To investigate the aetiology of the 2015 A(H1N1)pdm09 influenza outbreak in India, 1,083 nasopharyngeal swabs from suspect patients were screened for influenza A(H1N1)pdm09 in the state of Madhya Pradesh. Of 412 positive specimens, six were further characterised by phylogenetic analysis of haemagglutinin (HA) sequences revealing that they belonged to genogroup 6B. A new mutation (E164G) was observed in HA2 of two sequences. Neuraminidase genes in two of 12 isolates from fatal cases on prior oseltamivir treatment harboured the H275Y mutation.An epidemic of influenza A(H1N1)pdm09, affecting over 39,000 persons and causing more than 2,500 deaths occurred in India in 2015 [1]. We show that genotype 6B strains forming two sub-lineages circulated during the outbreak. Comparison of the sequences of six outbreak strains recovered in this work, to other published genotype 6B sequences, also reveals a unique combination of previously-reported mutations in the haemagglutinin (HA) gene. Two of the six sequences additionally display a E164G mutation in HA2, which has not been reported to date, moreover a N129D mutation in HA1 is observed for two sequences derived from patients with severe disease. Among strains analysed from 12 fatal cases on prior oseltamivir treatment, two harbour the H275Y mutation in the neuraminidase (NA) gene, which confers resistance to this antiviral.

Aim:This study was aimed to detect Mannheimia haemolytica in lung tissues of sheep and from a bacterial culture.Introduction:M. haemolytica is one of the most important and well-established etiological agents of pneumonia in sheep and other ruminants throughout the world. Accurate diagnosis of M. haemolytica primarily relies on bacteriological examination, biochemical characteristics and, biotyping and serotyping of the isolates. In an effort to facilitate rapid M. haemolytica detection, polymerase chain reaction assay targeting Pasteurella haemolytica serotype-1 specific antigens (PHSSA), Rpt2 and 12S ribosomal RNA (rRNA) genes were used to detect M. haemolytica directly from lung tissues and from bacterial culture.Materials and Methods:A total of 12 archived lung tissues from sheep that died of pneumonia on an organized farm were used. A multiplex polymerase chain reaction (mPCR) based on two-amplicons targeted PHSSA and Rpt2 genes of M. haemolytica were used for identification of M. haemolytica isolates in culture from the lung samples. All the 12 lung tissue samples were tested for the presence M. haemolytica by PHSSA and Rpt2 genes based PCR and its confirmation by sequencing of the amplicons.Results:All the 12 lung tissue samples tested for the presence of PHSSA and Rpt2 genes of M. haemolytica by mPCR were found to be positive. Amplification of 12S rRNA gene fragment as internal amplification control was obtained with each mPCR reaction performed from DNA extracted directly from lung tissue samples. All the M. haemolytica were also positive for mPCR. No amplified DNA bands were observed for negative control reactions. All the three nucleotide sequences were deposited in NCBI GenBank (Accession No. KJ534629, KJ534630 and KJ534631). Sequencing of the amplified products revealed the identity of 99-100%, with published sequence of PHSSA and Rpt2 genes of M. haemolytica available in the NCBI database. Sheep specific mitochondrial 12S rRNA gene sequence also revealed the identity of 98% with published sequences in the NCBI database.Conclusion:The present study emphasized the PCR as a valuable tool for rapid detection of M. haemolytica in clinical samples from animals. In addition, it offers the opportunity to perform large-scale epidemiological studies regarding the role of M. haemolytica in clinical cases of pneumonia and other disease manifestations in sheep and other ruminants, thereby providing the basis for effective preventive strategies.

Aim:This study was aimed to detect ovine pulmonary adenocarcinoma (OPA) in sheep flocks affected with pulmonary disorders at organized farm.Materials and Methods:A total of 75 sheep died naturally were thoroughly examined for the lesions of OPA during necropsy. Tissue sections from affected portion of the lungs from each animal were collected aseptically and divided into two parts; one each for polymerase chain reaction (PCR) and another for histopathology.Results:On PCR examination of lung tissues, six sheep (8%) were found to be positive for JSRV. Two of them were 3-6 months of age and did not show clinical signs/gross lesions of OPA. Four adult sheep positive on PCR revealed characteristic lesions of OPA on gross and histopathological examination.Conclusion:In the absence of known specific antibody response to the infection with JSRV, there is no diagnostic serological test available. The PCR assay employed in this study on lung tissues, using primers based on the U3 region of the viral long terminal repeat for JSRV would be helpful in the screening of preclinical and clinical cases of OPA in sheep.

Canine parvovirus (CPV) is the leading viral cause of enteritis in dogs and occurs mainly in 6- to 8-week-old pups. Rapid diagnosis of CPV under field conditions is now possible due to commercially available immunochromatographic (IC) assays. However, these commercial kits are somewhat expensive because they utilize a minimum of two monoclonal antibodies (mAbs) targeting different epitopes as capture and detector antibodies. Using only a single mAb for both capture and detection purpose may reduce the sensitivity of the assay. In the present study, efforts were made to develop an economical assay that can be utilized for diagnosis of CPV under Indian conditions with a high level of confidence. Rabbit polyclonal antibodies (pAbs) generated against recombinant truncated VP2 proteins of CPV were used as capture antibodies because they can be produced economically, while a commercial anti-CPV mAb was used as the detector antibody. The detection limit of the test strip was 6.6×10 TCID/ml, and it specifically detected CPV-2, CPV-2a and CPV-2b while displaying no cross-reactivity with other common canine enteric pathogens. The relative sensitivity/specificity of pAb based strip test was 71%/92% and 71%/100% in relation to the hemagglutination test and a commercial IC kit, respectively, with substantial agreement. In addition, two commercially available mAbs targeting different epitopes were also used for development of another IC assay, which showed sensitivity, and specificity of 82%/87% and 90%/98% in relation to the hemagglutination test and commercial kit. Hence, the present strip test based on a combination of mAb and pAb provides an acceptable alternative for onsite and cost-effective diagnosis of CPV infection.

Crimean-Congo hemorrhagic fever (CCHF) is a re-emerging zoonotic viral disease prevalent in many parts of Asia, Europe, and Africa. The causative agent, Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV), is transmitted through hard ticks. Tick vectors especially belonging to the Hyalomma species serve as the reservoir and amplifying host. The vertebrate animals including sheep, goat, and bovine act as a short-lasting bridge linking the virus and ticks. CCHFV causes fatal hemorrhagic fever in humans. Humans are usually infected with CCHFV either through the bite of infected ticks or by close contact with infected animals. Immunological assays, primarily enzyme-linked immunosorbent assay (ELISA) using whole viral antigen, are widely used for serosurveillance in animals. However, the whole virus antigen poses a high biohazard risk and can only be produced in biosafety level 4 laboratories. The present study focuses on the development and evaluation of safe, sensitive, and specific IgG indirect enzyme-linked immunosorbent assay (iELISA) using recombinant nucleoprotein (NP) of CCHF virus as an antigen. The codon-optimized NP gene sequence was synthesized, cloned, and expressed in pET28a+ vector. The recombinant NP was purified to homogeneity by affinity chromatography and characterized through Western blot and MALDI-TOF/MS analysis. The characterized protein was used to develop an indirect IgG microplate ELISA using a panel of animal sera. The in-house ELISA was comparatively evaluated vis-à-vis a commercially available ELISA kit (Vector-Best, Russia) with 76 suspected samples that revealed a concordance of 90% with a sensitivity and specificity of 79.4 and 100%, respectively. The precision analysis revealed that the assay is robust and reproducible in different sets of conditions. Further, the assay was used for serosurveillance in ruminants from different regions of India that revealed 18% seropositivity in ruminants, indicating continued circulation of virus in the region. The findings suggest that the developed IgG iELISA employing recombinant NP is a safe and valuable tool for scalable high-throughput screening of CCHFV-specific antibodies in multiple species.

An early diagnosis of Japanese encephalitis (JE) is important for timely clinical management and epidemiological control in areas where multiple flaviviruses are endemic. The NS1 antigen has an advantage over IgM enzyme-linked immunosorbent assay (ELISA) for early confirmatory diagnosis of Japanese encephalitis virus (JEV) infection due to its proliferation on the surface of the host cells in the acute phase of infection. In this study, the development and evaluation of JE-specific NS1 antigen capture ELISA is described using high-affinity monoclonal antibody specific to the recombinant NS1 protein for early diagnosis of JE. The gene encoding NS1 protein was cloned and expressed in the pQE30UA expression vector followed by purification of the recombinant protein by affinity chromatography. A sandwich ELISA for antigen detection was developed using purified rabbit IgG antibody and mouse monoclonal antibody as the capture and detector antibody, respectively. The application of JE NS1 antigen ELISA for early diagnosis was evaluated with 120 acute phase sera and 80 CSF samples. The comparative evaluation of the JE NS1 antigen ELISA by real-time RT-PCR revealed 97% concordance with a sensitivity and specificity of 97% and 98%, respectively. The JE NS1 antigen was detectable in the blood from the first day up to day 9 after the onset of symptoms. These findings suggest that the JEV NS1 antigen capture ELISA may help early diagnosis of JE infection.

Despite the fact that Chikungunya resurgence is associated with epidemic of unprecedented magnitude, there are challenges in the field of its clinical diagnosis. However, serological tests in an ELISA format provide a rapid tool for the diagnosis of Chikungunya infection. Indeed, ELISAs based on recombinant proteins hold a great promise as these methods are cost effective and are free from the risk of handling biohazardous material. In this study, the performance of recombinant CHIKV antigens was compared in various ELISA formats for the diagnosis of Chikungunya. Two recombinant antigens derived from the envelope proteins of Chikungunya virus were prepared and evaluated by comparing their competence for detecting circulating antibodies in serum samples of patients infected with CHIKV using MAC-ELISA and indirect IgM-ELISA. The efficacy of the recombinant antigens was also compared with the native antigen. The indirect antibody capture IgM microplate ELISA revealed ≥90% concordance with the native antigen in detecting the CHIKV specific IgM antibodies whereas the recombinant antigen based MAC-ELISA showed 100% specificity. The recombinant antigens used in this study were effective and reliable targets for the diagnosis of CHIKV infection and also provide an alternative for native antigen use which is potentially biohazardous.

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