Comparison of lateral flow assay and polymerase chain reaction for diagnosis of canine distemper.

In present study, 50 nasal and conjunctival pooled swab samples from 48 dogs, one each from leopard and cat were collected, which were suspected for canine distemper from Anand and Ahmedabad districts during period of January-21 to June-21. Out of the 50 samples, 18% were positive for CD by Lateral flow assay (LFA). Using LFA, one each sample from leopard and cat samples were found negative for CD by LFA. Out of the total 50 (48 dogs, one each from leopard and cat) samples, 14 and 4% were positive by N-gene and H-gene based RT-PCR respectively. Comparative analysis of N and H genes, both the samples positive by H-gene based RT-PCR were also positive by N-gene based RT-PCR, which detected 5 more samples positive than H-gene based RT-PCR. Comparative analysis of N-gene based RT-PCR and LFA, relative sensitivity and specificity were 55.55% and 95.12% respectively. Comparative analysis of H-gene based RT-PCR and LFA relative sensitivity and specificity were 22.22 and 100% respectively.

Pathotypic and Sequence Characterization of Newcastle Disease Viruses from Vaccinated Chickens Reveals Circulation of Genotype II, IV and XIII and in India.

Newcastle disease virus (NDV) causes a highly contagious disease which continuously haunts the global poultry industry. The nature and molecular epidemiology of NDVs prevalent in recent outbreaks in India is poorly understood. This study aimed to characterize NDVs prevalent in vaccinated flocks in India using whole-genome sequencing and biological pathotyping. Twelve field isolates were collected from outbreaks which occurred in different parts of India and characterized as velogenic based on their intracerebral pathogenicity index (ICPI) and amino acid sequence at the F protein cleavage site. All 12 of the field isolates and five commonly used vaccine strains were selected for whole-genome sequencing using Ion Torrent PGM technology, yielding complete genome sequences for ten field isolates and all vaccine strains. The genome of all isolates was found to be 15 192 nt long with a high level of conservation across multiple genomic features with APMV-I viruses. Phylogenetic analysis and evolutionary distance calculations placed the isolates in genotypes II, IV and XIII. Revisiting other recently reported strains provided preliminary evidence of genotypes VI, VII and XVIII circulating in India. Comparison between the field and vaccine virus sequences revealed unique genomic and amino acid differences in important antigenic regions of the F and hemagglutinin-neuraminidase (HN) genes which can be targeted for site directed mutagenesis to evaluate the impact of these substitutions on virus pathogenicity. This study highlights the requirement to evaluate current vaccines through systematic protection assays to determine protection efficacy against field isolates.

Evaluation of phenotypic factors for anti-rabies antibody in vaccinated pet dogs.

The aims of this study were to investigate various factors associated with protective anti-rabies antibody status (0.5 EU/ml) in vaccinated pet dogs and anti-rabies antibody status in unvaccinated stray dogs. One hundred and seven serum samples were collected from vaccinated pet dogs, out of these 58 (62.36 %) dogs showed antibody titre above 0.5 EU/ml. All the dogs were divided into different groups based on age, sex, breed, vaccine brand and time of vaccination after last vaccine to assess the relationship of these factors with vaccinal immune response. One way analysis of variance was performed in graphpad prism software to check the effect of all these factors. Statistical analysis of ELISA titres of pet dog serum samples suggested that age, sex, breed and vaccine brands have no significant effect on the anti-rabies antibody titres. To check anti-rabies antibody status in stray dogs 53 serum samples were collected and only one out of 53 (1.88 %) stray dogs showed anti-rabies antibody titre above 0.5 EU/ml indicating susceptibility to rabies infection and thereby posing possible threat to surrounding human and animal populations.

Isolation and molecular characterization of nephropathic infectious bronchitis virus isolates of Gujarat state, India.

Infectious bronchitis (IB) is a common, highly contagious, acute, and economically important viral disease of chickens caused by Infectious bronchitis virus (IBV, sp. Avian coronavirus). Five pooled tissue suspensions of 50 layer birds and one reference Massachusetts vaccine strain were inoculated into specific pathogen free (SPF) chicken egg for isolation of IBV. Reverse-transcription polymerase chain reaction (RT-PCR) was carried out using post inoculated allontoic fluid to amplify the spike (S) glycoprotein of S1 subunit of IBV. All the eggs inoculated with five pooled tissue samples and vaccine sample showed dwarfing and curling of SPF embryos indicative of IBV. All the five samples and the vaccine sample produced the expected amplicons of 466 bp by RT-PCR. The sequencing of five isolates revealed that all the five sequences were 99.09-100 % similar among themselves and showed 99.10-100 % nucleotide identity with the vaccine strain. On multiple sequence alignment it was found that our isolates were more similar at S1 subunit nucleotide level with the reference Ma5 and H120 vaccine strains than the reference Mass41 strain. The sequences of Anand isolates revealed further genetic changes in the circulating IBV in comparison to previous isolate of Gujarat as well as higher differences with the strains isolated in other states showing substantial changes at genetic level in Indian IBV isolates, which may partially explain the increasing incidences of IB in the country in spite of the vaccination.

Differentiation of Staphylococcus aureus and Staphylococcus epidermidis by PCR for the fibrinogen binding protein gene.

Mastitis is one of the most common and burdensome diseases afflicting dairy animals. Among other causes of mastitis, staphylococci are frequently associated with clinical and subclinical mastitis. Although Staphylococcus aureus is the predominant species involved, Staphylococcus epidermidis and other coagulase-negative staphylococci are increasingly being isolated from cases of bovine mastitis. Although Staph. aureus and Staph. epidermidis can be easily differentiated based on their biochemical properties, such phenotypic identification is time consuming and laborious. This study aimed to rapidly identify Staph. aureus and Staph. epidermidis. Accordingly, a multiplex PCR was developed and we found that a single gene encoding the adhesin fibrinogen binding protein could be used to identify and differentiate the two species. Consequently, a multiplex reaction combining a triplex PCR for Staph. aureus and a duplex PCR for Staph. epidermidis was standardized, first using bacterial cultures and then with pasteurized milk spiked with live organisms or DNA extracted from the organisms. The test could specifically detect Staph. aureus and Staph. epidermidis even in the presence of a dozen other organisms. The limit of detection for detecting Staph. aureus and Staph. epidermidis separately was 10 to 100 cfu/mL for simplex PCR and 10(4)cfu/mL for multiplex PCR. Conversely, the limit was 10(6)cfu/mL by multiplex PCR for simultaneous detection of both the organisms when spiked into culture medium or pasteurized milk. Overnight enrichment enhanced the assay sensitivity 100-fold. The assay had a high diagnostic sensitivity and specificity. The application of the test was verified on 602 field isolates of staphylococci that had been characterized earlier by phenotypic methods. Importantly, 25 coagulase-negative isolates were identified as Staph. aureus by the multiplex PCR. The test could be adapted for use in clinical diagnostic laboratories.

Genome sequence of Pasteurella multocida subsp. gallicida Anand1_poultry.

We report the finished and annotated genome sequence of Pasteurella multocida gallicida strain Anand1_poultry, which was isolated from the liver of a diseased adult female chicken. The strain causes a disease called "fowl cholera," which is a contagious disease in birds. We compared it with the published genome sequence of Pasteurella multocida Pm70.

Characterization of 132 bp Repeats BamH1-H Region in Pathogenic Marek's Disease Virus of Poultry in Gujarat, India, Using PCR and Sequencing.

A total of 34 clinical samples and four Marek's disease virus (MDV) vaccines were tested using primer BamH1/BamH2 in layer birds of poultry. Out of 34 samples tested for detection of MDV, 32 samples produced approximately 434 bp product. All the three HVT vaccines as well as SB-1 (MDV-2) vaccine failed to produce the expected amplicons, there by proving negative for the targeted 132 bp repeats of MDV genome by the primers BamH1/BamH2. Resultant PCR products of the field samples were purified and sequenced and resulted in 378 bp long sequences. PCR was found very satisfactory in detecting the presence of MDV either in feather follicle or in tissue samples. Sequencing study has proved beyond doubt that the two representative samples contained two 132 bp repeats indicating the virulent nature of the field virus.

Genetic characterization of Indian peste des petits ruminants virus (PPRV) by sequencing and phylogenetic analysis of fusion protein and nucleoprotein gene segments.

Peste des petits ruminants (PPR) is an important viral disease of sheep and goats, endemic in India. The study was undertaken to characterize the local PPRV by sequencing fusion (F) protein and nucleoprotein (N) gene segments and phylogenetic analysis, so as to focus on genetic variation in the field viruses. Selected regions of PPRV genome were amplified from clinical samples collected from 32 sheep and goats by RT-PCR and the resulting amplicons were sequenced for phylogenetic analysis. The phylogenetic tree based on the 322bp F gene sequences of PPRV from five different locations clustered them into lineage 4 along with other Asian isolates. While the 425bp N gene sequences revealed a different pattern of branching, yielding three distinct clusters for Nigerian, Turkey and Indian isolates. Thus, classification of PPRV into lineages based on the N gene sequences appeared to yield better picture of molecular epidemiology for PPRV.