Nadia Mukhtar (2008-VA-718)

Isolation And Molecular Characterization Of Rotavirus From Calf Diarrhea And Preparation Of Vaccine - 2016. - 138p.;

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
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
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.
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
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.

Phd. Thesis


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