Molecular Characterisation, Pathogenicity and Immunological Studies of Chicken Anaemia Virus Isolated In Malaysia

A comprehensive study was carried out to isolate, identify and characterise chicken anaemia virus (CAV) isolated in Malaysia. The study resulted in the isolation of five CAV isolates from broiler chickens, designated as BL-1, BL-2, BL-3, BL-4 and BL-5. These isolates together with three isolates (SMSC-1, SMSC-2 and 3-1) provided by Veterinary Research Institute (VRI), Malaysia and a reference Cux-1 isolate were analysed by different restriction endonuclease enzymes. The whole genome of each CAV isolate was amplified by PCR into four fragments: Fragments A, B, C and D. Fragment A was digested with Styl, fragment B with StyI, Hpall and Mbol, fragment C with Haelll, and fragment 0 with EcoRI. The overall analysis revealed that the four isolates, BL-1, BL-2 , BL-4 and BL-5, exhibited the same restriction profiles in all enzymatic reactions and they are placed in one group, whereas, the other five isolates (SMSC-1, SMSC-2, 3-1, BL-3 and Cux-1) were found to be different from each other and also from the group of four isolates mentioned above. The pathogenicity studies in specific pathogen free (SPF) chickens inoculated with SMSC-1, 3-1 and BL-5 isolates at 1-day old showed that, the isolates produced clinical signs and characteristic lesions suggestive of CAV infection at 14-16 days post inoculation (p.L). The histopathological lesions in infected chicks showed severe depletion of lymphocytes from thymus, bursa and spleen and aplastic changes in bone marrow. The repeated passages of two VRI isolates, SMSC-1 and 3-1, in MSB1 cell line until passage sixty (P60), and passage 123 (P123), produced attenuated viruses (SMSC-1/P60, 3-1/P60. SMSC-1/P123 and 3-1/P123) which showed significantly reduced level of pathogenicity in SPF chickens compared to the pathogenic parent isolates. The whole genome of two non-attenuated isolates (SMSC-1 and 3-1) and two attenuated isolates (SMSC-1/P60 and 3-1/P60) were sequenced using the Perkin Elmer's BigDye Terminator Cycle Sequencing Kit. The high G:C regions of the CAV genome were sequenced using the same kit by the development of a modified method. The results showed that the complete genome of all isolates consisted of 2298 nucleotides. Three major ORFs of 1347 bp, 648 bp and 363 bp long were found in the plus DNA strand in all isolates, coding for putative proteins of about 52 kDa (VP1). 24 kDa (VP2) and 13 kDa (VP3). respectively. The alignment and antigenic index of VP1 sequence revealed the appearance of a hypervariable region from amino acid positions 139 to 157. The results showed that 76 nucleotide changes in SMSC-1/P60 and 43 nucleotide changes in 3-1/P60 isolates compared to their parent isolates, were thought to contribute to virus attenuation. Among these nucleotide changes, only one nucleotide difference (T-C) at position 816 resulted in changes of amino acid residues at positions 153 in VP2 from V to A, and 118 in VP3 from C to R. This single nucleotide change is probably important for the change in virus pathogenicity or attenuation. The phylogenetic analysis showed that the SMSC-1 isolate is close to the Australian 704 and Japanese TR20, the 3-1 isolate is close to the German Cux-1 isolate and the attenuated cloned isolate 10 (derived from the Cux-1). The attenuated SMSC-1/P60 and 3-1/P60 isolates were very close to the Japanese isolate A2. The apoptosis study carried out with electron microscopy and DNA fragmentation analysis, detected apoptosis both in infected thymocytes and infected MSB1 cells. The immunological studies with P1, P60 and P123 isolates of SMSC-1 and 3-1, and also with BL-5 isolate, after inoculation into 1-day-old SPF chickens showed that each of the isolates elicited CAY antibody responses, both at 14-16 days and 30 days p.i. Based on the findings of antibody response and pathogenicity studies, the attenuated isolates of P60 and P123 are potential candidates for live vaccines

Antibody responses in chickens experimentally infected with low and high passaged chicken anemia virus isolates

It has been shown that chicken anemia virus (CAV) which had undergone 60 and 123 passages in cell cultures (SMSC-1IP60, SMSC-I/P123, 3-1IP60 and 3-l/P123) were less pathogenic compared to low passaged CAY (SMSC- 1 and 3-1) isolates. In this study, the ability of the isolates to induce antibody responses was studied using enzymelinked immunosorbent assay (ELISA). All the isolates regardless of the number of cell culture passages that they had undergone elicited CAY antibody responses both at 16 and 30 days post inoculation. A CAY isolate, BL-5 that was not passaged in cell culture elicited higher antibody response than the cell culture passaged isolates. However, the differences in the average ELISA titres and the percentage of positive sera between the isolates were not statistically significant (P>0.05). The study showed that CAY isolates which had undergone repeated passages in cell culture are still immunogeni

Isolation and characterization of Staphylococcus aureus from raw cow milk in Bangladesh

The study was intended for identification and characterization of Staphylococcus aureus isolated from raw cow milk. A total of 47 milk samples were collected from Sheshmore, Shutiakhali and Bangladesh Agricultural University Dairy Farm, Mymensingh. Using bacteriological, biochemical and PCR-based identification schemes, 12 (25.53%) isolates were confirmed as S. aureus. All the isolates showed ?-hemolysis on 5% sheep blood agar. S. aureus specific nuc gene (target size 279-bp) was amplified in the cases of all isolates. The isolates were found as resistant to Penicillin (100%), Erythromycin (75%) and Amoxicillin (100%). On the other hand, the isolates were sensitive to Ciprofloxacin (83.33%), Oxacillin (100%), Cloxacillin (100%) and Neomycin (100%). The isolated S. aureus showed increased resistance to broad spectrum antibiotic (e.g., Ciprofloxacin). As many people have a tendency to drink raw milk and raw milk products, there is high risk of S. aureus infection in human

Factors associated with repeated outbreak of anthrax in Bangladesh: qualitative and quantitative study

Anthrax, caused by Bacillus anthracis is an acute, febrile disease of warm blooded animals including humans. Social norms and poverty in addition to climatic factors such as soil conditions, seasons of year, ambient temperature and rainfall influence the persistence of the B. anthracis and anthrax outbreaks. The present study was designed to reveal the factors influencing the repeated outbreak of anthrax in Bangladesh. Considering the previous outbreaks of anthrax, Sirajganj, Bogra, Kushtia, Tangail and Mymensingh districts of Bangladesh were selected for this study. To elucidate the factors, qualitative data relating to the animal management, knowledge and behavior of the people; and quantitative data relating to soil conditions, ambient temperature and rainfall were acquired, and analyzed critically. Based on the outbreak histories, a year was divided into two seasons, anthrax prone season (May-November) and anthrax dry season (December-April). Anthrax spores could be isolated from 11.67% (n=14/120) of the soil samples collected from the study areas. The present study revealed that poor knowledge, lack of awareness, improper carcass disposal, inadequate vaccination, high Ca content and moisture in the soil along with high ambient temperature and rainfall during the anthrax prone season were the possible influencing factors of repeated outbreaks of anthrax in the study areas. Intensive propaganda to create public awareness of anthrax together with proper vaccination may reduce anthrax outbreaks in Bangladesh

Molecular identification of Mycoplasma synoviae from seroprevalent commercial breeder farms at Chittagong district, Bangladesh

Aim: Worldwide, Mycoplasma synoviae (MS) is an important pathogen of poultry, especially for chicken and turkey. It causes respiratory tract infection and infectious sinusitis. The study was conducted to determine the seroprevalence of MS infection with associated risk factors and identification of MS organism in unvaccinated flocks of commercial breeder farms of the Chittagong district, Bangladesh. Materials and Methods: A total of 365 serum samples were collected and tested for MS using serum plate agglutination (SPA) test for determination of MS seroprevalence. On the other hand, tracheal swabs were collected from each seropositive flocks for polymerase chain reaction (PCR) to determine the presence of MS organism. Results: Among the farms, the highest prevalence was found to be 69% and the lowest prevalence was 28% with the average 60%. The seroprevalence of MS infection in breeder farms was highest 70% with the flock size >10,000 birds, whereas it was lowest 57% in the flocks ranging from 4000 to 7000. According to age group, the prevalence was found highest 70% in >60 weeks age group of birds and lowest 42% in 10-19 weeks group. The seroprevalence of MS in winter season was found as highest as 64%, whereas it was found lowest 60% in the summer season. There was a statistically significant difference (p0.05) difference in the winter, summer, and rainy season. To confirm the presence of MS in the samples, PCR test was applied using specific primers to amplify a 214 bp region of the 16S rRNA gene of the organism. In PCR, all seropositive flocks showed a positive result for MS. Conclusion: As the plate agglutination test result showed 100% similar with PCR result, it can be suggested that agglutination test is better than molecular and culture techniques for MS detection and it is also cheaper and less time-consuming method