1.
Effect Of Temperature And Relative Humidity On The Survival Of Newcastle Disease Virus Isolates Using Germ Carrier Techniques
by Tayyeba Sohail (2009-VA-209) | Dr. Jawad Nazir | Prof. Dr. Khushi Muhammad) | Prof. Dr. Asim Aslam.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Newcastle Disease (ND) is a highly contagious viral disease that affects almost all avian species including poultry, cage and wild birds around the globe (Terregino et al. 2003; Vidanovic et al. 2011). ND is economically important disease and included as list-A disease of Office des International Epizootics (OIE) (Anonymous). Mortality of infected birds ranges from negligible to as high as 100 % depending on the pathotype of the virus involved and health status of the birds (Alexander and Manvell 2004). NDV is an enveloped virus with single stranded, non-segmented, negative sense RNA genome (Makoui et al. 2013). The virus belongs to Avulavirus genus of Paramyxoviridae. There exist only one serotype of NDV designated as avian paramyxovirus-1 (Kapczynski et al. 2013) however, different virus strains do vary in their pathogenicity. There are 3 pathotypes of NDV; velogenic (highly virulent), mesogenic (moderate virulent), and lentogenic (mild virulent) based upon diseases producing potential and severity of signs in the infected birds (de Leeuw and Peeters 1999).
NDV is primarily transmitted to the susceptible birds through aerosol and fecal oral route (Martin 1992). Infected birds secrete high amount of the virus in their feces, saliva, mucous and nasal secretions which might contaminate the premises. Inanimate objects or fomites are a potential reservoir of viruses outside the host and might play an important role in the transmission of pathogens (Nicas and Sun 2006). Several factors can influence the survival of viruses outside the host (Sobsey and Meschke 2003; Weber and Stilianakis 2008; Stallknecht and Brown 2009). A number of studies show that respiratory pathogens can survive from hours to months on fomites (Abad et al. 2001; Kramer et al. 2006). Certain physical factors like temperature, humidity, pH, salinity, exposure to ultraviolet (UV) rays etc drastically affects the
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virus persistence in the environment. Effect of such physical insults is more pronounced on enveloped viruses than non-enveloped ones (Mbithi et al. 1991; Schaap et al. 2012; Tuladhar et al. 2012). High humidity and temperatures not only reduces the survival of influenza viruses on contaminated surfaces but also modulates their transmission to the susceptible birds (Shaman and Kohn 2009; McDevitt et al. 2010; Paynter 2014). Similarly lower temperature and less humidity promote the survival of NDV in the environment (Dat and Chuc 1985; Kournikakis et al. 1988).
ND is endemic in Pakistan but since last few years several new virus strains are circulating in commercial and rural poultry of the country (Munir et al. 2012; Shabbir et al. 2013). Central Punjab region is densely populated with commercial poultry and serve as disease epicenter every year. It has been observed that the disease outbreaks usually start in December, attain peak in the late winter and spring season, start decline in June and disappear in the rainy season. Apart from several other contributing factors, environmental survival of the viruses might contribute to the disease outbreaks. Availability: Items available for loan: UVAS Library [Call number: 2227-T] (1).
2.
In Process Quality Control Factors Affecting The Quality Of Locally Prepared Salmonella Gallinarum Antigen
by Zahra Malik (2009-VA-245) | Dr. Arfan Ahmad | Prof. Dr. Aftab Ahmad Anjum | Prof. Dr. Asim Aslam.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Fowl typhoid is a septicaemic disease caused by S. gallinarum biovar gallinarum has major economic significance in many parts of the world. It is an acute or chronic septicaemic disease that usually affects the birds (mostly adult birds). Eradication of disease is normally done by identifying the infected flocks and eliminating the reactor birds by using serological tests, but diagnosis of the disease is much expensive because antigen used for this purpose is imported. The study, therefore, has been proposed to prepare and evaluate the stained antigen of S. gallinarum using local isolates.
A total of 15 isolates were procured from Poultry Research Institute (PRI) Rawalpindi, University Diagnostic Lab (UDL) and Department of Microbiology, UVAS Lahore, which were identified by Biochemical testing and further confirmed by Polymerase Chain Reaction. Among all 15 isolates two isolates were confirmed as S. gallinarum and proceeded to prepare local antigen of S. gallinarum. Locally prepared antigen was checked with known positive and negative sera, Effect of different preservatives (Sodium azide and Thiomersal sodium) and different storage temperatures (4°C, 25°C and -20°C) was also studied after every fifteen days post storage upto 6 months to observe the stability and shelf life of local antigen. On the end of study both preservatives i.e. Sodium azide and Thiomersal sodium was found equally effective for antigen activity, whereas 4°C proved best storage temperature to be used for the antigen preservation.
Activity of locally prepared antigens was also compared with the imported antigen (Charles, River, USA) stored at different temperatures regularly throughout the six months, which showed that local antigens was almost as good as the imported antigen.
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CONCLUSION
Locally prepared S. gallinarum antigen was found as effective as imported antigen. Both the test preservatives (Sodium azide and Thiomersal Sodium) had the same effect on antigen preservation. Among all three test temperatures, 4°C was accepted as best storage temperature for the long term preservation of local antigen with either of the preservative. Availability: Items available for loan: UVAS Library [Call number: 2278-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).
