1.
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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2.
Immune Response Of Buffaloes To Foot And Mouth Disease Virus Vaccine
by Munir Ahmad Tariq | Prof.Dr.Khushi Muhammad | Prof.Dr.Muhammad Akram Muneer | Faculty of Veterinary Sciences.
Material type: ; Format:
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; Nature of contents: ; Literary form: Publisher: 2007Dissertation note: Foot and Mouth Disease (FMD) is a highly contagious infection of cloven-footed animals such as buffalo, cattle, sheep, goats and camels and FMD is characterized by high rise of temperature, salivation, smacking of mouth, vesicular lesion in the buccal cavity, inner flares, coronary band and interdigital spaces, memory glands etc. In Pakistan FMI) disease is caused by "0", "A" or "Asia-i" type of the virus of an Aphthovirus of Picornaviridae. The vaccinal serotypes of FMD virus were characterized as "A", "0" and "Asia-i" by virus neutralization test using imported mono-specific rabbit antiserum. Each of the serotypes multiplied rapidly on monolayer of Baby Hamster Kidney -21 (BHK-21) cells.
The BHK-2 I cells were propagated in carrel and roux flasks in MEM 199 containing 10% fetal bovine serum. Heat treated goat serum was equally effective as growth promoter for BHK-21 cell line. The cells rapidly multiplied and formed a monolayer within 72 hours at 37 °C. The cells were harvested using trypsin (0.025%) without affecting the cell viability that was observed by cytometeric as well as by colorimetric assays. The cells were stored in cryogenic containers and revived successfully on 12 months post storage.
The FMD virus isolate ("0", "A" and "Asia-i") grew well on the monolayer of BHK-21 cells and produced more than 106, and i04 units of the Tissue Culture Infective Dose-50 (TCID50) on 5th passage, respectively. Each of the virus serotypes was effectively inactivated using 0.12 % formaldehyde, or 0.004 M of Binary Etyhieneimine (BET).
The inactivated virus suspension was admixed with either oil base, lanolin or aluminium hydroxide gel and homogenized to get stable vaccine preparation. The adjuvant containing vaccines induced detectable level anti-FMDV-VN antibodies titer in buffalo calves on 19 days post-priming. Oil and gel based FMD vaccines induced detectable geometic mean titer (GMT) of the anti-FMDV-CFT antibodies (2-3 and 7-8) on 19 days post vaccination, respectively. The oil and gel based vaccines induced 1: 64 and 1:80 GMT titer of the anti-FMDV-CFT antibodies on 128 and 64 days post-vaccination, respectively and the titer declined there after as 1: 9 and 1: 3.3 on 258 days post vaccination. From this study it can be concluded that oil based vaccine induces the antibody response in buffalo latter than that of gel adsorbed vaccine. Higher titers of the antibodies are retained for comparably longer period of time by oil based vaccines. Moreover, age of buffaloes, animal species and vaccine storage at 4 C exhibited undetectable effects on the antibody response to the vaccine.
The study has indicated that vaccination programs against field infection of FMD in all the domestic cloven footed animal species could be effective way of immunoprophylaxis.
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3.
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:
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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.
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4.
Effect Of Various Stress Factors On The Immune Response(Ph.D)
by Muhammad Yasser Mustafa Butt | Prof.Dr.Muhammad Akram Muneer | Prof.Dr.Khushi Muhammad | Faculty of Veterinary Sciences.
Material type: ; Format:
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; Nature of contents: ; Literary form: Publisher: 2009Dissertation note: Pakistan has vast population of dogs belonging to different breeds. Most of the dogs have no pedigree record which is a great threat to conservation of different breeds. No study on DNA fingerprinting of dogs has been conducted in Pakistan. DNA fingerprinting of dogs is necessary to overcome the problems like forensic cases, sale & purchase, individual identity in case of fertilization by more than one male and ownership disputes. Microsatellite markers have been proved as an efficient and powerful tool for parentage testing and breed characterization of dogs. In this study, a panel of microsatellite markers, having high polymorphism information content (PlC) values, was developed. Blood samples were taken from cephalic vein of two breeds of dogs (German shepherd and Labrador retriever). DNA was extracted by Inorganic method. Primers of microsatellite markers were optimized for successful amplification conditions in the Bio-Rad thermocycler. Multiplex PCR was performed, for amplification of these microsatellite markers on 46 samples belonging to 20 families. Genotyping analysis was performed for the PCR products of microsatellite markers on non denaturing polyacrylamide gel. These results were analyzed statistically software "POPGENE 3.3 and POWER STAT". Allele frequency, heterozygosity, homozygosity, polymorphism information content (PlC), power of discrimination and power of exclusion of all microsatellite markers were calculated. Average power of discrimination among non parents, average hetrozygosity, average observed homozygosity and average polymorphism information content (PlC) value for all alleles was 0.809, 0.6345, 0.29 13 and 0.724 respectively. Moreover combined power of exclusion reached a significant value of 0.9998. Almost all of the microsatellite markers showed significant variations in both German shepherd and Labrador retriever breeds. Microsatellite "REN41D2Ob" showed maximum variation i.e. 17 alleles and microsatellite"REN49F22b" showed the least variation among all microsatellite markers i.e. 4 alleles. Genotyping results of microsatellite markers were clearly different for two different breeds showing a distinct genetic distance between German shepherd and Labrador retriever breeds.
Results of this study lead to development of a panel of microsatellite markers which can be used for parentage analysis and breed characterization of dogs. This was a preliminary study on dogs in Pakistan. This facility can be provided on commercial basis to pet owners and kennel clubs. Moreover this study can become the basis for further research investigations in canines in Pakistan.
To evaluate effects of various stress factors on immune response and growth performance of broiler chicks, a total of five experiments using 2000 broiler chicks were conducted. In each experiment, chicks were divided into five groups (A, B, C, D and E), and each group consisted of 80 day-old-chicks. In each experiment, the chicks were exposed to stress factors, such as temperature, stocking density, feed deprivation, water restriction and light. Each chick in groups A, B, C, and E was vaccinated against IBV, NDV, IBDV and HPSV, but chicks in group D were kept as unvaccinated controls. Blood samples from each group were collected on 36 day of age, at 18 hrs for determining TLC, DLC and H/L ratio. The antibody titers of chicks in different groups were analyzed using HI test at days 36th and 56. The cellular response was analyzed by injecting PHA-P in the wattles of bird during post stress period. The effects of each stress on lymphoid organs were determined. The potential to resist virulent NDV challenge and effect of stress factors on body weight gains and FCR of chicks was also determined.
In experiment 1, conducted to determine the effect of various temperature ranges on broiler chicks, it was observed that the heat stressed (HS) birds showed non-signilicant dilYerence in TLC values. The HS effect on lymphoid organs indicated that the mean weight of thymus of chicks in group B (0.29±0.02) and C (0.59±0.13) was significantly (P<0.05). lower than those in groups A (4.11±3.26), D (4.50±0.77) and E (4.35±0.21). The mean bursa weight of heat stressed chicks in goups A (0.94±0.59) and B (0.20±0.01) were significantly (P0.05) lower as compared to non- heat stressed chicks in groups D (1.42±0.22) and E (1.33±0.18). The mean spleen weight of groups A (1.21±0.13) and B (1.30±0.11) was significantly (P0.05) lower than groups D (L93±0.16) and E (1.52±0.10) indicating the adverse effect of increased temperature. The FCR valueswere significantly (P0.05) different among groups in 6th1 week and effect of Vitamin C was found significantly (P<0.05) improved than Vitamin E and glucose treatment. At 36 day of age the HI titer was recorded significantly (P<0.05) lower in group A (GMT 61) than B (GMT 144) and C (GMT 109) groups while group E (GMT 186) showed significantly (P<0.05) higher HI titer. At the age day 56 (06 days post challenge) the HI antibody titers in all groups registered a rise except in group D. All the chicks in group D died indicating clinical signs of Newcastle disease. The post challenge GM HI titers recorded in groups A, B, C and E at the age of 56 days was 79, 156, 122 and 216, respectively. There was nonsignificant (P>0.05) differences in wattle thickness (cm) among groups A (1.51+0.06), B (1.77±0.26), C (1.2±0.25) and D (1.52±0.22) but increased in group E (1.83+0.08). The mortality was found significantly (P<0.05) higher in groups A (14) and D (40) on challenge with NDV virulent virus.
In experiment 2, conducted to determine the effect of various levels of stocking densities on broiler chicks, it was observed that stressed birds had showed non-signilicant (P>0.05) difference in TLC values. The effect on lymphoid organs found that the mean thymLls weight (grn) of chicks in groups A (0.37±0.04) and B (0.74±0.17) were significantly (P<0.05) lower than groups C (1.50±0.35), D (4.43±0.72) and E (4.40±0.23). The mean bursa weight (gm) of groups A (0.33+0.03) and B (0.57+0. 1 7) were significantly (P0.05) lower than those of groups D (1.76±0.05) and 13(1.33±0.08) indicating that less space effect the bursa development in the chicks. The mean spleen weight (gm) of groups A (1.18±0.07) and B (1.52±0.20) was significantly (P<0.05) lower than group D (2.37±0.28). The FCR values were sign ilicantly (P<0.05) different among groups in 6thi week and there was non-signiflcant (P>0.05) difference among groups with treatment of Vitamin C, Vitamin E and glucose. At 36th day of age the I-Il titer was recorded significantly (P<0.05) lower in group A (GMT 67) than groups B (GMT 9.6) and C (GMT 102). The chicks in group D (GMT 07) showed negligible HI titer while group E (GMT 185) showed significantly (P<0.05) higher HI titer. At the age day 56 (06 days post challenge) the 1-Il antibody titers in all groups registered a rise except in group D. All the chicks in group D died indicating clinical signs of Newcastle disease indicating that the titers in the birds were not enough to resist the virulent challenge. The postNDV-challenge GM HI titers recorded in groups A, B, C and Eat the age of 56 days were 86, 121, 132 and 210. There wa non-significant (P0.05) differences in wattle thickness (cm) in groups A (1.44±0.07) and C (1.43±0.10) while group E (1.64±0.31) showed significantly (P0.05) increased wattle thickness. The mortality was found significantly (P0.05) higher in groups A (25) and D (40) on challenge with NDV virulent virus. This indicated that less floor space decreased the immune response of the birds which leads to the infection/death.
In experiment 3, effect of feed deprivation at different time intervals on broiler chicks was studied, it was observed that feed deprivation stressed birds had showed non-significant (P>0.05) differences in blood cells population. The effect of feed deprivation on the mean thymus weight (gm) of chicks in group C (0.96±0.29) was adversely affected as the chicks in this group had significantly (P<0.05) lower weight than groups B, 1) and E. The bursa mean weight (gm) of groups A (0.48±0.11) and C (0.52±0.06) was significantly (P0.05) lower than those of groups D (1.40±0.18) and E (1.28±0.11) indicating, that 24 hrs and morning off-feed effect the bursa development in the chicks. The mean splen weight(gm) of groups A (I. 19±0.07) and C (1.21±0.06) vcre significantly (P0.05) lower than groups D and E indicating adverse effect ol24hrs and day off feed on chicks lead to infection. The FCR values were significantly (P0.05) different among groups in 6hhl week and there was significant (P<0.05) differences among groups with treatment of glucose than Vitamin C and Vitamin E treated. groups. At 36th day of age, the HI titer was recorded significantly (P<0.05) lower in group B (GMT 15) than C (GMT 74) and group D (GMT 07) showed negligible HI titer while group E (GMT 140) showed significantly (P<0.05) higher HI titer. At the age day 56 (06 days post challenge) the HI antibody titers in all groups registered a rise except in group D. All the chicks in group D died indicating clinical signs of Newcastle disease. The post challenge GM HI titers recorded in groups A, B, C and Eat the age of 56 days were 68, 54, 115 and 165. There was non-significant (P0.05) difference in wattle thickness (cm) among groups while group E (I .78±0.06) showed significantly (P<0.05) increased wattle thickness. The mortality was Found significantly (P<0.05) higher in Groups C (12) and D (40) on challenge with NDV virulent virus. This indicated that 24 hrs off feed decreased the immune response of the birds which leads to the infection.
In experiment 4, studied the effect of water restriction at different time intervals on broiler chicks, it was observed that water restricted birds had nonsignificant (P0.05) differences in blood cells population except lymphocytes percentage was found higher in groups A (43.7±2.47), C (43.7±1.16) and D (53 .3±1 .30) than group B (39.1±1.06). The effect of water restriction on the mean thymus weight (gm) of chicks in group C (0.60±0.07) was adversely effected as the chicks in this group had significantly (PO.O5) lower weight than group D (4.09±0.70) indicating that increased in the period of water restriction in chicks adversely affected the mean thymus weight and chicks reared on ad-flbituni water had higher mean thymus weight. The mean bursa weight (gui) of group C (0.07±0.02) was significantly (P<0.05) lower as compared to group D (1.37±0.88) indicating that water restriction of 24 hr had affected the bursa development in the chicks. The mean spleen weight (gm) of group C (2.64±1.49) was significantly (P0.05) higher than groups A, B and E. The FCR values were significantly (P<0.05) different among groups in 6th week and there was non-significant (P0.05) difference among groups with treatment of Vitamin C, Vitamin E and glucose treated groups. At 36th day of age the HI titer was recorded significantly (P<0.05) lower in group D (GMT 09) than A (GMT 54) and group E (GMT 109) showed significantly (P0.05) higher HI titer. At the age day 56 (06 days post challenge) the HI antibody titers in all groups registered a rise except in group D. All the chicks in group D died indicating clinical signs of Newcastle disease. The post challenge GM HI titers recorded in groups A, B, C and E at the age of 56 clay was 78, 63, 48 and 134. There was non-significant (P0.05) difference in wattle thickness (cm) among groups B and E and group A (1.28±0.08) showed significantly (P0.05) lower wattle thickness. The mortality was found significantly (P<0.05) higher in groups B (14) and C (21) on challenge with NDV virulent virus. This indicated that 18 and 24 hrs water restriction decreased the immune response of the birds.
In experiment 5, effect of light stress at various time intervals on broiler chicks was studied. It was observed that light stressed birds had showed non-significant (P>0.05) difference on blood cells population except lymphocytes percentage was found higher in groups D (6 1.4±1.16) than group C. The effect on lymphoid organs studied and the mean thymus weight (gin) old chicks in group B (I .90±0.53) was adversely effected as the chicks in this group had significantly (P0.05) lower mean thymus weight than groups D (4.64±0.74) and E (4.34±0.25) indicating that increase in the period of oil-light in chicks adversely effected the mean thymus weight and chicks reared on 24hr light had higher mean thymus weight. The bursa mean weight (gm) 01' group 13(0.43±0.05) was significantly (P0.05) lower as compared to group D (1.59±0.17). The spleen mean weight (gun) of group D (1.88±0.15) was significantly (P<O.05) higher than group B (1.18±0.08). The FCR values were significantly (P().O5) different among groups in 6th week and there was non significant (P0.O5) difference among groups with treatment of Vitamin C, Vitamin E and glucose. At 36° day of age the HI titer was recorded significantly (P<0.05 lower in group C (GMJ 83) and group D (GMT 06) showed negligible HI titer while group E (GMT 128) showed significantly (P0.05) higher HI titer. At the age day 56 (06 days post challenge) in HI antibody titers in all groups registered a rise except in group D. All the chicks in group D died indicating clinical signs of Newcastle disease. The post challenge GM HI titers recorded in groups A, B, C and B at the age of 56 days was 122, 116. 108 and 133. There was non-significant (P>0.05) difference in wattle thickness (cm) among groups while group B (1.53±0.15) showed significantly (P<0.05) increased wattle thickness. The mortality was found significantly (PO.05) higher in groups A (18) and D (40) on challenge with NDV virulent virus. This indicated that 24 hr off-light decreased the immune response of the birds.
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5.
Identification And Genotyping Of Vp1 Genses Of Fmd Viruses
by Atia Bukhari | Prof. Dr. Irshad Hussain | Prof. Dr. Khushi Muhammad.
Material type: ; Format:
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Publisher: 2009Dissertation note: Within two decades after its first report in 1954 from Pakistan, Foot and mouth disease has become endemic in the country and poses a serious threat to large as well as small ruminant population. Foot and Mouth Disease (FMD) is prevailing in cattle and buffaloes and is caused by either 0, A, Asia-i serotype of the FMD virus in Pakistan. The present study was undertaken to study the mutation rate of FMD virus and also molecular typing of the strains prevalent in Pakistan was done.
A total of 60 samples from buffalo and cattle were collected from five districts of Punjab including Lahore, Faisalabad, Sialkot, Okara and Sheikhupura. Soon after extraction of their RNA, all of them were reverse transcribed and then subjected to amplification by using different sets of the primers including universal as well as serotype specific primers. Then their VPI portions were amplified by using VP1 specific primers. Among 60 samples, 48 were positive with universal primers. Other 12 samples were not amplified with these primers hence not processed.
Among 48 FMD positive samples, 24 were positive with serotype 0 specific primers, 16 with serotype Asia-i and remaining 8 were positive with serotype A specific primers. After their amplification, the amplicons were run on the gel. These amplicons were extracted by using DNA extraction kit. After their purification, they were sent to Macrogen® (Seopl, Korea) and Centre of Excellence for Molecplar Biology, Pakistan (CEMB) for sequencing. Each amplicon was sequenced thrice and the consensus sequence was established eliminating sequencing errors.
Sequence identity and multiple sequence alignment of molecular sequences (nucleotide and amino acids) were performed with Clustal W algorithm (Thompson et al., 1994). Neighbour joining trees were constructed by using MEGA version 4.0 (Kumar et al., 2004). Nucleotide distance matrices were computed by Kimura two parameter algorithm based on the total nucleotide substitutions and evolutionary trees for VP1 genes were constructed.
For FMDV serotype '0' phylogenetic analysis, 14 VPI sequences from various field isolates were compared with some previously published Pakistani FMD 0 type VP1 specific sequences available with GeneBank and some recently published VP1 sequences reported by countries bordering with Pakistan including India, Iran and Afghanistan Similarly, 12 VP 1 sequences of FMDV serotype Asia-I isolates of this study were compared with previously published sequences and their phylogenetic relationship was established. However, the sequencing results of serotype A were inconclusive and were not included for phylogenetic analysis. Three sequences of three locally available FMD vaccines were also studied and compared with the outbreak strains.
Polymerase chain reaction was optimized with respect to MgCI2, buffer pH, annealing temperature, primer concentration, template concentration, and Taq polymerase. A concentration of 2.5 mM of MgCl2 resulted in the best amplification of the target sequences (Figure 1). The buffer with pH 8.8 yielded the best results (Figure 2) Although, the suggested annealing temperatures for various primers (of various serotypes) ranged from 48 °C to 63 °C, however, a temperature of 56 °C was found to be the best with all sets of primers (Figure 3). The best intensity DNA bands were observed with 0.3 pM concentration of the primers (Figure 4). Moreover, the best cDNA template concentration giving optimum amplification was found to be 3.0 p1 per reaction (Figure 5). Lastly, a concentration of 0.5 U of Taq polymerase was not sufficient for amplification of cDNAs, however, 1.0 U of enzyme was found to yield better amplification (Figure 6).
VP 1 DNA sequences of six previously published Pakistani FMD serotype 0 strains were analyzed phylogenetically with VP 1 DNA sequences of 14 isolates of the study. Serotype 0 isolates of this study distributed themselves into two distinct clusters (Figure 19). First cluster comprised of Sheikhupura 1 and 2, Muridkey 1, Raiwind 1, Nankana 1, Gujranwala 1 and Gujrat I isolates (Figures 19 and 20), whereas the second cluster included Depalpur 1, Sahiwal 1, Okara I, Multan 1, Toba 1, Faisalabad I and Pattoki 1 isolates (Figures 19 and 21). The first cluster was found to be associated with previously published Pakistani isolates of 2006 mostly. However, it also showed association with Afghanistan's isolates of 2004 (Figure 20). The second cluster seemed to be mostly related to previously published Pakistani isolates of 2003 (Figure 21). The overall grouping of the 14 sequences, when compared with each other, depicted a three clustered phylogram (Figure 22). Serotype 0 isolates from Depalpur, Sahiwal, Okara, Multan, Pattoki, Toba Tek Singh and Faisalabad grouped together into a clan and had more than 85% sequence similarity with each other. The second cluster consisted of isolates of Sheikhupura, Nankana, Raiwind and Muridkey. These sequences had more than 86% similarity with each other. The third cluster consisted of only two isolates which were 100 % similar to each other. However the third cluster had only 74 % sequence similarity to cluster I and 73 % sequence similarity when compared with cluster 2.
When the phylogenetic relationships with previously reported isolates of Asia 1 was evaluated, FMD Asia I isolates of this study were found to be scattered into two distinct groups (Figure 16). Group one consisted of isolates of Lodhran, Toba and Hafizabad that were more closely related to Indian isolates sharing more than 98% identity with each other and more than 94 % sequence identity with isolates of Indian 2001 to 2004 (Table 5 and Figures 16 and 17). However, they shared more than 86% sequence similarity with Pakistani isolates of 2002-2005 (Table 5). Group two comprised of isolates of kasur, Lahore, Pakpattan, Okara, Faisalabad, Jhang, Rahim Yar Khan, Bahawalpur and multan alongwith vaccine A and B (Figure 16). The isolates of group 2 were found to be closely associated with previously published isolates of Pakistani and Afghani origin of year 2003 and 2004 (Figures 16 and 18). Collectively, they shared an overall 70% sequence identity with each other. However, isolates of Bahawalpur, Rahim Yar Khan and Multan shared more than 98% similarity with each other, a measurement of close relationship denoting a likely common origin as one clan or dade. Similarly, isolates of Pakpatan, Faisalabad, Okara, Kasur, and Lahore shared 88% sequence identity with each other and qualified as one clade.
Although, overall amino acid sequence similarity of our isolates was not strikingly different from that of the published isolates, however, amino acid substitutions with dissimilar properties were found with a scattered pattern of distribution. For example, 15th amino acid residue which is hydrophilic in the previously published isolates had a substitution with a hydrophobic amino acid residue in our three isolates namely Sheikhupura 2, Muridkey I and Raiwind I (Figure 25). Similarly, 14th amino acid residue which is hydrophobic in nature was found to be replaced with a hydrophilic one in our last five isolates. Amino acid residue number 13 (Figure 25) had a substitution with a hydrophobic residue in some of our isolates etc. etc. It is interesting to note that such substitutions with amino acids having dissimilar properties have also been found, albeit at lower rate, in previously published sequences by many researchers (Figure 25).
A comparison of the deduced amino acid sequences in the critical VP I region of FMD serotype Asia I revealed that most of this study isolates shared very high homology with sequences of Vaccine A. However, the sequences of isolates of Lodhran, Hafizabad and Toba did not match much with that of either vaccines, A or B (Figure 23). Sequences of Vaccine A had a "K" which seemed to be replaced by a "T" in the sequences of most of the isolates. Considering the properties of various amino acids, this change does not signify a major shift in the three dimensional picture of the protein as K is a lysine, a positively charged amino acid, whereas a T is threonine, a hydrophilic amino acid in nature. Next substitution in most of the isolates is a "P" for "A" in comparison to the vaccines. Again, it is not a significant change as both P and A share the same property, hydorphobicity. Similarly a K with an R can be substituted without much change in the overall shape of the protein molecule. Next amino acid substitution is a leucine instead of methionine. Again both are hydrophobic in nature; hence their impact on the overall picture is minute, if at all. However, glycine and arginine are two very different amino acids; the former is a hydrophobic amino acid whereas the latter is positively charged one. Such amino acid substitutions may have the potential to make a major impact in terms of the epitopic differences in the capsids of vaccinal and field viruses. A comparison of the deduced amino acids of FMD serotype 0 isolates also exhibited such changes with the vaccinal virus (Figure 24).
Of the three hyper immune sera raised against three different vaccines in rabbits, only one vaccine induced a measureable immune response yielding good precipitation line against various FMD virus antigens.
In summary, RT-PCR for diagnosis of serotypes A, 0 and Asia 1 of FMDV was optimized and could be used for prompt and precise diagnosis of FMD in the country. Although, RT-PCR data pertains to bovines in the current project, but PCR optimization parameters are equally applicable to FMDV infections in other FMD susceptible animal species such as sheep and goat. The combination of PCR and sequencing of the VP1 gene to detect and analyze FMDV in disease outbreaks is fast (less than 6 hours for PCR and about 24 hours for sequencing), and it can give an accurate immunologic characterization of the virus, thus providing a rational basis for choice of vaccine. In fact, the molecular epidemiology of field isolates is a powerful tool to monitor the circulation of viruses (Saiz et al., 1993).
Secondly, various isolates of serotypes 0 and Asia 1 were sequenced along with some vaccinal strains. Sequence similarity tree analysis indicated that most of our isolates were closely related to previously reported Pakistani isolates and to those of neighboring countries such as India, Afghanistan and Iran. Additionally, amino acid sequence similarity data of major immunogenic site that forms 13G-13H loop in FMDV serotypes revealed that serotype Asia 1 vaccinal strain and Asia 1 isolates of this study possessed high degree of similarity suggesting a likely host immune response against the vaccine that may afford some protection against most field isolates of serotype Asia 1 type. Lastly, of three vaccines tested, only one was found to afford protection against field isolates of FMDV suggesting more work on vaccine issue in the country.
Availability: Items available for loan: UVAS Library [Call number: 1179,T] (1).
6.
Isolation And Molecular Characterization Of Antimicrobial Resistant E-Coli Isolation From Retail Meats
by Ali Ahmed | Prof.Dr.Khushi Muhammad | Dr.Mueen Aslam | Prof.Dr.Masood.
Material type: ; Format:
print
; Nature of contents: ; Literary form: Publisher: 2010Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1252,T] (1).
7.
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).
8.
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.
Availability: Items available for loan: UVAS Library [Call number: 1536,T] (1).
9.
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).
10.
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).
11.
Characterization Of Mycoplasma Gallisepticum Isolates And Their Use In The Production Of Indigenous
by Mushtaq Ahmad | Prof. Dr. Masood Rabbani | Prof. Dr | Prof. Dr. Tahir Yaqub.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2012Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1564,T] (1).
12.
Biomass Production Of Pasteurella Multocida By Using Biofermentor For Preparation Of Montanoid Based Vaccine
by Noreen Sarwar | Prof. Dr. Khushi Muhammad | Dr. Atif Hanif | Prof. Dr. Muhammad.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2012Dissertation note: Hemorrhagic septicemia is a contagious bacterial disease of large ruminants principally in cattle and buffalo with high morbidity and mortality. The disease is endemic in nature and outbreaks are common during hot, humid and wet season. The acute and fatal nature and brief duration of the disease limit the antimicrobial therapy. In Pakistan, the disease causes heavy economic losses to dairy industry. Vaccination therefore, is an option for controlling the disease. For a quality vaccine, biomass production of P. multocida along with well developed capsule (immunogen) is necessary. The problem associated with the production of a quality vaccine is poor biomass production of P. multocida when grown in ordinary or routine media.
Present study was designed to isolate P. multocida from sick animals and its molecular characterization in the laboratory and study factors (temperature, media composition, pH incubation time and agitation or shaking) affecting its immunogen production and "in process quality control" factors (biological titer, dry mass, adjuvant and storage time) that affect antibody response. Finally, biomass production of the organism using biofermentor and monitoring of the antibody response of buffaloes to inactivated Montanide ISA-70 based P. multocida vaccine.
Each of the field isolates showed grey, viscous, mucoid, translucent and non hemolytic colonies on blood agar. There was no growth on MacConkey's agar. It was Gram negative coccobacilli or thin rods and bipolar when stained with Leishman's stain. The isolates were positive for Catalase, Oxidase, Hydrogen sulphide and Indole production along with nitrate reduction while it was negative for urease production, citrate utilization and gelatin liquefaction. The bacteria fermented glucose, sucrose, mannitol, mannose, but failed to ferment arabinose, maltose, salicin, lactose, dulcito and inositol.
Polymerase chain reaction (PCR) was performed on isolated colonies by using P. multocida specific and HS causing serotype B specific primers. P. multocida specific PCR gave product of 465 bp while HS causing serotype B specific primers amplified a product of approximately 590 bp.
Growth of the bacteria in casein yeast sucrose broth was optimized under different conditions. CSY broth showed dense growth of P. multocida during incubation for 18 hours. A temperature in between 35°C and 40°C showed its optimum growth. Poor growth was observed below 30°C and no growth was detected at 50°C and above. No growth occurred at pH 0.5 and 10.0 but best growth was obtained at pH 7.0 and 8.0. There was positive correlation between shaking in terms of rpm and growth. There was optimum growth at 500 rpm for 24 hours.
Inactivated HS Vaccine was prepared from dense growth in biofermentor on the basis of dry mass and bacterial count. The effect of biomass, adjuvant, storage of the vaccine, priming alone or with boosting on its potency was also studied along with boosting effect of montanoid ISA 70 oil based vaccine. Dry mass 1.7 mg/dose produced protective antibody titer while bacterial count 10-14/ml was sufficient to produce the protective antibody titer. Montanoid ISA 70 based vaccine provided immunity to buffalo calves better than aluminium hydroxide gel and bacterins. Boosting with oil based vaccine can help to keep the animal immunized for whole year. For better results of vaccine, it can be stored at 4oC for six months.
It is concluded that the proposed study improved quality of the vaccine and reduced volume of the vaccine dose, cost of its production and frequency of vaccination.
Availability: Items available for loan: UVAS Library [Call number: 1581,T] (1).
13.
Production And Evaluation Of Peste Des Petits Ruminants Virus Vaccine
by Muhammad Aness | Prof. Dr. Khushi Muhammad | Prof. Dr. Masood Rabbani.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2013Dissertation note: PPR is an acute highly contagious viral disease of small ruminants which is endemic in Pakistan. Present study was aimed to evaluate the freeze dried PPRV vaccine with variable biological titer to induce protective immune response in beetal goats. The comparative immune response of animals to adjuvant and non-adjuvant vaccine and duration of immunity was also studied. Effect of boosting on the humoral immune response of animals as well as shelf life of various vaccines was also evaluated. Each of the vaccines was inoculated in a group of five animals. Serum samples were collected at specified time intervals and antibody levels were detected through cELISA as PI values and neutralization test as MNA titer.
The virus was propagated on the Vero cells. It was estimated that infecting 2 x 107cells with 104.00 TCID50 virus concentrations added to a T-175 cell culture flask at the time of subculture yielded maximum virus titer in the cell culture harvest following three freeze thaw cycles of the contents. The freeze dried vaccine with a biological titer of 105.00 TCID50 per dose provoked maximum antibody titer followed by the ones with a titer of 104.00 or 103.00TCID50 which provoked nearly equivalent protective immune response while the animals inoculated with a vaccine having 102.00 TCID50virus concentrations developed minimum antibody titer. The oil adjuvant PPRV vaccines elicited significantly higher antibody titer in comparison to gel based vaccines but however minimum antibody titers were detectable in response to freeze dried vaccines. Although protective antibody level (? 10 neutralizing antibody units) was detectable in the animals vaccinated with either oil based, gel based or freeze dried vaccine containing biological titer of 104.00 TCID50 but however the extent and duration of immunity was found to be most superior in response to oil based vaccines. It can be concluded that a single shot of either gel or oil based vaccine can provide protection in the vaccinated animals for a minimum of one year duration. Goats receiving a booster dose of the vaccines had a significantly higher antibody tier in comparison to the ones who received single dose of the vaccines. The freeze dried and wet vaccine kept at 4 °C did not show any significant drop in the biological activity of the virus even after 12 months of storage. Immunogenicity of the both adjuvant and non-adjuvant vaccines, as measured through the immune response in the vaccinated animals, also remained unaffected after 12 months of storage at 4 °C.
Availability: Items available for loan: UVAS Library [Call number: 1731,T] (1).
14.
Isolation And Molecular Identification Of H9N2 Avian Influena Virus From Human In Punjab Province Pakistan
by Abdul ahad | Prof .Dr, Masood rabbani | Prof. Dr. Rana | Prof. Dr. Tahir yaqub.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2012Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1926,T] (1).
