1.
Antibody Response Of Buffalo To Inactivated Foot And Mouth Disease (Aphtho) Virus Vaccine
by Nadeem Murtaza | Prof. Dr. Khushi Muhammad | Prof. Dr. Masood Rabbani | Faculty of Veterinary Sciences.
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Publisher: 2005Dissertation note: Buffaloes when vaccinated against Foot and Mouth Disease (FMD) vaccine containing serotype "O" of the virus, showed detectable level of complement fixing (CF) antibodies. Buffaloes vaccinated with Aftovaxpur vaccine showed undetectable level of CF-antibodies when tested through complement fixation test using local vaccinal serotype "O" of FMD virus. However, buffaloes irrespective of age and sex when vaccinated with aluminum hydroxide adsorbed FMD vaccine containing serotype "O" of the FMD virus, showed detectable level of CF-antibodies, when tested through CFT using the local serotype "O" Of FMD virus. These antibodies disappeared fourth month post boosting. Buffalo calves immunized with oil based FMD vaccine showed high-level GMT titer (17.6) of the CF-antibodies. Rabbits immunized with the oil based FMD vaccine showed high level GMT (31.2) of the CF-antibodies. Low level of CF-antibodies might be sufficient to induce resistant to field exposure of the FMD virus serotype in the presence of blood complement. Sera of buffalo, cattle, sheep, goat and guinea pigs contained complement titer 35.2, 32.6, 19.2, 20.8, 614.4, respectively. Moreover, it was observed that complement activity remained stable when stored at -200C for 24 hours. The complement activity decreased from 1:512 to 192, 70.4, and 13.6 when stored at 40C, 250C and 370C, respectively. The complement activity was detectable when diluents containing Ca++and Mg++ ions were used.
Availability: Items available for loan: UVAS Library [Call number: 0877,T] (1).
2.
Development Of Standard Protocols For Preparation And Evaluation Of Liver Homogenate Vaccines Against
by Sahidullah | prof. Dr. Masood Rabbani | Prof. Dr. Khushi Muhammad | Faculty of Veterinary Sciences.
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Publisher: 2005Dissertation note: Twelve vaccines were prepared from HPS infected liver homogenate by using two different virus concentrations (1x102 &1x103 LD50) and two virus inactivants (1%formalin and 0.001%Binary ethyleneimine) with and with out different adjuvants. These vaccines were evaluated in 13 groups of day old broilers (105 chicks) for their comparative immunogenicity and protection. At day 14 of age, groups A1, B1, C1 & D1 were vaccinated with 4 oil base vaccines (OB-HPSV) with different virus concentration and different inactivants. Similarly groups A2, B2, C2 & D2 were vaccinated with aluminized vaccines (AH-HPSV) and groups A3, B3, C3 & D3 with non adjuvant vaccines (NA-HPSV). Groups E was kept as unvaccinated control group. All the vaccinated birds were found sero-positive 7 days post vaccination (PV). IHA GMT results indicated no difference for different virus concentrations and different virus inactivants but same adjuvant. The IHA GMT recorded weekly during 0-28 days post vaccination was the highest and more consistent (52-181) for OB-HPSV followed by AH-HPSV (52-147) and then NA-HPSV (73.3-104). All the birds vaccinated with OB-HPSV resisted the virus challenge 21 days PV (100% protection). While protection percentage recorded for AH-HPSV and NA-HPSV was 87.5 % & 62.5% respectively. It was concluded that 1x102 LD50 oil base vaccines inactivated with either formalin or binary amine may be recommended for commercial use being the best in experimental trails.
Availability: Items available for loan: UVAS Library [Call number: 0878,T] (1).
3.
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).
4.
Effects Of Different Disinfectatnts On Pathogens In Poultry
by Asif Abbas Malik | Prof. Dr. Masood Rabbani | Prof. Dr. Khushi Muhammad | Faculty of Veterinary Sciences.
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Publisher: 2006Dissertation note: Poultry sector is the second largest industry after textile in Pakistan. It is threatened by various diseases i.e; Newcastle disease (ND), Avian Influenza (Al), Colibacillosis, Hydro-pericardium syndrome (HPS) and Infectious bursal disease (IBD, Gumboro). The efficacy of various available disinfectants (Hygen 275 — 2000 H, Virkon S and Aldekol) was tested at 2x, lx and Y2 x dilution against Staphylococcus aureus, Escheria Coil, Newcastle Disease virus and Avian Influenza virus. Each dilution of all the disinfectant was divided into 4 aliquots i.e; a, b, c and d (each of the
aliquot, for each pathogen). Each aliquot were mixed with equal volume of either of the pathogen. The mixture of the disinfectant and the pathogen was incubated at 37°C for a period of 15, 30 and 45 minutes of interaction. The contents were collected aseptically and processed to evaluate the effectiveness of the disinfectants. Disinfectant A (Hygen 275-2000H) showed good bactericidal as well as virucidal activity at 1% dilution. Disinfectant B (Virkon S) was able to kill all the bacteria and viruses even at 0.25 % dilution. While, disinfectant C (Aldekol) effectively killed the bacteria and viruses at 0.5 % and I % dilutions. Results of the study will help the farmers to adopt effective biosecurity measures to minimize the challenges at farm level.
Availability: Items available for loan: UVAS Library [Call number: 0966,T] (1).
5.
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.
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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.
Availability: Items available for loan: UVAS Library [Call number: 1355,T] (1).
6.
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.
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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).
7.
Molecular And Serological Characterization Of Avian Influenza Viruses In Domestic And Wild Birds
by Mobeen Sarwar | Prof. Dr. Khushi Muhammad | Prof. Dr. Masood Rabbani.
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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.
Availability: Items available for loan: UVAS Library [Call number: 1536,T] (1).
8.
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.
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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).
9.
Transovarian Transmission And Molecular Characterization Of Hydropericardium Syndroe Virus In Experimentally Infected Poultry Birds
by Rabia Tahir | Prof. Dr. Khushi Muhammad | Prof. Dr. Masood Rabbani.
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Publisher: 2013Dissertation note: Poultry is one of the important and growing industries in Pakistan. It has now become the tremendously growing sub-sector of livestock. But the major constraint in growth of this industry is infectious diseases of poultry which leads to high mortality and morbidity rate resulting in heavy economic losses. The present study was conducted on one of the infectious poultry diseases named Hydropericardium syndrome which commonly affects broilers between 3-5 weeks of age. The causative agent is highly infectious virus belonging to Avian Adenovirus serotype 4. These are non-enveloped DNA viruses. It is an acute, infectious disease characterized by high mortality and excess pericardial fluid and multifocal hepatic necrosis. The incubation period ranges from 2-5 days followed by inoculation with liver homogenate or purified virus. This disease is characterized by the accumulation of straw coloured jelly like fluid in the pericardial sac, discoloured and inflamed liver with basophilic intranuclear inclusion bodies, congested kidneys and mortality up to 70%.
The present research was planned to prove that hydropericardium syndrome virus besides horizontal transmission is also transmitted by vertical transmission through transovarian route. For this purpose Liver sample was used as source of this virus. These liver samples were processed for further propagation of virus in live birds. Moreover, Virus neutralization test was also conducted for the confirmation of virus. To prove the transovarian transmission of this disease liver homogenate of infected birds was injected in 22 wk old breeder. Eggs were collected at 7-14, 15-21 and 22-28 days post infection. The day old hatched chicks were slaughtered to obtain liver and spleen sample for confirmation of virus through PCR. For confirmation, DNA was extracted using KIT method followed by polymerase chain reaction and amplified genomic material was visualized through gel doc system.
After validation of PCR 90 samples of liver and spleen were processed for DNA extraction followed by PCR. None of the samples of extracted DNA processed for each tissue from chicks produced a visible band of DNA in agarose gel after ethidium bromide staining. The possible reason for these negative results may be that viral load was below detectable limit or the presence of high titre of neutralizing antibodies. The implications of these findings are that vertical transmission via transovarian route does occur in poultry birds but the exact mechanism and establishment of latent infections further need to be investigated.
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10.
Production And Evaluation Of Peste Des Petits Ruminants Virus Vaccine
by Muhammad Aness | Prof. Dr. Khushi Muhammad | Prof. Dr. Masood Rabbani.
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Publisher: 2013Dissertation note: PPR is an acute highly contagious viral disease of small ruminants which is endemic in Pakistan. Present study was aimed to evaluate the freeze dried PPRV vaccine with variable biological titer to induce protective immune response in beetal goats. The comparative immune response of animals to adjuvant and non-adjuvant vaccine and duration of immunity was also studied. Effect of boosting on the humoral immune response of animals as well as shelf life of various vaccines was also evaluated. Each of the vaccines was inoculated in a group of five animals. Serum samples were collected at specified time intervals and antibody levels were detected through cELISA as PI values and neutralization test as MNA titer.
The virus was propagated on the Vero cells. It was estimated that infecting 2 x 107cells with 104.00 TCID50 virus concentrations added to a T-175 cell culture flask at the time of subculture yielded maximum virus titer in the cell culture harvest following three freeze thaw cycles of the contents. The freeze dried vaccine with a biological titer of 105.00 TCID50 per dose provoked maximum antibody titer followed by the ones with a titer of 104.00 or 103.00TCID50 which provoked nearly equivalent protective immune response while the animals inoculated with a vaccine having 102.00 TCID50virus concentrations developed minimum antibody titer. The oil adjuvant PPRV vaccines elicited significantly higher antibody titer in comparison to gel based vaccines but however minimum antibody titers were detectable in response to freeze dried vaccines. Although protective antibody level (? 10 neutralizing antibody units) was detectable in the animals vaccinated with either oil based, gel based or freeze dried vaccine containing biological titer of 104.00 TCID50 but however the extent and duration of immunity was found to be most superior in response to oil based vaccines. It can be concluded that a single shot of either gel or oil based vaccine can provide protection in the vaccinated animals for a minimum of one year duration. Goats receiving a booster dose of the vaccines had a significantly higher antibody tier in comparison to the ones who received single dose of the vaccines. The freeze dried and wet vaccine kept at 4 °C did not show any significant drop in the biological activity of the virus even after 12 months of storage. Immunogenicity of the both adjuvant and non-adjuvant vaccines, as measured through the immune response in the vaccinated animals, also remained unaffected after 12 months of storage at 4 °C.
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11.
Comparative Analysis Of Respiratiory Microbiota From Clinically Healthy And Deseased Broiler Breeders
by Husnain Ahmed | Prof. Dr. Masood Rabbani | Prof. Dr. Khushi Muhammad.
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Publisher: 2014Dissertation note: The pulmonary system of birds is a considered a reservoir of innumerable bacterial pathogens including those which are subject to public health significance. This scenario makes respiratory tract of birds prone to many bacterial infections as well. There are many respiratory outbreaks in poultry that causes huge economic losses and a number of bacterial pathogens either acting as primary or secondary pathogen can be held responsible for these losses. A very small fraction of (<1%) of bacterial species are culturable, limited as well as highly variable and time consuming conventional microbiological procedures have typically excluded the normal flora present in the respiratory tract or have restricted the analysis to potential pulmonary pathogens. Due to unculturable nature of many bacterial species there is a very little room left for discovering or determining novel organisms or pathogens and their association with clinical outcomes through conventional microbiological procedures. With the advancement of technology metagenomic analysis of a given sample has emerged as a major culture independent technique for identification of many pathogens, by reading the hypervariable region of the 16S rRNA gene, culture-independent technique such as 454-pyrosequencing, can provide species specific sequence of any bacteria in a given sample.
A total of 12 T-BAL samples from breeder birds were selected based upon the quality and quantity of the double-stranded gDNA. Using 454 bar-coded pyrosequencing, the hypervariable regions of the 16S rRNA gene corresponding to V1 – V5 (~ 1,000 bp) were sequenced. Of the high-quality reads obtained (296,811) using the MOTHUR platform, the sequences were processed for sequence alignment with the 16S RDP database via BLASTn, and subsequent taxonomic analysis through MEGAN programs using a homology-based method to bin sequence reads. The results of study indicate that birds harbor a diverse microbial community including number of phyla, families, genera and characterized bacterial species. The bacterial communities were relatively conserved at the phylum level; however, at lower taxonomic levels, differences were observed in the phylotypes and abundance between the clinically diseased and healthy birds. As indicated by the rarefaction plot and the Shannon-Wiener and Simpson-Reciprocal diversity indices, the biodiversity and richness in the taxonomic content was higher in the clinically healthy birds compared with the diseased birds. Regardless of the bird health status a number of new species were identified. A number of these bacterial species have been found to be associated with infectious diseases in humans and other species, although the clinical importance of these bacteria as part of the respiratory microbiome of birds has not been established.
As the nature of bacterial species is to constantly act with one another and, potentially, with the birds themselves provides an interesting avenue for continued research. There is a need to conduct further clinico-pathological studies to establish the link between causes versus effects.
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12.
Molecular And Serological Characterization Of Soilborne Francisella Tularensis
by Javed Muhammad (2010-VA-65) | Prof. Dr. Masood Rabbani | Prof. Dr. Khushi Muhammad | Dr. Muhammad Wasim.
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Publisher: 2016Dissertation note: Tularemia, caused by Francisella tularensis (F. tularensis ), is a zoonotic disease transmitted through contact with infected animals and contaminated environment. The disease has been reported from many countries of the world but no study has been done in Pakistan. In the current project, a total of 2280 soil samples representing 456 villages of eight districts of Punjab province were collected from way-points having human-animal interaction, processed for genomic extraction and tested through real time PCR for presence or absence of F. tularensis. Association of risk factors was determined from data such as gender and age of animals, plough method, irrigation system, fertilizer type used, availability of veterinary services, level of farmer education, physical and chemical composition of the soil. Moreover, sero-prevalence against F. tularensis in cattle, buffaloes, sheep and goats was determined using ELISA. Seventy four soil samples (3.24 percent) were found positive for F. tularensis. Phylogenetic analysis showed 100 percent similarity index with F. tularensis sub specie holarctica reported from other regions like USA, Sweden, Spain, Turkey and Germany. Presence of F. tularensis in soil showed negative association with increase in number of human density (0.7159; 0.3834-0.2054). Prevalence of anti- F. tularensis ELISA antibodies were significantly higher (p<0.05) in large ruminants (cattle and buffalo) as compared to small ruminants (goat and sheep). Age and gender-wise analyses showed non-significant differences (p>0.05) between small and large ruminants. Whereas, rain-irrigation system (2.96: 1.35- 6.48), lack of veterinary services (4.77:1.26-18.03) and use of organic fertilizer (5.3: 11.38- 20.39) have positive association with prevalence of anti- F. tularensis ELISA antibodies in the serum. Sero-prevalence of Ft in the animals has significant association with quantity of clay in soil (p<0.05). A conventional PCR based test has also been optimized for
Summary
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detection of F. tularensis using tul4 gene specific primers. Specificity of primer showed F. tularensis detection in soil DNA in the presence of other cross-reactive organism. Sensitivity was determined in two fold dilutions with detection limit of up to 320 pg/μL. Utilizing pET28a vector, a construct was prepared containing transformed tul4 gene (450bp) showing 100 percent sequence homology to query gene sequence. For manufacturing diagnostic assays especially in developing countries where availability of BSL-3 facilities and positive control reagents is an issue, provision of tul4 gene based constructs in vector can act as positive control and biosafe to use.
It is recommended that similar studies may be done in other parts of Pakistan to have spatial distribution of F. tularensis all over Pakistan. In future studies, other sources of transmission like water, ticks and rodents may be considered with soil for complete analysis. Transportation of whole genome of F. tularensis has been prohibited by Russian government, ATCC and CDC, WHO is working on designing a complete protocol for transportation of this bacteria or genome to other countries. Under such situation, conventional PCR optimization can be done for diagnosis of F. tularensis and pET28+tul4 constructs, developed in this study, can be used as a PCR positive control reagents. Availability: Items available for loan: UVAS Library [Call number: 2640-T] (1).
