1.
Immuno-Modulatory Effects Of Beta-Glucans And Nucleotides Based Commercial Products In Broiler Chicken Experimentally Infected With Velogenic Newcastle Disease Virus
by Hafiza Zain ul Fatima (2008-VA-232) | Prof. Dr. Asim Aslam | Dr. Raheela Akhter | Dr. Aamir Ghafoor.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Immune-potentiation effect of commercially available products containing beta-glucans (betaplex and catalyzer) and nucleotides (electroimmune and avimmune) in the broilers was evaluated. A total of 120 broiler chicks were reared under standard husbandry conditions. The birds were vaccinated by live NDV vaccine at 5, 14 days and killed Newcastle disease virus (NDV) vaccine at 7 days of age. Additionally, live infectious bronchitis and infectious bursal disease vaccines were also given through water at 5 and 10 days of age, respectively. The birds were randomly divided into six groups each comprising 20 birds. Four groups (treatment groups) were offered four commercial immune-booster products at 7, 14 and 21 days of age for 72 hours. Fifth group (untreated vaccinated) was not offered any drug while sixth group (untreated and unvaccinated) was not given any drug and also remained unvaccinated against NDV. Anti NDV antibodies were measured at 7, 14, 21 and 28 days of age and heterophil/lymphocyte (H/L) ratio was measured at 14, 21 and 28 days. Weight of immune organs and performance of birds was also measured from each group. Finally, 15 birds from each group were inoculated with virulent NDV through intranasal route at 28 days of age to assess the protection against challenge infection.
Mean GMT anti NDV antibody titers in vaccinated groups were 3.65 (±0.59) at one week of age which were raised to the levels of 5.85 (±0.88), 5.50 (±1.00), 6.10 (±0.64), 5.90 (±0.91) and 5.45 (±1.05) in the betaplex, catalyzer, electroimmune, avimmune, and untreated control groups, respectively. Maximum antibody titer was achieved in electroimmune treated group while minimum in the catalyser treated ones. In non-vaccinated control group the antibody titers were minimum in the beginning that reduced to negligible level at four weeks of age. The H:L ratio of
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all treatment groups did not significantly vary at 14 days of age while at 21 and 28 days of age, the birds receiving electroimmune have significantly low H:L ratio in comparison to other treatment groups. Weight of liver and spleen in nucleotide treated groups was significantly higher than other treatment and control groups while there was no significant difference in the weight of bursa of fabricious in all treatment groups. Relative weight of all immune organs to the live body weight did not significantly vary in all treatment and control groups. Performance of the birds as a measure of feed conversion ratio (FCR) was highest in electroimmune treated birds. The challenge protection data shows that minimum mortality of 40 % (6/15) was observed in nucleotides treated groups while betaplex and catalyzer treatment groups have 47 % (7/15) and 53 % (8/15) mortality ratios in comparison to 60 % (9/15) mortality in untreated birds. Results of present study suggest that nucleotide or beta glucans supplementation in the diet have beneficial effects to enhance the humoral immune response and better bird performance in broilers. Use of these products not on delay the onset of clinical disease but also helpful in providing better protection against challenge to velogenic NDV. Availability: Items available for loan: UVAS Library [Call number: 2516-T] (1).
2.
Amelioration Of Pathological Changes Due To Infectious Bursal Disease By The Administration Of Mentofin And Asi-Mirus In Broiler Chicken
by Muhammad Umair Shah (2011-VA-15) | Prof. Dr. Asim Aslam | Dr. Ghulam Mustafa | Dr. Ali Ahmed Sheikh.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Poultry industry is the second largest industry in Pakistan but despite of its rapid growth rate it is facing huge economic losses due to many infectious diseases. Infectious Bursal Disease is one of them. Huge economic losses in case of infectious bursal disease are due to immunosuppression and high mortality.
In Pakistan, commercially available vaccines are abruptly used to control different viral diseases but unfortunately failure of these products occur from time to time. Hence, current study was designed to determine the immunostimulatory effect of two commercially available products (Mentofin and ASI-MIRUS) against IBD vaccine.
A total 300 broiler chicks were taken, divided into six groups each having 50 birds and were replicated under controlled conditions. A, B and D groups were vaccinated with the IBD live virus vaccine. B and C groups were treated with Mentofin. D and E groups were treated with ASI-MIRUS while F group served negative control. To detect antibody titer against IBDV at every week (0-42 days of age), a commercial ELISA kit, IDEXX Flock Chek standard (IDEXX Corporation, Westbrook, ME, USA) was used. In order to analyze gross and microscopic changes in bursa, postmortem examination and histopathology of bursa was done.
The volatile oils in Mentofin and ASI-MIRUS have effective immunomodulatory effects on humoral immune response in broiler chicks. Eucalyptus and peppermint oils increase bursa to body weight (B/BW) ratio as compared to untreated birds. Results of present study indicated the highest antibody titer in group D supplemented with ASI-MIRUS and vaccinated as compared to group B supplemented with Mentofin and vaccinated. Significantly high bursa to body weight ratio also observed in vaccinated group D (ASI-MIRUS treated) comparing with other
CHAPTER 6
SUMMARY
Summary
35
vaccinated groups A and B. In Group B (Mentofin treated), bursal samples showed necrosis at medullary region of bursal follicle. Group D (ASI-MIRUS treated) showed the active follicle consist of lymphoid cells and shown no obvious histopathological lesion. So present study showed that ASI-MIRUS is reduced the severity of IBDV which has more beneficial effect on immune response against IBD vaccinated Broiler Chicken as compared to Mentofin. Availability: Items available for loan: UVAS Library [Call number: 2557-T] (1).
3.
Isolation And Molecular Characterization Of Rotavirus From Calf Diarrhea And Preparation Of Vaccine
by Nadia Mukhtar (2008-VA-718) | Prof. Dr. Tahir Yaqub | Dr. Jawad Nazir | Prof. Dr. Asim Aslam.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: The main contribution of the thesis “Title” is threefold. First, rotavirus was isolated and identified from calf diarrhea samples from 10 districts in Punjab. Second, optimization of molecular diagnostics and genome sequencing was done of the positive bovine rotavirus isolates from Pakistan. And thirdly, the preparation as well as evaluation of killed vaccine against bovine rotavirus isolates was performed.
The above three objectives of this study were created due to the distribution of rotavirus all over the world as an enteric pathogen in both human as well as animal species. In developing countries where cases of malnutrition are very common in young children and animals, this virus has a special importance as an etiologic agent. It causes severe diarrhea, when accompanied with severe dehydration, leads to high rate of mortality. Among the rest of the infectious diseases present in calves, neonatal diarrhea is a dire threat as it has a major impact on economic viability. Calf diarrhea is the most important problem in dairy calves that causes more financial losses to the calf producers than any other. Although numerous etiological agents may be implicated, Rotaviral diarrhea is one of the main infections causing calves to scour between five to fourteen days of age.
The cattle and buffalo calves’ population in Pakistan is devastatingly affected by the neonatal calf diarrhea due to rotavirus outbreaks. Neonatal calf mortality varies from 8.7 to 64 per cent throughout the world accounting for 84 per cent of the total mortality in the first month of age and is particularly high in the third week. While vaccination is available for the disease, it is being imported in Pakistan from other countries. The importation of the said vaccine thus, leads
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to extra expenses for the farm managers. As mentioned above one of the aims of this study is to develop an effective vaccine against bovine rotavirus and cut down expenses for farm managers.
To fulfill the objectives proposed in this thesis, rectal swabs and fecal samples were collected from public/private sector buffalo and cattle farms from 10 districts of the Punjab: Lahore, Faisalabad, Okara, Sahiwal, Sargodha, Chakwal, Bhakkar, Bahawalnagar, Multan and Bahawalpur. The samples were selected on the basis of agro-ecological zones of the province. As sampling based on agro-ecological zones allow for better data collection for recording incidence rate of the disease. Samples (n=10) from each diarrheic and apparently healthy cattle and buffalo calves from all of the districts were collected. In this way a total of 200 samples from buffalo calves and 200 samples from cattle calves were collected for this study.
Antigen of bovine rotavirus was screened from calf feces through Direct Sandwich ELISA. Bovine rotavirus samples were further confirmed through the amplification of the VP4 and VP6 genes through Rt-PCR. Homology and phylogenetic analysis of the sequenced samples was also performed. The data gathered through this analysis was helpful in collecting important data regarding the similarities as well as differences of the bovine rotavirus strain present in Pakistani isolates when compared to local regions as well as international ones. The data is also valuable when it comes to production of effective vaccines again rotavirus. RNA viruses are known to mutate unpredictably and it is safe to assume that a particular vaccine might not work effectively against all strains of a particular virus. That’s why analysis of data pertaining to all possible BRV strains is important for creation of an effective vaccine of import quality in order to help the economy of Pakistan.
Rotavirus isolate, after adaptation on MDBK cell line, was further propagated to determine TCID50 for vaccine preparation purposes. Final dose of the vaccine was adjusted to
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approximately 3ml, containing 40% culture and 60% adjuvant. Final vaccine contained 1ml of inactivated bovine rotavirus harvested culture, 1.8ml of Montanide ISA 70, 0.2ml of PBS and 0.05% of Thiomersal sodium. Efficacy of the vaccine was checked in rabbits.
For vaccine efficacy testing twenty one month old rabbits were procured. Rabbits were reared in individual isolator units in the shed facility of Quality Operations Laboratory, UVAS, Lahore. The collected rabbits were divided into two groups, vaccinated and unvaccinated rabbit groups. Each group had 10 rabbits. One ml of rotavirus vaccine was administered intramuscularly in vaccinated rabbits group. In unvaccinated rabbits group 1ml of normal saline was injected intramuscularly. The second dose of vaccine was administered at 24 days post-vaccination of first dose. The rabbits from both groups were bled at 0, 14, 28 and 42 days post-vaccination. The antibody response of rabbits to rotavirus vaccine was determined through using Antibody detection kit. The rabbits were challenged on day 42 post-vaccination using live field strain of rotavirus having TCID50 1 × 108.5. The rabbits were observed daily up to 14 days post-vaccination for appearance of diarrheic signs. The stool samples of ELISA positive were further confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR) at least 14 days post-vaccination.
The field trials were conducted at Livestock Production Research Institute, (LPRI) Bahadurnagar, Okara. The field study was done to evaluate the prepared rotavirus vaccine for prevention of neonatal calf diarrhea. For this trial, 100 dams were selected. The dams were divided into two groups and each group consisted of 25 pregnant cows and 25 pregnant buffalos. A total of 50 dams (25 cattle and 25 buffalo) were vaccinated intramuscularly with 3ml of prepared inactivated rotavirus vaccine. The 50 remaining dams (25 cattle and 25 buffalo) were kept unvaccinated.
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The blood samples were collected for serum separation after 0, 14, 28 and 42 days post vaccination in dams. The antibody titers were measured using antibody detection ELSIA kit.
After calving, newborn calves were fed with the colostrum obtained from the vaccinated dams daily for 5 consecutive days. Similarly, the calves from unvaccinated dams were fed on colostrum from their unvaccinated dams.
The 5 calves from vaccinated and 5 from the unvaccinated dams were isolated in individual isolators. These calves were challenged orally with 1ml of live field strain of rotavirus having 1 × 108.5 TCID50 and the animals were observed for diarrheic signs for 7 days.
All of the collected data was subjected to statistical analysis of (one way) ANOVA and t-test using SPSS. The <0.05 p-value determined the significance of the results through this study.
The data collected through this study allowed for the creation of valuable inferences. According to the current results of this study, the prevalence of bovine rotavirus was shown to be 6% in Punjab. This 6% included 40% and 20% from the districts of Lahore and Faisalabad respectively. Keeping these results in mind, it is to be noted that the recorded prevalence percentage from this study is higher than the prevalence of 2% in Lahore according to a previous study done in the country. It is to be noted that while the 6% prevalence of rotavirus in Punjab detected through ELISA is lower than the prevalence of 16.83% which was detected by ELISA in diarrheic calves from pervious researches, the 12% prevalence detected by ELISA in this research is higher than the prevalence of 7.25% detected by ELISA in diarrheic calves from past data.
In the present study of this thesis it was observed that the use of killed vaccine for bovines produced more efficient immune response in calves. It also enhanced the clostral rotavirus antibody titers as compared to previous studies where the use of the same strain of modified-live virus in a commercial vaccine administered IM with or without adjuvant did not significantly
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elevate colostrum antibody titers. The results collected from the present research showed that the average antibody titers in the 25 cattle dams at 0, 14, 28 and 42 days post vaccination were 0%, 57%, 68% and 78% respectively. In a similar manner the average antibody titers in the 25 buffalo dams at 0, 14, 28 and 42 days post vaccination were 0%, 55%, 70% and 82% respectively. These results indicated
the protective maternal antibody level against the rotavirus which will be transferred passively to calves. The results indicate that vaccinated dams were able to provide passive immunity to both buffalo and cattle calves in order to provide protection against the deadly virus. Availability: Items available for loan: UVAS Library [Call number: 2570-T] (1).
4.
Pathogenesis Of Aflatoxin B1 In Quails Under Experimental Conditions And Detoxification By Biological And Chemical Means
by Sakhra Mahmood (2005-VA-251) | Prof. Dr. Muhammad Younus Rana | Prof. Dr. Asim Aslam | Prof. Dr. Aftab Ahmed Anjum.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Secondary metabolites of certain fungi produce toxins under favorable conditions especially while growing on different food grains. Mycotoxins are among major threats to growing poultry industry and human beings. Aflatoxins are closely related, biologically active fungal metabolites and commonly produced by Aspergillus species.
A research was carried out to evaluate the ability of Aspergillus flavus for Aflatoxin B1 production using rice, wheat and maize as substrates. Lethal effects on growth performance parameters, hematological and histopathological of graded doses of aflatoxin B1 in quails under experimental conditions were observed. Effect of Aflatoxin B1 on humoral immune response to Newcastle Disease virus vaccine in quails were determined. Biological detoxification of Aflatoxin B1 by Saccharomyces servisiae was evaluated in quails. Comparative evaluations of different commercially available toxin binders were checked. All these experiments were carried out till the six weeks (42 days).
Aspergillus flavus was identified on the basis of macroscopic and microscopic characteristics. Rice, wheat and maize grains was used as substrate to check the level of Aflatoxin B1 produced by inoculating an aqueous suspension of 106 spores/ml. Aflatoxin B1 checked by Thin Layer Chromatography (TLC) and quantified by High Performance Liquid Chromatography (HPLC).
Quails were reared under standard management conditions in five groups (A, B, C, D and E) having sixty each. Each group was further divided in two independent units. Diets offered to groups were control (without toxins), 0.25, 0.50, 1 and 2 mg Aflatoxin B1/kg feed. One unit of
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each group was vaccinated with Newcastle Disease Virus (NDV) vaccine while other was not and studied the lethal effects on growth performance, blood parameters, immune response and histopathology of vital organs. At the end of the experiment, it was found that the deleterious effects of Aflatoxin B1 were dose and duration dependent. As the level of the toxin was increased, the lethal effects were prominent. The growth performance parameters including gain in body weight, feed intake and feed conversion ratio was adversely affected at high doses. The body weight gain was significantly reduced in Aflatoxin B1 treated groups as compared to control group. Similarly feed intake and feed conversion ratio were significantly different from the control group. The hematological studies exhibited that aflatoxin B1 significantly reduced the hemoglobin, packed cell volume and total leukocyte count whereas the erythrocyte sedimentation rate was significantly increased as compared to control group. The immune response against NDV vaccine was adversely effected in Aflatoxin B1 treated groups and values of Antibody titer in AFB1 were significantly low as compared to group A( control) In the second experiment, Saccharomyces cervisae (SC) dried powder was mixed in basal quail diet having 0.5mg Aflatoxin B1 for all experimental groups and control was without toxins. SC was added at levels of 0.5 gm, 1.0 gm and 2.0 gm /kg of feed. It was recorded that Saccharomyces cervisae (yeast) have the potential to remove the deleterious effects of Aflatoxin B1. Yeast effectively detoxified the Aflatoxin B1. The results recorded of growth performance and other parameters were non-significantly different from the control group. Chemical detoxification of Aflatoxin B1 was evaluated in quails using commercially available toxin binders. Toxin binders used were activated charcoal, kaoline, Myco AD and selenium plus vitamin E and mixed in basal quail diet having 0.5mg Aflatoxin B1 for all experimental groups and control was without toxins. The Myco AD and selenium plus vitamin E showed the highest detoxification potential as compared
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to other chemical toxin binders. Groups E and F showed the results of growth performance, hematological, immune response and histopathological were non-significantly different from the control group (A). Kaolin was moderately detoxifying the toxin.
Presence of aflatoxin B1 in soft tissues was checked by TLC and quantified using HPLC. The liver exhibited the residues of Aflatoxin B1 at high doses of toxin. Group D and E rearing on feeds having 1mg AFB1 /Kg feed and 2mg AFB1 /Kg feed of toxin showed the residues of AFB1 in liver and kidney.
Statistical means for growth performance parameters, hematological, immune response and histopathological scores in each subunit of quails were analyzed by applying one way ANOVA and Duncans‟s Multiple Range (DMR) test at 95% probability. Aflatoxin B1 is lethal and lowers the performance of birds. The lethal effects can be detoxified by biological and chemical means to lower the economic losses to poultry industry. It can be concluded that biological detoxification is preferably better as compared to chemical detoxification. Availability: Items available for loan: UVAS Library [Call number: 2670-T] (1).
5.
Pathobiological Investigations Of Peste Des Petits Ruminants (Ppr) Virus With Reference To Antiviral Activity Of Nigella Sativa (Black Seed)
by Kiran Aqil (2008-VA-456) | Dr. Muti Ur Rehman Khan | Prof. Dr. Asim Aslam | Dr. Aqeel Javeed.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Peste des Petits Ruminants (PPR) is a highly contagious, infectious, acute or sub-acute transboundary viral disease of domestic and wild small ruminants. It is an economically important viral disease of sheep and goats causing varying degree of morbidity and mortality in susceptible animals which may be as high as 100 and 90 per cent, respectively. PPR is responsible for serious socioeconomic problems. There is no data available regarding pathogenesis and field virus characterization to compare it with vaccinal strain for any difference. Nigella sativa(Black Seed) has antiviral activity against many viruses. Therefore present studywas undertaken to investigate the antiviral effect of Black Seed in vivo and in vitro against PPR virus. Further more time course detection of virus is still needed to be studied.
Nigella sativa (Black seed) has antiviral activity against PPR virus.
Pathogenesis can better be studied through histopathology, necropcy findings and morphometric changes.
A total of 250 clinically positive samples suspected for PPR virus were included in the study. Samples were consisted of nasal, ocular and anal swabs; whole blood in EDTA were collected from suspected animals. In case of mortality morbid material included lungs, liver, spleen and mysenteric lymph nodes were included in the study. Samples were subjected to immune capture Elisa for detection of viral antigen in suspected samples. Samples which found positive foe IC – Elisa were then subjected to RT-PCR for confirmation of virus. After confirmation of virus through IC – Elisa and RT-PCR the positive samples were subjected to virus isolation on vero cell. After isolation of virus, the TCID 50 of the virus was calculated for preparation of inoculum for further use. In this experiment mesenteric lymph nodes and spleen found to be major organ for isolation of PPRV.RT-PCR found to be most reliable and confirmatory diagnostic test for PPRV. Field Virus adaptation on vero cells found to be difficult to optimize.
In this experiment antiviral activity of black seed was checked on vero cells infected with PPRV. Three extracts of N. Sativa were prepared to check the in vitro antiviral activity of black seed. In this study poly saccharides extracted from black seed found to be more effective against PPRV. Adaptation of field virus was done on Vero cell line. Antiviral activity of Black Seed extract was determined in vitro on Vero cell on bases of CPE (Cytopathic effect). The ethanolic and aqueous extract were found to be more toxic to consistency of monolayer of vero cells. The TCID50 of virus was calculated after treating cells with different extracts. In this study poly saccharides extract exhibit lower TCID50‘s as compared to ethanolic and aqueous extract which showed higher TCID50’s.So less cytopethic effect was observed in vero cells treated with black seed extracts. Antiviral activity was determined on base of CPE.
Pathogenesis of virus in natural host was studied through time course detection of virus in body secretions, blood, organs. Histopathological changes were studied.20 goats were procured from market divided into four groups (n=5) A,B,C and D. In animals of group A prophylactic effect of N.Sativa was studied. In group B complete pathogenesis of PPR virus was studied without any prophylactic or therapeutic measure. In group C therapeutic effect of N. Sativa was studied after onset of clinical picture of disease. At the end of this experiment, clinical picture, gross pathology, histopathology, and morphometric changes revealed that N. Sativa has noticeable prophylactic effect on PPR infected goats. It can be used as a therapeutic agent in PPR infected goats but it can’t control pathological effect of virus after onset of infection.
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Data collected were statistically analyzed by using Microsoft Excel (Microsoft Excel, 2007) and SPSS (for Windows, Version 16.0). The data were put the descriptive analysis and Chi square test was employed to test the significance and test of hypotheses
It was concluded that Black Seed therapy possessed marvelous prophylective effect against PPR virus and RT-PCR was the most efficient methodology to confirm the virus. Availability: Items available for loan: UVAS Library [Call number: 2890-T] (1).
