kept control in each factor in which viral suspension without physical or chemical treatment was inoculated.
The project was designed to study the effect of physical and chemical factors on the survival of Newcastle disease virus. The physical factors were temperature, p11 and UV light and chemical factors included five disinfectants like Formaline, Iosan, Phenol Aldekol and Bromosept (QAC). It was noted that at 56°C temperature virus lost its haemagglutinating activity after 45 minutes, but survived this temperature at 15 and 30 minutes exposure. It was observed that virus survived at pH 4 and 9 for 6, 12, 18 and 24 hrs but was killed at pH 1 and 13 for all the said time periods. After exposing virus to UV light, it was examined that Newcastle disease virus survived at UV light exposure for 45 minutes.
As far as the chemical factors were concerned, the results showed that 0.48% concentration of formalin inactivated virus in 30 minutes but not in 15 minutes. Other two concentrations i.e. 0.12% and 0.24% could not inactivate the virus. Phenol and Bromosept showed good antiviral activity against ND virus. 0.4% and 0.6% concentrations of Phenol inactivated the virus within 15 minutes but virus retained its HA activity at 0.2% phenol concentrations for 15, 30 and 45 minutes. The virus survived at 0.1% Bromosept concentration for 45 minutes and at 0.5% concentration for 15 minutes time but its haemagglutinating property was lost at 0.5% concentration in 30 minutes and at 1% concentration, the virus was killed within 15 minutes time. 0.1% concentration of Aldekol could not inactivate the virus in 15, 30 or 45 minutes. At its 0.5% concentration virus was inactivated after 45 minutes exposure but not at 15 and 30 minutes. However 1% Aldekol inactivated virus after 30 minutes but not within 15 minutes time. losan with 0.5% and 1.0% concentrations killed the mesogenic strain of Newcastle disease virus in 15, 30 and 45 minutes respectively. So the results of this study show that losan shows excellent antiviral activity against ND virus and is the best for disinfection of this virus at the farm. Bromosept (QAC) and Phenol should be the other two options for farmers to disinfect their sheds and hatcheries to minimize the chances of infection from Newcastle disease virus.
Availability: Items available for loan: UVAS Library [Call number: 0519,T] (1).
2.
A Study On The Effect Of Synthetic Pyrethroid Insecticide Talstar (Bifenthrim) On Immune Response In Broiler Chicken
by Fida Hussain | Prof. Dr. A.R. Rizvi | Dr. Muhammad Naeem | Prof. Dr. Rashid | Faculty of Veterinary Sciences.
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Publisher: 1995Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 0520,T] (1).
3.
Effect Of Various Growth Promoting Antibiotics On The Immune And Digestive Systems Of Broiler Chickens
by Shahan Azeem | prof. Dr. Muhammad Akram Munir | Dr. Sameera Akhtar | Dr. Talat | Faculty of Veterinary Sciences.
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Publisher: 2002Dissertation note: This project was designed to study the effects of growth promoting antibiotics on immune and digestive systems of broiler chickens. This study indicated that un-medicated un-vaccinated chickens had the higher body weights than the vaccinated un-medicated or medicated chickens.
Flavomycin, Lincomycin and Zinc bacitracin did not adversely affect the mean weights of spleen, thymus and livers of experimental chickens. However, the use of lincomycin, adversely affected the weight of bursa of' Fabricius. Furthermore, the use of Flavoinycin, Lincomycin and Zinc bacitracin did not have any adverse effects on the development of antibody titers against NDV and AIV. The total viable microflora counts of different treatment groups were not different from each other.
Evaluation of the economics of flocks at the end of the experiment indicated that un-medicated, un-vaccinated groups had higher profit returns and the Lincomycin medicated, vaccinated groups demonstrated lowest profit.
Availability: Items available for loan: UVAS Library [Call number: 0738,T] (1).
4.
Bacteriological Examination Of Camel (Camelus Dromedarius) Milk With Particular Reference To Public Health
by Muhammad Ishaq | Dr. masood Rabbani | Dr. Muhammad | Prof. Dr. M. Akram Muneer | Faculty of Veterinary Sciences.
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Publisher: 2002Dissertation note: The present research was envisaged to study the bacteriological profile of raw camel's milk. A total of 50 milk samples were collected directly from the udders of healthy she-camels from various areas of Punjab and were examined for total viable counts (TVC), coliform counts (CC), effect of storage period on total viable counts and coliform counts, using milk ring test (MRT) for brucellosis and In-vitro antibiotic sensitivity tests for the isolates.
All the samples were found negative for milk ring test (MRT) and hence for Brucella abortus. Standard plate count was in the range of 1 .39x 10 to 2. 13x107 c.f.u./ml. The mean standard plate count remained 2. 1x106 C. f. u. /ml. The coliform count was in the range of 3 . 2x iO to 5 . 9x104 c . f.u.Iml. The overall mean for coliforms count remained 3 . 9x104 c . f.u . /ml.
The effect of storage period on standard plate count upto 12 hours was zero. At 24 hours, increase was not very high and it remained in the range of 0.008 % to 1.72% organisms per ml of milk. At 36 hours increase was in the range of 0.008% to 4.95%. Similarly the effect of storage period on coliform count was studied and it showed no increase in the number of organisms per ml upto 12 hours of storage. At 24 hours coliform count increase was in the range of 1.75% to 6.06% organisms/mi. At 36 hours, increase was in the range of 2.38 % to 9.09% organisms/mi. It showed that the storage period had no serious effect on the standard plate count and coliform count. Standard plate count (SPC) showed that 48 % samples gave between 1 .01x105 - 9.5x105 organisms per ml. which was not according to international standard of good quality raw milk. Of the total samples, 42 % gave the coliform count between 3 .2x103 - 6.2x103 organisms per ml which fulfilled the international standard of good quality raw milk.
Different types of colonies on milk agar, nutrient agar and MacConkey's agar were purified and identified. The species isolated from all the milk samples included; Staphylococcus aureus (14 strains), Staphylococcus epidermidis (8 strains), Escherichia coli (16 strains), Lactobacillusfermentum (4 strains) , Lactobacillus casei (12 strains), Bacillus cereus (10 strains), Bacillus subtilis (6 strains), Enterobacter aerogenes (4 strains) and Neisseria mucosa (4-strains).
In-vitro antibiotic sensitivity of different antibiotics with known concentrations was studied. Results showed that all of the isolated organisms were resistant to oxytetracycline, ampicillin and followed by penicillin while most of the organisms were sensitive to gentamycin, followed by chioramphenicol, kanamycin and streptomycin.
Escherichia coli was resistant to all the antibiotics used while gave intermediate results by gentamycin and penicillin.
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5.
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.
Availability: Items available for loan: UVAS Library [Call number: 0877,T] (1).
6.
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.
Availability: Items available for loan: UVAS Library [Call number: 0878,T] (1).
7.
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.
Availability: Items available for loan: UVAS Library [Call number: 0879,T] (1).
8.
Immunomodulatory Effects Of Flumequine And Enrofloxacin On Newcastle Disease Virus Vaccinated
by Waseem Abbas | Prof. Dr. Muhammad Akram Muneer | Dr. Mansur-ud-Din Ahmad | Faculty of Veterinary Sciences.
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Publisher: 2005Dissertation note: An experiment was conducted to determine whether Flumequine and Enrofloxacin supplementation has any immunomodulatory effects on broiler chicks. A total of 192 one day old broiler chicks were randomly divided into four groups, each consisting of 48 chicks. Each group was further divided into 2 subgroups of 24 chicks. The chicks in group 1st were administered Flumequine, those in group 2 were treated with, Enrofloxacin and those in group 3 were treated with cyclophosphamide. Chicks in group 4 were not given any treatment. The parameters of investigation included the effects of Flumequine and Enrofloxacin treatment on live weight gain, feed conversion ratio, effect on various lymphoid organs (bursa of Fabricius, thymus, spleen and liver) and immune response of treated chicks to NDV-vaccination, post field NDV challenge mortality. Data presented in this study indicated that the Flumequine treated chicks had higher mean body weights, better FCR, higher NDV HI antibody, lesser overall mortality, no NDV post challenge mortality and no detrimental effects on their lymphoid organs, compared to the cyclophosphamide treated, and untreated chicks. The overall findings of this study clearly demonstrate that the use of Flumequine has good effect on growth and performance of the treated chickens. (Key words: Flumequine, Enrofloxacin, Immunomodulation, Broiler)
Availability: Items available for loan: UVAS Library [Call number: 0881,T] (1).
9.
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.
Availability: Items available for loan: UVAS Library [Call number: 0882,T] (1).
10.
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.
Availability: Items available for loan: UVAS Library [Call number: 0883,T] (1).
11.
Comparative Efficacy Of Five Diffrent Brands Of Commercial Newcastle Disease Lasota Viurs Vaccines In Broilers
by Tariq Abbas | Prof. Dr. Akram Muneer | Dr. Mansur-ud-Din Ahmad | Faculty of Veterinary Sciences.
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Publisher: 2005Dissertation note: The aim of the study was to compare five major commercial NDV (LaSota strain) vaccines being used in Pakistan with respect to potency, efficacy, thcrnioslability and influence on production performance in briolier chicks. The representative vials of the live NDV LaSota strain vaccines namely A, B, C, D and E were procured from local market. The vaccines were assayed for 50% infectivity (BID50) and Haemagglutinative ability (HA). A 3-log10 difference oF EJD50 and two- to-eight fold difference of HA activity was found was found among the various vaccines. Onc hundred and fifty day- old broiler chicks were divided into six groups and managed separately. The birds in group I, II, Ill, IV and V were actively immunized against ND on day 7 and 21 using vaccines A, B, C, D and E respectively. The birds in group VI served as unvaccinated control. The serum 1-Il antibody response of the different vaccines was determined 7, 14, 21, 28 days post-vaccination. The birds (n= 15) in all tile groups including unvaccinated control was challenged at day 35 with local virulent field isolate. The HI serum antibody profile and post-challenge mortality pattern revealed a dose- response relation between the virus content, humor-al antibody response and clinical protection. To compare the heat stability, the vaccines were incubated at 4C, 25CC and 40C for a period of 24 hours. There was no remarkable reduction in I IA liter, however slight dips (less than 2 logarithmic units) in LID50 values were found in all the vaccines. All the vaccines caused siginifcant suppression in weight gain leading to a poor performance in terms of Feed Conversion Ration (FCR) and European Efficiency Factor (EEF)
Availability: Items available for loan: UVAS Library [Call number: 0909,T] (1).
12.
An Epidemiological Study Of Nosocomial Infections At Mayo Hospital, Lahore
by Tayyaba Ijaz (Phd) | Prof. Dr. Muhammad Akram Muneer | Dr. Mansur-ud-Din Ahmad | Faculty of Veterinary Sciences.
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Publisher: 2005Dissertation note: The present study was designed to investigate the Prevalence of Etiological Agents of Nosocomial Infections in Mayo Hospital, Lahore-Pakistan of the 32,620 patients studied during 1997-2001; a total of 4502 (13.80%) patients acquired various types of nosocomial infections during their stay at Hospital. Clinical samples collected from various types of patients consisted of 1040 samples of Pus & Wound Swabs, 109 samples of blood; 115 of Pleural Fluids, 286 of Ascetic Fluids, 37 of Cerebrospinal Fluid, 1398 of Urine, 988 of Sputum; 329 of Burn Swabs, 99 of Patient Body Devices and 101 of Fecal and Drainage Material. The routine techniques for isolation. Identification through Biochemical, Serological and Antibiotic Sensitivity Testing were used for studying the Bacteriology of the selected samples. The present findings revealed that from a total of 4502 samples, 1287 Strains of Staphylococci, 429 Strains of Streptococci, 328 Strains of Enterococci, 781 Strains of Pseudomonas, 349 Strains of Enterobacter, 41 Strains of Acinetobacter, 26 Strains of Klebsiella, 140 Strains of Proteus, 1031 Strains of Escherichia, 67 Strains of Serratia, 93 Strains of Haemophilus, 119 Strains of other types of Gram Positive Bacteria, 13 Strains of other types of Gram Negative Bacteria, and 189 Strains of Yeast and Fungi were found as Etiological Agent for Nosocomial Infections.
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13.
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).
14.
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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15.
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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16.
Studies On The Physico-Chemical Factors Affecting In Vitro Replication Of Foot And Mouth Disease Virus (Serotype"O")
by Muhammad Taslim Ghori | Prof. Dr. Khushi Muhamamd | Prof. Dr. Masood Rabbani ( | Faculty of Veterinary Sciences.
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Publisher: 2007Dissertation note: Effect of physical (temperature, U.V light, and pH) and chemical factors such as sodium carbonate, sodium hydroxide, potassium permanganate, formaldehyde, citric acid, fin-virus, iodine and sodium hypochlorite on the replicating ability of"O" type of FMD virus on BHK cell line was determined.
The FMD virus of known TCID was exposed to 37, 57 and 77°C for 15, 30 and 45 minutes. Each of the virus's aliquot exposed to temperature was inoculated to 8 wells of the 96-well cell culture plate containing adherent monolayer of BI-IK cell line. The plates were incubated at 37 °C for 48 hours. The results showed that the temperature more than 57°C can inactivate the virus within 15 minutes. The virus was admixed in the MEM-199 maintenance media at pH 3, 5, 7, 9 and 11. The results showed that the virus was survived at pH 7 but virus was inactivated at pH 3, 5, 9 and 11. The FMD virus of the known TCID o was exposed to U.V. light (1 foot distance) for 15, 30 and 45 minutes. The results indicated that the virus tolerated UV light of 252.7nrn as it showed cytopathogenic effects (CPE).
The FMD virus of known TCID was exposed to 0.5 x, lx, and 2x concentration of each of the iodine, sodium carbonate, sodium hydroxide, formalin, finvirus, potassium permanganate, sodium hypochlorite and citric acid for 30, 60 and 90 minutes. Each of the virus's aliquot exposed to either of chemical factors was inoculated to 8 well of the 96-well culture plate containing adherent monolayer of the BI-IK cell line. The plates were incubated at 37 °C for 48 hours. Development of CPE indicated the virus inactivating ability of the chemical factor. The results showed that formalin, iodine, finvirus and sodium hypochiorite are more effective against FMD virus.
The results of the study are helpful to curtail the spread of virus, to implement the effective bio-security measures in our local conditions and in processing of animal products fit for export.
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17.
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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; Literary form:
not fiction
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.
Availability: Items available for loan: UVAS Library [Call number: 0960,T] (1).
18.
Immmunobiolotical Observations On Avian Influenza Virus Types H7 And H,
by Shahid Iqbal | Prof. Dr. Muhammad Akram Muneer | Dr. Mansur-ud-Din Ahmad | Faculty of Veterinary Sciences.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2007Dissertation note: The present study was designed to 'find the prevalence of Avian Influenza disease in and around Lahore in commercial and household poultry. A total of 1000 blood and 500 cloacal swabs were collected from Broilers, Broiler-Breeders, Ducks, Pigeons, Sparrows, Quails and Desi Chickens. The blood samples from all the flocks showed non-significant titers while vaccinated flocks showed protective titers. All the cloacal swabs were negative for virus isolation.
The final conclusions from this study were the following.i.e.
- Avian influenza caused by H7 & H9 type is not prevalent in broiler and broiler breeders in and around Lahore.
- The vaccinated poultry flocks showed higher titers of antibodies as compared to non-vaccinated flocks which means that vaccine can play a vital role in protection of bird from H7 & H9.
Availability: Items available for loan: UVAS Library [Call number: 0963,T] (1).
19.
Seroprevalence Of Bovine Brucellosis In District Quetta, Balochistan
by Muhammad Shafee | Prof. Dr. Masood Rabbani | Dr. Mansur-ud-Din Ahmad | Faculty of Veterinary Sciences.
Material type: ; Format:
print
Publisher: 2007Dissertation note: The sero-epidemiological study of bovine brucellosis was carried out to observe the incidence of brucellosis in slaughterhouse and Government and private dairy farm, (GDF, PDF) Quetta, Balochistan. The prevalence of this disease out of 780 serum samples of cattle and buffalo in slaughterhouse was recorded 3% by Rose Bengal Plate Test (RBPT) and 3.20% by indirect enzyme linked immunosorbent assay (i-ELISA), respectively. The zoonotic natures of this disease was also checked by screening 20 serum samples of slaughterhouse workers butchers and veterinarians and were found (5%) 01 positive out of 20 by RBPT but no positive case was found by i-ELISA. Similarly the disease was also checked in 200 milk samples of Government and Privatly owned Dairy Farm, Quetta.
The overall prevalence observed in the area by screening 1000 serum and milk samples of the target human, cattle and buffalo, was 4.2 % through i-ELISA.
The prevalence observed in Government Dairy Farm (GDF), Quetta was 14.8% (11 positive out of 74) while the Private Dairy Farm (PDF), exhibited 4.76% (6 positive out of 126 milk samples) by screening through i-ELISA. At GDF, Quetta, out of total of 74 cattle, no case were found positive by MRT, although 03 cases were found doubtful while i-ELISA show 11 positive cases in cattle (14.8%) while in private dairy farm 4 out of 15 cattle (26%) were found positive and 01 was considered doubtful by MRT and ELISA detected 06 cases of cattle out of 15(40%). Similarly 2 out of Ill (1.8%) buffalo were positive and 02 were doubtful by MRT but ELJSA did not detect any positive case and the prevalence of bovine brucellosis was higher in animals with reproductive disorders especially in cases of abortion.
The present study also revealed that the disease is more prevalent in cattle than buffalo both in slaughterhouse and organized dairy farm (Govt and private). In slaughterhouse 12 out of 23 cases were found positive by RBPT and 22 out of 23 were found positive by i-ELISA while in organized dairy farm all of the 17 milk samples were found positive from cattle population.
The efficacy of the i-ELISA both for milk and serum samples was found higher than other two conventional tests (MRT and RBPT), as it detected higher percentage of brucellosis cases both in serum and milk samples in comparison to other two tests.
The results of this study have revealed an alarming situation of bovine brucellosis in our dairy animals, which needs an emergent response from policy makers, as the disease is a potential threat to the human and animal health.
Availability: Items available for loan: UVAS Library [Call number: 0964,T] (1).
20.
Effects Of Different Disinfectatnts On Pathogens In Poultry
by Asif Abbas Malik | Prof. Dr. Masood Rabbani | Prof. Dr. Khushi Muhammad | Faculty of Veterinary Sciences.
Material type: ; Format:
print
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.
Availability: Items available for loan: UVAS Library [Call number: 0966,T] (1).
21.
Factors Affecting Hemagglutination Potential Of Avain Influenza Viuruses (H5, H7, H9 Subtypes)
by Mubashir Hussain | Prof. Dr. Muhammad Akram Muneer | Dr. Mansur-ud-Din Ahmad | Faculty of Veterinary Sciences.
Material type: ; Format:
print
Publisher: 2007Dissertation note: The objective of this study was to standardize hemagglutination and hemagglutination inhibition tests for AIV H5, H7 and H9 subtypes. These subtypes were propagated in 09-day old chicken embryonated eggs and after 72 hours post incubation the allantoic fluid (AF) was harvested and confirmed by spot agglutination test and by AGPT. While standardizing HA test maximum titers were recorded using 1% RBCs of chicken, human blood group Qe and dog using phosphate buffer saline (PBS) as a diluting agent for washing suspension of erythrocyte and by incubating the micro titer plates at 22c or 37C for 30 minutes or 40 minutes time period. The AIV subtypes eluted rapidly with increase in temperature with maximum elution observed within the time period of 8 hours. The live AIV provided much higher HA titer when compared with the titers obtained from AJV subtypes inactivated with formalin or Binary ethylene imine (BET). The BET was found to have little effect on HA activity as compared to formalin. While standardizing the HI test the best titers were obtained using 4 HA units of AIV antigen as compared to 1 HA and 8 HA units of antigen and by incubating the micro titer plates for 60 minutes period (time given for antigen-antibody reaction before the addition of erythrocytes suspension).
Availability: Items available for loan: UVAS Library [Call number: 0984,T] (1).
22.
Isolation And Characterization Of Clostridium Perfringens From Domestic Animals An Man In Punjab
by Waheeda Raana | Prof. Dr. Muhammad Akram Muneer | Dr. Khushi Muhammad | Prof. Dr | Faculty of Veterinary Sciences.
Material type: ; Format:
print
Publisher: 2007Dissertation note: The objectives of present investigation were to isolate the Cl. perfringens from the domestic and zoo animals and human beings; characterize it through biotyping and pathogencity observation, and to develop a vaccine- from the common CI. perfringens isolate. For this purpose a total of 1240 samples of morbid tissues (faecal samples from animals and gangrenous tissues from humans). From cattle (n=180), goats (n=180), horses (n=250), camel (n=250), deer (n=28), wild beast (n=07), monkeys (n16), zebra (n10), elephant (n01), yaks (n=09), foxes (n07), jackals (n=08), baboons (n=08), and bears (n08) were collected and processed for isolation of CI. perfringens. In addition a total of 100 human cases; 83 wound swabs and 17 gas gangrene were also collected and analyzed bacteriologically. This study has indicated that Clostridium (Cl.) perfringens causes multiple clinical problems in animals and human beings as was indicated by good rate of its isolation from the examined morbid tissues and fecal samples. Of the total 1240 samples from various types of animals 297 (23.95%) indicated the presence of CI. perfringens. The overall isolation percentages of various types of CI. perfringens from the cattle, sheep goat, horses, camel, wild beast, deer, bear, jackal, zebra, monkeys, yak, elephant, baboon, foxes, and humans were 22.2, 12.2, 57.2, 8.0, 21.6, 57.1, 30.76, 37.50, 50.0, 50.0, 37.50, 33.33, 100.00 75.00, 57.14 and 18.00, respectively. Of the tested population of domestic animals, goats indicated the highest Cl. perfringens (57.2%) infection rate. In the zoo animal population, the elephant, baboons, wild beast, jackals, and foxes were shown to be heavily infected with various CI. perfringens types compared to other wild life animals species. Of the 298 isolates obtained through this investigation Cl. perfringens type D was obtained from 118 (39.7%) morbid samples of the domestic and zoo animals; CI. perfringens type A from 63 (21 .21%) samples, Cl. perfringens type B from 95 (31.98%) samples; and the CI. perfringens type E was isolated from 21(7.07%) samples. None of the samples indicated the presence of CI. perfringens type C. Of the total 100 samples from the humans, CI. perfringens type A was isolated from 14 (14%) and Cl. perfringens type D was isolated from 04 (4%). None of the human samples showed the presence of Cl. perfringens types B, C, or E. Of the 17 human gangrene tissue samples, Cl. perfringens type A was isolated from 09 (52.94%) samples and the Cl. perfringens type D was recovered from 02 (11 .76%) samples. However, all attempts to isolate Cl. perfringens types B, C or E from the human gangrene tissue/material samples were unsuccessful.
The overall findings indicated that of the total 297 samples positive for various Cl. perfringens types 63 indicated the presence of Cl. perfringens type A. Of those 63 Cl. perfringens type A isolates, 49 were recovered from the animals; and 14 were isolated from the wound swabs and gangrene tissue material samples from humans. Of the 63 Cl. perfringens type A isolates from the animals, 5 were isolated from cattle; 3 from sheep, 20 from goats; 5 from the horses; 10 from camels, 01 from the deer; 01 from the zebra, 01 from baboon, 01 from fox, 01 from the monkey, and 01 isolate was recovered from yak. Of the 14 isolates of Cl. perfringens type A from humans, 05 were recovered from the open wound swabs, and 09 strains of the organism were isolated from the gangrenous tissue material.
Of the 297 samples positive for various Cl. Perfringens types, 95 animal samples indicated the presence of Cl. perfringens type B. These 95 isolates were obtained from cattle (n=22), sheep (n=10), goats (n=30), horses (n=03), camel (n=14), deer (n03), wild beast (n=02), monkey (n=02), zebra (n=02), yak (n=01), fox (n01), jackals (n02), baboon (n02) and bear (n=02). None of the human samples was positive for Cl. perfringens type B. Isolation of C/. perfringens type B from the zoo animals is a matter of concern for the human health, as the zoo visitors have the possibility to get infected with this organism.
Of the total 297 positive samples of faecal and morbid tissues from various types of animals and human being Cl. perfringens type D isolates were recovered from 118 (39.7%) samples. Of these 118 isolates of Cl. perfringens typeD, 114 were obtained from various types of animals, and 04 isolates were from the humans. Of the 114 animal isolates, 10 from the cattle, 5 from the sheep, 44 from the goats, 9 from the horses, 27 from the camel, 4 from the deer, 02 from the wild beast, 02 from the monkey, 02 from the zebra, 01 from the elephant, 01 from the yak, 02 from the fox, 02 from the jackals, 02 from the baboon, and 01 isolate the bear. A total of 04 CI. perfringens type D isolates were recovered from gangrenous tissue and open wound samples from human beings.
During this investigation 21 isolates of CI. perfringens Type E were obtained from domestic and zoo animals. Of the 21 isolates, 03 were from cattle, 04 from sheep, 09 from goats and 03 from horses, 01 from monkey, and 01 from the baboon. All the 21 isolations were from the fecal material of above mentioned animals. None of the human samples was positive for CI. perfringens type E.
Alpha toxin was produced by all of the 63 Cl. perfringens type A isolates. Within the toxin producing isolates, there was no difference in the quality of toxin in respect to its lethality for mice, dermonecrosis effects for guinea pigs and cytotoxicity in the HeLa cells. The 07 fecal isolates were hemolytic, lecithinase (+), and positive for all biochemical characteristics of Cl. perfringens. Those isolates were not lethal for mice, indicated no dermonecrotic activity in guinea pig, and produced mild degree of cytotoxicity in the cell cultures.
The activity of beta toxin obtained from 95 isolates of CI. perfringens type B isolates was determined using standard toxin-antitoxin test carried in mice and the standard serum neutralization test with antitoxin raised in rabbits. Within the toxin producing isolates, no difference was seen in the potential of toxin based on its lethality for mice.
Epsilon () toxin activity of the 114 isolates of CI. perfringens type D from animals and 4 of the human isolates was also determined. Of the 114 animal isolate, 110(96.49%), and all the 4 human isolates produced E-toxin. There was no difference in the lethal potential of toxin for mice, dermonecrotis action in guinea pig and production of CPE in VERO cells.
Iota (i) toxin activity of the 21 isolates of Cl. perfringens type E was also determined serum neutralization test in mice. Many isolates produced more than one major toxin. Ci. perfringens (CP) type
A produced Alpha (a) toxin; CP type B produced Alpha (a), Beta (3) and Epsilon (E) toxins; OP type D isolates produced Alpha (a) and Epsilon (E) toxins, and OP type E isolates produced Alpha (a) toxin + Iota (i) toxin.
The immunobiologic studies of isolates showed that many of the isolates were quite antigenic. Isolates of CI. perfringèns type D and B were found highly immunogenic as those isolates producing SN titer of 1:320.
Availability: Items available for loan: UVAS Library [Call number: 0998,T] (1).
23.
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.
Material type: ; Format:
print
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.
Availability: Items available for loan: UVAS Library [Call number: 1148,T] (1).
24.
Staphylococcal Coagglutination Test For Rapid Detection Of Foot And Moth Disease Virus
by Baitullah Khan | Dr. Atif Hanif | Prof. Dr. Masood Rabbani | Faculty of Veterinary Sciences.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2010Dissertation note: Foot and mouth disease is a highly contagious, viral disease of cloven hoofed animals and causes high economic losses. Rapid detection of FMD is necessary to control the disease from spreading. Although several reliable tests like ELISA, CFT' and PCR already exit but none of them applicable in field conditions. The aim of this study was to optimize rapid, economical and sensitive test for the detection of FMD. For this purpose rabbits were used to raise immune sera against FMD virus with one uninnoculated control. Immune sera collected from these rabbits at different time interval and presence of antibody was determined by using AGPT. Immune sera were then conjugated with satbalized and inactivated staphylococcus aureus cell using different dilutions. Cell wall of S. aureus contains Protein A which naturally binds with Fc portion of IgG leaving the Fab portion to interact with antigen. In presence of homologues antigen causing agglutination was seen with nacked eyes. Light blue background was found best while observing results.
The coagglutination test was applied on FMD known antigen. Clear agglutination on slide was observed by mixing equal quantity of COAT reagent and its respective antigen. Total 40 vesicular fluid samples from FMD infected animals were tested with COAT, in which 38 yielded positive results and the remaining two yielded negative results. COAT reagents were also tested against PPR virus depicting negative results. COAT was found specific for FMD antigens. This test is quick and generates results within five minutes.
This reagents of CAOT also applied on two fold dilution of vesicular fluid from FMD infected animal and positive result were observed up to 1:32 dilution. This test is sensitive, specific, economical and rapid for detection of FMD. This test was successfully used for detection of FMD in filed.
Availability: Items available for loan: UVAS Library [Call number: 1176,T] (1).
25.
Identification And Genotyping Of Vp1 Genses Of Fmd Viruses
by Atia Bukhari | Prof. Dr. Irshad Hussain | Prof. Dr. Khushi Muhammad.
Material type: ; Format:
print
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.
Availability: Items available for loan: UVAS Library [Call number: 1179,T] (1).
26.
Development And Optimization Of Multiplex Pcr For The Identification Of A, O And Asia 1 Strains Of FMDV In Pakistan
by Muhammad Ikram | Dr. Atif Hanif | Dr. Imran Najeeb | Prof. Dr.
Material type: ; Format:
print
Publisher: 2010Dissertation note: Foot and mouth disease (FMD) is highly infectious disease of cattle, buffalo, sheep and goats. It is caused by genus Aphthovirus of Picomaviradae family. FMDV is RNA virus having seven serotypes A, 0, C, Asia I, SAT1, SAT2 and SAT3.
Foot and mouth disease is endemic in Pakistan and causes high economic losses to livestock industry. So priority is to develop quick and efficient methods for detection of FMDV and to limit the spread of disease outbreak. Although CFT, VNT and ELISA are already being used for the diagnosis of FMDV in Pakistan but these diagnostic techniques are time consuming and their specificity and sensitivity is low. Multiplex PCR for the identification of FMDV is very much sensitive and specific, can be done with in three hours after the receipt of samples.
Present study has been designed to optimize multiplex RT-PCR for rapid detection of FMD virus. RNA was extracted from virus stock obtained from QOL, UVAS Lahore and from field samples. After RNA extraction the samples were subjected to synthesize cDNA by the use of Reverse Transcriptase enzyme. After cDNA synthesis PCR reaction was carried out. The amplified products were resolved on 1.5% Agarose Gel. A multiplex RT-PCR strategy was optimized and developed for the detection of virus serotypes A, 0 and Asia l.
Restulst of this study helped to develop an efficient and economical method for rapid detection of FMD virus and also helpful in differential diagnosis from other vesicular diseases.
Availability: Items available for loan: UVAS Library [Call number: 1189,T] (1).
27.
Microbial Evaluation Of Raw Meat At Abattoirs And Retail Outlests (Lahore)
by Abid Sarwar | Prof. Dr. Mansur ud Din Ahmad | Dr. Imran Najeeb | Prof. Dr. Azhar.
Material type: ; Format:
print
Publisher: 2010Dissertation note: The objective of this study was to evaluate the microbial quality of meat. The present study was planed to determine the aerobic plate count on meat obtained from the abattoirs and local market. A total of 90 meat samples that were collected for determining the microbiological quality of meat. Half of the meat samples (n=45) were collected from various abattoirs and half of the meat samples (n=45) were collected from retail outlets in Lahore City to get an idea of contamination from slaughtering point to retail outlets.
These samples were processed for Aerobic plate counts, E.coli, S.aureus and Salmonella counts. Overall, this study revealed that the level of contamination on meat carcasses was higher in retail meat shops compared to the abattoir. However, the microbial contamination in the abattoir were high if we compare these results to the reports from developing countries like India, Iran and Bangladesh.
Bacterial isolates identified and counted from this study were Staphylococcus aureus (44) out of 90 samples was the most abundant as 48.88%, followed by E. coli (43) 47.77% and Salmonella (26) 28.88%.
Statistical analysis revealed that analysis of variance between various abattoir and the retail meat shops for E.coli, Salmonella and S.aureus showed significant differences with some exceptions. E.coli counts were significantly higher (P < 0.05) in the meat shops and abattoirs. For E.coli most of the data were significant at 5% level (P < 0.05) with some exception in case of beef and goat samples taken from abattoirs which were non significant because of the unhygienic environments. Analysis of variance for Salmonella between various abattoir and the retail outlets were significant at 5% level (P < 0.05). For S.aureus between various abattoir and the retail outlets showed non significant at 5% level (P > 0.05) with some exceptions in case of beef abattoir and goat retail outlet samples taken which were significant at 5% level (P < 0.05).
The higher incidence of microbial load in fresh meat obtained in this study might be attributed to unhygienic and improper handling of animals during slaughter, dressing, evisceration, transportation and unhygienic environments at the retail shops. The usual practice of washing the carcass with the same water in which intestines and offal had been washed was considered as one of the predominant reasons for increased microbial counts of the carcasses. A complete ignorance on the part of the meat handlers/ butchers in hygienic handling of carcasses during slaughter and retailing processes might be the main factors for producing meat with high microbial load.
Levels of microbial contamination in Pakistani abattoirs and traditional retail meat shops reflect the hygiene status of meat production in the developing world. Education of the meat retailers' community which runs the traditional meat shops, in terms of the importance of hygienic and sanitary precautions would go a long way towards providing wholesome and safe meat to the consumers.
Availability: Items available for loan: UVAS Library [Call number: 1196,T] (1).
28.
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.
Material type: ; Format:
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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).
29.
Characterization Of Indigenious Species Of Mycotoxins Producing Aspergilli
by Gull Naz | Dr. Aftab Ahmad Anjum | Prof. Dr. Khushi Muhammad.
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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.
Availability: Items available for loan: UVAS Library [Call number: 1217,T] (1).
30.
Detection Of Hazardous Organism In Raw And Pasteurized Milk With Particular Reference To 3Enterobacteriaceae
by Ayesha | Prof. Dr. Mansur ud Din Ahmad | Dr. Aftab Ahmad Anjum | Prof. Dr.
Material type: ; Format:
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; Literary form:
not fiction
Publisher: 2010Dissertation note: The present study was carried out to detect the hazardous organisms in raw milk from public health point of view. In total one hundred twenty (120) milk samples were collected from milk retail shops in and around Lahore. Out of these 120, one hundred samples were of raw milk and rests of the twenty samples were of pasteurized milk. Their microbiological quality was studied by performing standard plate count (SPC), coliform count and identification of hazardous bacteria belonging to the family Enterobacteriaceae. The micro flora of milk was also studied for the prevalence of multiple drug resistant (MDR) bacteria.
Milk supplied in Lahore city was found to have poor microbiological quality. Bacterial load was determined by SPC and coliform count. The standard plate count (S.P.C) of the raw milk ranged from 4.2x106 to 7.7xl07 c.f.u/ml. The coliform counts ranged from 3.4x 104 c.f.u /ml to 6.9x105 /ml. A total of 81 isolates were identified from raw milk samples. These included Yersinia (3 strains), Klebsiella (16 strains), Escherichia coli (14 strains), Enterobacter (11 strains), Shigella (3 strains), Salmonella (19 strains) and' Proteus (15 strains).The standard plate count for pasteurized milk ranged from 1.45x104 c.f.u/ml to 3.8x 105 c.f.u/ml. The minimum and maximum coliform count was 7.2x102 to 8.4xl03 c.f.u/ml respectively for pasteurized. All samples were outside the international standard for coliform bacteria. A total of 13 isolates were identified from pasteurized milk samples. These included Yersinia (2 strains), Klebsiella (1 strains), Escherichia coli (6 strains), Enterobacter (2 strains), Shigella (1 strains) and Proteus (1 strains).
All the isolates showed multiple drug resistance to various commonly used antibiotics in veterinary practices. Escherichia coli were resistant to all antibiotics used except Gentamicin (10µg). Enterobacter was sensitive to all the antibiotics used except to Ampicillin (10µg). Shigella was sensitive to Gentamicin (10µg), Kanamycin (30µg), Choloramphenicol( 25µg), but showed resistance to Ampicillin (10µg), Oxytetracycline ( 25µg), Streptomycin (10 µg), Pencillin (10 µg) and Tribrissin (25µg)., Salmonella was resistanct to Ampicillin (10µg), Oxytetracycline ( 25µg), Streptomycin (10 µg), Pencillin (10 µg) and Tribrissin (25µg). But sensitive to Gentamicin (10µg). .All the isolates showed greatest resistance to Penicillin (10 ug.) whereas, most of the isolates were sensitive to Gentamycin, Kanamycin and Chloramphenicol.
Finally, it is recommended that the members of the public should always boil raw milk before consumption because of their microbial content. Therefore, it is highly recommended that hygienic practices and regulations, such as on-site pasteurization and implementation of HACCP following established standards, should be introduced to facilitate the production of raw milk of high quality and safety.
Availability: Items available for loan: UVAS Library [Call number: 1219,T] (1).
31.
Prevalence Of Multiple Drug Resistant (Mdr) Bacteria In Intestinal Infections Of Dogs
by Iffat Habib | Dr. Aftab Ahmad Anjum | Prof. Dr. Masood Rabbani.
Material type: ; Format:
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Publisher: 2011Dissertation note: Antimicrobial resistance is a complex problem involving various bacterial species, resistance mechanisms, transfer mechanisms and reservoirs. Cats and dogs are the potential sources for spread of antimicrobial resistance in humans due to their close contact with them. The horizontal transfer of antimicrobial resistance genes through plasmids, integrons and transposons has been found to play an important role in the dissemination of antimicrobial resistance genes. Canine antimicrobial resistant genes had been identified in bacteria isolated from human clinical infections suggesting the spread of resistance mechanisms from canine to human bacteria.
The present study has been designed to study the prevalence of multiple drug resistant strains causing enteritis in dogs. 100 Samples were collected from different Pet clinics in and around of Lahore city. These samples were cultured for identification of MDR bacteria. Antibiotic resistance profile was studied by the standard Disk diffusion method (Kirby-Bauer Method) for commonly used antibiotics. These MDR bacteria were isolated and identified as per standard protocols described in the Bergey's Manual of Determinative Bacteriology. Different combinations of antibiotics were also evaluated for in-vitro antibiotic sensitivity for an effective treatment of these cases so that the load of MDR bacteria could be reduced.
From the collected samples E. coli, Salmonella enterica, Proteus vulgaris, Citrobacter diversus and Psedomonas spp. were identified. Among all of these E.coli was most prevalent followed by Salmonella enterica, Citrobacter diversus, Proteus vulgaris and Psedomonas spp. Out of 127 E.coli isolates 52 40.94%) were declared as MDR-Bacteria following 50 Salmonella enterica isolates 17 (34.00%), 17 Citrobacter diversus 6 (35.29), 12 Proteus vulgaris isolates 06 (50%). It was concluded that MDR isolates were most sensitive to antibiotic combination (Amoxicillin + Clavulanic Acid), followed by (Oxytetracyclin + Tylosin), (Gentamycin + Ceftriaxone), and (Penicillin + Streptomycin). Out of 52 MDR E.coli isolates 23 (44.23%) were found to be invasive. Recommendations are made on prudent use of antimicrobial drugs in dogs, as well as on the need to develop science-based infection control programs in veterinary hospitals.
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32.
Uterine Microbial Flora Of Sahiwal Cattle During Oestrus And Its Relayionship With Pregnancy Rate
by Habib- Ur- Rehman | Prof. Dr. Masood Rabbani | Dr. Ali Ahmad Sheikh | Prof. Dr. Nasim.
Material type: ; Format:
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; Literary form:
not fiction
Publisher: 2011Dissertation note: In the present study uterine microbial flora of Sahiwal cattle during oestrus and its relationship with pregnancy rate was determined. According to the results a total of 11 bacterial species were isolated from 50 uterine samples of estrus Sahiwal cattle, maintained at Livestock Production Research Institute (LPRI), Bahardur Nagar, district Okara, Punjab province, Pakistan. The isolates include E. coli, Micrococcus spp., Bacillus spp., Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus spp., Pseudomonas spp., Citrobacter diversus, Salmonella spp., Proteus spp. and Corynebacterium spp. Tabulation of results showed that prevalence of these isolates was different among pregnant and non-pregnant animals. Moreover, E .coli, Micrococcus spp., Bacillus spp., Staphylococcus aureus and Citrobacter diversus are found to be thriving in uterus as normal microbial flora, whereas, Streptococcus spp. isolate as abnormal microbial flora appearing to be having some role in decreasing pregnancy rate. While, Pseudomonas spp., Corynebacterium spp. Staphylococcus epidermidis, Salmonella spp., and Proteus spp. Isolates could not be differentiable as normal and abnormal uterine microbial flora due to insignificant available data. Furthermore, complete blood counts of 50 blood samples of these same animals indicated that those animals harboring isolates like Streptococcus spp., Pseudomonas spp., and Corynebacterium spp. in their uterus, had more likelihood of abnormally increased value of Mean Corpuscular Volume (MCV) than to presence of any other bacteria. But due to lower data of Pseudomonas spp., and Corynebacterium spp isolated from total samples, only Streptococcus spp. seemed to be ranked as abnormal in Pakistani Sahiwal cattle cows. Interestingly all those animals from where Corynebacterium spp. was isolated, were showing increased values both of MCV and HCT (Hematocrit) which is indicative of their pathogenic role in causing uterine infections.
On the basis of this study it can be modestly concluded that uterine microbial flora identification may serve as a better tool in assessing and foretelling the reproductive health status of the breeding animals. After necessary assessment, presence of any harmful microbial flora or pathogen can be effectively treated through either selecting an appropriate antibiotic by using culture sensitivity testing or by using any suitable bactericidal agent thereby help in boosting conception and pregnancy rates.
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33.
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:
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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®.
Availability: Items available for loan: UVAS Library [Call number: 1294,T] (1).
34.
Uterine Microbial Frlora Of Nili Ravi Buffalo During Estrus And Its Relationship With Pregnancy Rate
by Sohail Raza | Prof. Dr. Masood Rabbani | Dr. Ali Ahmad | Dr. Muhammad Iqbal.
Material type: ; Format:
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Publisher: 2011Dissertation note: The low conception rate has been reported as one of the major cause of poor productivity of livestock. Beside other reasons, presence of different types of microflora inside the uterus of breeding animals, play a key role in the failure of pregnancy. All these microbes results in the infection of uterus ultimately affecting drastically the animal's conception rate. To study the impact of microbial flora on conception rate, 50 Nili Ravi buffalos were selected from Buffalo Research Institute, Pattoki. The breeding animals in heat just before artificial insemination were used to collect bacterial samples with the help of especially prepared and sterilized AI rod with some accessories. The samples were processed for the identification of bacterial microflora by doing number of conventional tests for final characterization. In this study seven different bacterial isolates were identified from all the samples. These include: Escherichia coli, S. aureus, S. epidermidis, Citrobacter species, Proteus species, Lactbacillus species, and Micrococcus species. After elapse of proper period of time the pregnancy statuses of all these buffaloes were determined and correlated with the presence or absence of isolated microbes. The results indicated that Escherichia coli and Staphylococcus aureus isolates were the most prominent bacteria in all the samples collected from pregnant, non pregnant and aborted animals. These two isolates could be designated as normal uterine microbial flora of Nili-Ravi buffaloes because of their presence during all the physical and pathological conditions. Proteus species and Micrococcus species were mostly isolated in pregnant animals. Statistical analysis also confirmed the above statement. Previous reports corroborate the present study and confirm that these bacteria are ranked as normal uterine microbial flora of bovines. So the previous study and present results confirm that both are the normal uterine microbial flora of pregnant Nili-Ravi buffaloes In the present study the prevalence of the Citrobacter spp. only in the aborted animals is supported by the previous studies which show that Citrobacter spp. Is only present in the diseased animals and it also cause the sporadic abortion. Statistical analysis of the data also proved the significance of Citrobacter spp. in aborted animals. So this concludes that Citrobacter spp. are the abnormal uterine microbial flora of Nili-Ravi buffaloes in Pakistan which leads to abortion. The present study has been able us to find the normal and the abnormal uterine microbial flora of Nili-Ravi buffaloes. This information will help to understand the infection process in breeding buffaloes and through corrective actions may decrease the infection rate / abortion rate in Nili-Ravi buffaloes.
Availability: Items available for loan: UVAS Library [Call number: 1306,T] (1).
35.
Phylogenetic Analysis Of Newcastle Disease Virus On The Basis Of Fusion Protein Gene Isolated From Poultry In Lahore District
by Saleem Hassan Shahzad | Dr. Tahir Yaqub | Prof. Dr. Masood Rabbabi.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2011Dissertation note: Among the destructive and vastly communicable viral infections of poultry the most devastating disease is Newcastle Disease (ND) caused by a virus belong to genus Avulavirus of paramyxoviridae family, avian paramyxovirus-1. The NDV causes huge economic losses to the Poultry Industry. The available vaccines failed to protect the birds from the disease; this project is designed to find reasons of the vaccine failures. Keeping in view the importance of outbreaks reported due to NDV and adverse effects on Poultry Industry a study was conducted to examine the function of the cleavage site of Fusion protein sequencing, in Newcastle Disease Virus virulency through Reverse Transcriptase Polymerase Chain Reaction via objective to find the genetic variations among the different isolates of Newcastle Disease field viruses in Lahore District.
One hundred suspected samples of NDV from dead and morbid birds were collected from different sources and areas of Lahore district. Prepared inoculums were inoculated in the 9-11 days old embryonated hen eggs for virus isolation. The allontoic amniotic fluid (AAF) was harvested and tested for HA activity. Further confirmation of NDV was done by using Reference NDV antiserum (HI). Out of 100 samples, 63 showed Hemagglutination activity with washed chicken RBCs and only 16 samples were repressed with specific known NDV antiserum. The remaining samples showed inhibition with known H5, H7 and H9 specific antiserum (12, 13, and 22) respectively. The isolates that were found to be positive through Hemagglutination Inhibition Test (HI) were further tested for Intra Cerebral Pathogencity Index (ICPI). ICPI was performed to characterize the isolates into Lentogenic, Mesogenic and Velogenic forms. The ICPI values obtained after pathogencity test of 16 isolates showed that only 6 isolates have the pathogenicity index above 1.5, and the remaining isolates below 1.5 and above 1, the average higher ICPI value of 16 virus isolates was 1.78.
On the basis of Intracerebral Pathogencity index (ICPI) results only 5 samples were selected for RNA extraction and PCR amplification. The RNA extraction was performed by using kit method (High Pure RNA Isolation kit by Roche-Germany) as recommended by the manufacturer. The gene representing F protein was amplified through Reverse transcriptase polymerase chain reaction (RT-PCR).
Nucleotide sequencing of complete (1580 bp) F gene of 1 NDV isolate was performed. The sequencing results of 1580 bp were compiled and sequence alignment of the NDV isolates, based on a variable portion covering the F-gene site, was done by using, software, ClustalW. The Neighbor-joining phylogenetic Tree was constructed with bootstrap value 1000 using software, MEGA 4.1. The phylogenetic result showed that our isolate has been distinct from Pakistani isolate and has 96% similarity with SPVC/Karachi/33/2007 (velogenic) available in GenBank.
Availability: Items available for loan: UVAS Library [Call number: 1328,T] (1).
36.
Preparatuin And Evaluation Of Monospecific Anisera Against Hemagglutinin, Neuraminidase And Matrix Proteins of local Avian Influenza Strains H5 N1, H7 N3, H9 N2, for diagnostics
by Sumaira Ijaz | Dr. Atif Hanif | Dr. Aftab Ahmad Anjum | Prof. Dr.
Material type: ; Format:
print
Publisher: 2011Dissertation note: Avian Infuenza is an economically important disease of poultry worldwide. It has caused losses to poultry industry on larger scale. It is important due to its zoonotic nature. Studies were carried out to raise monospecific antisera against hemagglutinin, neuraminidase and matrix antigens. HA, NA and M proteins of each of the avian influenza strains were separated on Sodium Dodecyl Sulfate Poly Acrylamide Gel Electrophoresis (SDS PAGE). First virus was lysed to release the proteins. Virus was lysed by using 4% Triton X 100, 1mM KCl and 0.01M Tris buffer. Then the sample was dialyzed. Sample was run on gel to purify proteins. The protein bands of appropriate molecular weight were cut and triturated in 1ml of normal saline. Material was centrifuged to remove the gel content.
Each protein was confirmed by the Bradford's Reagent. Each protein was individually mixed with Montanide ISA 50 adjuvant in 1:1 ratio to make the vaccine. Vaccine of each polypeptide of AIV strains was injected in three groups of nine birds each. One group of birds was injected with HA, second group with NA and third group with Matrix proteins of H?, H? and H?. Three groups of birds served as control. The blood samples of all birds were collected before and after inoculating vaccine. The sera of birds before and after inoculating vaccine were checked for antibodies titre against HA antigen by HI test. Antibodies against Matrix antigen were detected by Agar Gel Precipitation Test. Antibodies titre was raised after inoculating polypeptides. In case of sudden outbreaks, antisera may be helpful to control disease.
Availability: Items available for loan: UVAS Library [Call number: 1351,T] (1).
37.
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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38.
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).
39.
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).
40.
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).
41.
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.
Availability: Items available for loan: UVAS Library [Call number: 1417,T] (1).
42.
Production, Purification And Evaluation Of Anti Tetanus Serum
by Mian Muhammad Khubaib Sattar | Prof. Dr. Tahir Yaqub | Dr | Mr. Muhammad Zubair Shabbir.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2012Dissertation note: To produce anti-tetanus serum, ten female sheep of about 3 years of age are immunized with commercially available tetanus toxoid vaccine (Imatet™, Amson vaccines and Pharma) for eighteen weeks to these experimental animals with two weeks interval. Serum samples of all the sheep were also collected fortnightly and stored at -20 ºC.
ELISA was performed to determine the antibody (IgG) titer of all the test samples. Out of 90 samples, 20 samples showed tetanus antibody (IgG) titer of 100.8 I.U. or more, while 8 samples presented tetanus antibody (IgG) titer of 160.9 IU or more. Out of these 8 samples, 3 samples had tetanus antibody (IgG) titer of 190.9 I.U. or more. Maximum tetanus antibody (IgG) titer was 195.4 I.U. at day 120.
Three samples exhibiting maximum antibody titer (190.9 I.U., 195 I.U. and 195.4 I.U.) were mixed in equal quantities for purification of Immunoglobulins (IgG). A volume of 15 mL of aggregated serum samples was mixed with 15 ml of saturated ammonium sulfate having final concentration of 45 % in the mixture which is continuously stirred at room temperature for 1 hour. Mixture was centrifuged at 10000 rpm for 30 minutes in refrigerated centrifuge machine and dialyzed against 10 changes of PBS at 4 ºC. Desalting is checked with BaCl2 solution.
The purified tetanus immunoglobulins (IgG) were treated with papain to produce Fab Fragments. Then the protein content of the purified tetanus immunoglobulins and Fab fragments was estimated with Bradford protein Assay. BSA standard curve was used to produce a regression equation [Y (OD Value) = 0.218 + 0.033 X (Protein Concentration)] which was used for calculation of the protein contents of the samples.
The purified tetanus immunoglobulins and Fab fragments were tested for purity with SDS-PAGE analysis. Then in vivo toxin neutralization test was performed in mice to check the tetanus toxin neutralization ability of the sera produced.
Availability: Items available for loan: UVAS Library [Call number: 1420,T] (1).
43.
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.
Availability: Items available for loan: UVAS Library [Call number: 1446,T] (1).
44.
Determination Of The Hepatitis C Virus Genotyping Prevailing In The Hepatitis Suspected Patients In District Mardan,
by Suliman Qadir Afridi | Prof. Dr. Tahir Yaqub | Dr. Fariha Akhtar | Dr. Yasin Tipu.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2012Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1469,T] (1).
45.
Characterization Of Antimicrobial Resistance In Escherichia Coli Recovered From Retail Chicken In Lahore
by Fayyaz Yasin | Dr. Ali AHmad Sheikh | Prof. Dr. Masood Rabbani.
Material type: ; Format:
print
Publisher: 2012Dissertation note: Escherichia coli is normal inhabitant of lower intestinal tract of all warm blooded animals. The background of drug resistant studies on E. coli represent that extensive and irrational use of antimicrobials in human and veterinary sector for treatment, prophylaxis and feed additive made this organism resistant to commonly used antimicrobials.
It is assumed that E. coli have the ability to transfer resistant genes via bacterial conjugation, transduction and transformation. As a result pathogenic organisms are also becoming resistant to commonly used antibiotics. And there is a need to check the extent of resistance to ensure the efficacy of antimicrobials used in public health.
The purpose of current study was to estimate the prevalence of antimicrobial resistant E. coli in retailed chicken meat samples of Lahore city. In current study hundred chicken meat samples were collected from local market in various areas of Lahore.These samples were processed for isolation of generic E. coli. Initial confirmation of generic E. coliwas made using standard culturing and biochemical reactions. The prevalence rate of E. coli was found 85%. The antimicrobial resistance against in the E. coli isolates was determined by disk diffusion method. All the E. coli isolates were found resistant to at least seven antimicrobials and 34 different antimicrobial patterns were found.
Availability: Items available for loan: UVAS Library [Call number: 1477,T] (1).
46.
Efficacy Of Commercial Disinfectants Against The Water Contaminating Bacteria At Commercial Broiler Farms
by Mian Muhammad Salman | Dr. Aftab Ahmad Anjum | Pfor. Dr | Prof. Dr. Tahir Yaqub.
Material type: ; Format:
print
Publisher: 2012Dissertation note: Water is an important constituent for poultry. Poor hygienic conditions of water are health hazard for poultry. Many outbreaks are caused by consuming poor quality water.. Fifty water samples from different broiler farms in and around Lahore were collected from drinkers in sterile containers. Bacterial load was assessed using total viable count and coliform count. The counts were above the threshold level (50cfu/ml for coliform and 100cfu/ml for total viable count) showing that water used at poultry farms was of low microbiological quality. Five Disinfectants PHMB20% (.75ml/lit, 1.5ml/lit, 3.0ml/lit), PHMB11% (1.5ml/lit, 3.0ml/lit, 6ml/lit), 0.2% chlorine dioxide (0.1ml/lit , 0.2ml/lit, 0.4ml/lit) Glutral 9.8%(1.5ml/lit, 3.0ml/lit, 6ml/lit), organic acid(1.5ml/lit, 3.0ml/lit, 6ml/lit) were used and they resulted in log reduction of TVC by PHMB20% (5.83±4.36, 6.14±3.98, 9.35± 0.68), PHMB11% (9.42±0.21), 0.2% chlorine dioxide (2.45±0.97, 3.19±0.73, 6.33±0.80 ) Glutral 9.8%(6.87±1.00, 9.73±1.00,9.73±1.00), organic acid(4.75±1.21, 6.62±1.26, 6.90±1.15).PHMB20%,PHMB11%, Glutral 9.8% and organic acid were effective at normal dose, while 0.25 chlorine dioxide was effective at normal dose against at normal dose. Log reduction in Coliform count at half, normal and double dose by PHMB20% (6.52±3.33, 6.96±2.46, 7.96±0.98), PHMB11% (7.89±1.01), 0.2% chlorine dioxide (3.65±0.73, 5.08±0.98, 6.27±0.97) Glutral 9.8%(8.48±0.99), organic acid(5.18±1.21, 5.93±1.26,6.46±1.15±) . PHMB20%, PHMB11%, 9.8% Glutral, organic acid were effective against coliform bacteria at half dose while 0.2% chlorine dioxide was effective at normal dose. Glutraldehyde was effective at normal dose amongst all disinfectants against Total viable bacteria and coli form bacteria.
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47.
In Process Quality Control Factors Affecting The Potency Of Indigenous Mycoplasma Gallisepticum (Mg)
by Muhammad Asim Raza | Dr. Arfan Ahmad | Prof. Dr. Masood Rabbani.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2012Dissertation note: Mycoplasma gallisepticum (MG) is cause of chronic respiratory disease (CRD) and is responsible for significant economic losses to poultry industry. In Pakistan, imported MG bacterin fails to induce immunoprophylaxis that could be due to subtle antigenic variation in the immunogen of the vaccine. Therefore present study was conducted to optimize inactivations( phenol, formalin and binary ethylenimine) concentration and exposure time to inactivate MG and their effect on potency of MG bacterin ( prepared from local isolates) along with different bacterial biomasses (0.5%, 1% and 1.5% PCV) and adjuvants (montanide oil ,gel and water ). It was observed that the MG bacterin containing 1.5% level of immunogen/biomass induced significantly higher anti-MG-ELISA antibody titer (p < 0.05) as compared to other bacterins containing lower concentrations of the immunogen.The formaldehyde inactivated the pathogen within shortest possible time and showed undetectable effect on its potency. The antibody response was significantly higher (p<0.05) as compared to that of bacterins prepared from the pathogens inactivated by either phenol or BEI. . Montanide ISA70 containing MG bacterin induced significantly higher anti-MG-ELISA antibody titer (p<0.05) in broilers than the other bacterins containing either water or aluminum hydroxide gel. It is concluded that formaldehyde inactivated oil based vaccine containing one percent immunogen (0ne percent PCV) induce antibody response in broilers that is comparable with the imported vaccine.
Availability: Items available for loan: UVAS Library [Call number: 1495,T] (1).
48.
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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49.
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.
Availability: Items available for loan: UVAS Library [Call number: 1537,T] (1).
50.
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).
