Subclinical circulation of Chicken infectious anaemia virus- A Sero-molecular study.

Chicken infectious anaemia-an important immunosuppressive viral disease of chicken-gained much attention in the recent past. Based on huge mortality and production loss observed in the fast-growing poultry sector, the present study aimed to find out the current status of the chicken infectious anaemia virus (CIAV), among chicken flocks in the Punjab state of India by sero-molecular study. The sera from the blood samples were tested for anti-CIAV antibodies by indirect ELISA and also compared with haematological parameters. DNA from sero-positive samples underwent PCR amplification, sequencing and phylogenetic analysis of the most conserved genomic region (VP3 gene) to detect viraemia in asymptomatic birds. The serological study using indirect ELISA showed a high sero-positivity of 77.27% in chicken flocks. Additionally, the present study also revealed the high molecular evidence (72.54%) of CIAV in apparently healthy birds. Genetic analysis showed that all CIAVs have conserved VP3 genes without any nucleotide substitutions, indicating presence of CIAV and its subclinical circulation among apparently healthy flocks. The wide distribution of CIAV among birds may be the reason for huge mortality and production loss. Further, it is suggested that studies be conducted to find out the co-involvement of CIAV with other immunosuppressive microbial agents and the immunosuppressive effect of CIAV in apparently healthy birds. Also, its role in vaccine failure and outbreaks of various other avian diseases needs to be explored.

Prevalence and molecular characterization of canine parvovirus.

BACKGROUND AND AIM: Canine parvovirus (CPV) belonging to family Parvoviridae causes hemorrhagic gastroenteritis in dogs and heavy mortality in young dogs. The virus has three structural (VP1, VP2 and VP3) and two non-structural proteins (NS1 and NS2), VP2 being highly immunogenic. This study aims to study molecular epidemiology of CPV by sequence analysis of VP2 gene to determine the prevailing antigenic type(s) in the northern regions of India. MATERIALS AND METHODS: A total of 118 rectal swabs collected from dogs exhibiting clinical signs of CPV infection were processed for the isolation of DNA and subjected to polymerase chain reaction (PCR) and nested PCR (NPCR). A total of 13 NPCR products selected randomly were subjected to sequence analysis of VP2 gene. RESULTS: The percent positivity of CPV was found 28% and 70% by PCR and NPCR, respectively. Dogs with vaccination history against CPV too were found positive with a percent positivity of 24.10%. Gene sequencing and phylogenetic analysis of VP2 gene from these isolates revealed that most samples formed a clade with CPV-2a isolates. CONCLUSION: Sequence analysis and phylogenetic analysis of VP2 gene in the studied regions of northern India revealed that CPV-2a was the most prevalent antigenic type.

Detection and molecular characterization of chicken infectious anaemia virus in young chicks in Punjab region of north-western India.

Chicken infectious anaemia (CIA) is an important viral disease of chicken causing significant immunosuppression and severe anaemia worldwide. Occurrence of severe disease and mortality is noticed in young chicks (2-3 weeks). Vertical mode of transmission increases chance of infection and persistence of virus among the infected flocks. The current study was conducted in Punjab state for confirmation and genetic characterization of CAV among chicken flocks of various poultry farms. DNA was extracted from the tissue samples and subjected to polymerase chain reaction (PCR) of VP1 gene and whole genome. PCR products were further sequenced for confirmation of chicken infectious anaemia virus (CIAV) genome in the clinical samples. PCR amplification of DNA from the tissue samples yielded expected product size of 1350 bases of VP1 gene and 2.3 kb of whole genome. Out of 16 commercial poultry farms, 11 were confirmed with presence of CIAV, and out of 65 birds, 39 were found positive (60%) for CIAV genes. Among the various organs, the presence of viral gene was detected at highest level in thymus when compared with other organs. It is concluded that chicken infectious anaemia virus detected from Punjab state is closely related to other Indian isolates and neighbouring countries which necessitates need of more intensive studies with a greater number of samples for implementing effective control measures.

Phylogenetic analysis of VP2 gene of canine parvovirus and comparison with Indian and world isolates.

Canine parvovirus (CPV) causes hemorrhagic enteritis, especially in young dogs, leading to high morbidity and mortality. It has four main antigenic types CPV-2, CPV-2a, CPV-2b and CPV-2c. Virus protein 2 (VP2) is the main capsid protein and mutations affecting VP2 gene are responsible for the evolution of various antigenic types of CPV. Full length VP2 gene from field isolates was amplified and cloned for sequence analysis. The sequences were submitted to the GenBank and were assigned Acc. Nos., viz. KP406928.1 for P12, KP406927.1 for P15, KP406930.1 for P32, KP406926.1 for Megavac-6 and KP406929.1 for NobivacDHPPi. Phylogenetic analysis indicated that the samples were forming a separate clad with vaccine strains. When the samples were compared with the world and Indian isolates, it was observed that samples formed a separate node indicating regional genetic variation in CPV.

Multiplex real-time PCR for identification of canine parvovirus antigenic types.

Canine parvovirus (CPV) is an important disease causing gastroenteritis and/or haemorrhagic gastroenteritis in dogs. There are four antigenic types of CPV reported worldwide viz. CPV 2, CPV 2a, CPV 2b and CPV 2c. The diagnosis of CPV with the identification of the antigen type responsible remains problematic. In the present study, identification as well as antigenic typing of CPV was done using a de novo multiplex real time PCR to combat the problem of antigenic type identification. From the study it could be concluded that the here developed multiplex real time PCR assay could be used for rapid detection of CPV as well as typing of its three antigenic types.

Isolation of Canine parvovirus with a view to identify the prevalent serotype on the basis of partial sequence analysis.

AIM: The aim of this study was to isolate Canine parvovirus (CPV) from suspected dogs on madin darby canine kidney (MDCK) cell line and its confirmation by polymerase chain reaction (PCR) and nested PCR (NPCR). Further, VP2 gene of the CPV isolates was amplified and sequenced to determine prevailing antigenic type. MATERIALS AND METHODS: A total of 60 rectal swabs were collected from dogs showing signs of gastroenteritis, processed and subjected to isolation in MDCK cell line. The samples showing cytopathic effects (CPE) were confirmed by PCR and NPCR. These samples were subjected to PCR for amplification of VP2 gene of CPV, sequenced and analyzed to study the prevailing antigenic types of CPV. RESULTS: Out of the 60 samples subjected to isolation in MDCK cell line five samples showed CPE in the form of rounding of cells, clumping of cells and finally detachment of the cells. When these samples and the two commercially available vaccines were subjected to PCR for amplification of VP2 gene, a 1710 bp product was amplified. The sequence analysis revealed that the vaccines belonged to the CPV-2 type and the samples were of CPV-2b type. CONCLUSION: It can be concluded from the present study that out of a total of 60 samples 5 samples exhibited CPE as observed in MDCK cell line. Sequence analysis of the VP2 gene among the samples and vaccine strains revealed that samples belonged to CPV-2b type and vaccines belonging to CPV-2.

Antigenic typing of canine parvovirus using differential PCR.

Canine parvovirus (CPV) is an enteric pathogen causing hemorrhagic enteritis in pups of 3-6 months of age and is mainly transmitted via feco-oral route. In the present study, a total of 85 animals rectal swabs suspected of CPV were tested using a PCR, nested PCR and a newly designed differential PCR. Using PCR 7 (8.23 %) animals were positive whereas 39 (45.88 %) were positive by using nested PCR and 40 (47.05 %) were positive for either one or more than one antigenic types of CPV using differential PCR. Using differential PCR it was found that CPV-2a and CPV-2b were the most prevailing antigenic types. Also it was found that dogs that were vaccinated too yielded positive CPV indicating a possible presence of additional CPV antigenic types. Thus, the primers used in differential PCR can be used in a single PCR reaction to detect various antigenic types of CPV.

Comparison of three techniques for the diagnosis of urinary tract infections in dogs with urolithiasis.

OBJECTIVES: To identify an appropriate sampling technique(s) to accurately detect the bacteria causing urinary tract infections in dogs with urolithiasis. METHODS: Twenty-one dogs with urolithiasis were included in the study. Three types of samples were taken from each dog. Urine was collected by cystocentesis, and a urinary bladder mucosal biopsy and urolith were retrieved during cystotomy. The samples were then cultured on blood agar and MacConkey's agar to identify the bacteria associated with urinary tract infections. RESULTS: Bacterial urinary tract infection was found in 16 cases (76.19 per cent). The most prevalent bacteria found to cause urinary tract infection were Escherichia coli (n=7), followed by coagulase-positive Staphylococcus species (n=4), Klebsiella pneumoniae (n=2), Pseudomonas aeruginosa (n=2) and Proteus mirabilis (n=1). In the case of a positive urine culture, the same bacteria were also cultured from the urinary bladder mucosal biopsy alone or from both the urinary bladder mucosal biopsy and urolith. However, in the case of a negative urine culture, bacteria were found to be present in the urinary bladder mucosal biopsy or urolith cultures in 23.81 per cent of dogs. The uroliths that gave positive culture results were either infection-induced uroliths composed of struvite and calcium carbonate phosphate, ammonium acid urate only or metabolic uroliths composed of calcium oxalate and calcium phosphate, or calcium phosphate only. All the uroliths that gave negative culture results were metabolic uroliths composed of calcium oxalate and/or calcium phosphate, and uric acid and calcium phosphate. CLINICAL SIGNIFICANCE: When the culture from the urine obtained by cystocentesis is negative, cultures of urinary bladder mucosal biopsy and urolith are recommended in dogs with urolithiasis in order to accurately assess the microbiological status of the urinary tract.

The effects of cyclosporin A and cyclophosphamide on the populations of B and T cells and virus in the Harderian gland of chickens vaccinated with the Hitchner B1 strain of Newcastle disease virus.

The cellular response to conjunctival vaccination with the Hitchner B1 strain of Newcastle disease virus was studied in the Harderian gland (HG) by immunohistochemistry. Bu-1+ cells and all subpopulations of T cells, (CD3+, CD4+, CD8+, TCR gamma delta, TCR alpha beta 1, and TCR alpha beta 2) were in the interstitial tissue between the ducts and the acini. Plasma cells with cytoplasmic IgM were more dispersed than the other cells and outlined the acini. Bu-1+ cells and all subpopulations of T cells increased at least three-fold after vaccination when compared to uninfected birds on the basis of the average cell counts in sections taken at 3, 5, 7, 10, 14, and 20 days after vaccination. The most marked increase was in the CD8+ cells which increased six-fold. Virus replicated for 10 days in cyclophosphamide (Cy) treated birds and for 7 days in cyclosporin A (CsA) treated birds compared with 5 days in untreated birds. Cy treatment prevented an antibody response to NDV and reduced Bu-1+ and IgM cells in the HG by 20-fold. Cy treatment resulted in a doubling of the number of T cells in the HG but these T cells may have been transiently disabled because it also caused a poor response of the lymphocytes in whole blood to the T cell mitogen concanavalin A (ConA). CsA reduced the T cell numbers in the HG and whole-blood responses to ConA by about 4-fold but T cell numbers rebounded to normal resting values after vaccination with NDV. The clearance time was prolonged either by T cells being less numerous than normal after CsA or being disabled after Cy. T cells, but not B cells, may therefore be essential for virus clearance. CD8+ cells expanded more than CD4+ cells after the vaccination of untreated and CsA-treated birds indicating that CD8+ cells may be key players in vaccinal immunity to NDV.