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L-Arginine (LA) is a well-known amino acid involved in vital physiological processes. However, the physiological function of its methylated form known as L-Homoarginine (LHA), is not well understood. The aim of this study was to study the comparative effects of LA and LHA treatment on selected metabolites and parameters of reproductive physiology in non-pregnant ewes. Twelve Deccani ewes were selected and randomly distributed into 3 groups: Control, LA treated group and LHA group. Blood samples were collected for the evaluation of blood indices and hormone levels. Overall, nitrite, ovary weight, surface follicle number, estrogen, insulin like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF) showed an increase in the LA and LHA treated animals. On the other side, the levels of progesterone, inteleukin-1 beta (IL-1β), IL-6, tumor necrosis factor-alpha (TNF-α) were found to be decreased in LA and LHA group. Ovary size was not affected by LA and LHA treatment groups. In comparison with LA, the ovary weight, surface follicle number, estrogen levels and IL-6 expression were higher in LHA treated animals. The expression of VEGF indicated improved angiogenesis in the treated animals. Further, the expression of heat shock protein-27 (HSP-27) and HSP-70 were differentially modulated by LA and LHA. This data reinforces the beneficial role of LA and its metabolites LHA on the ovarian physiology and functionality and also reveals the potent role of LHA as an alternative to LA treatment in enhancing the reproductive ability in non-pregnant ewes.

Pasteurella multocida is a Gram-negative bacterium that causes haemorrhagic septicaemia in cattle and buffaloes. These organisms are involved in the formation of biofilm and can evade treatment. There is no definitive study to screen the genes associated with biofilm production associated with Pasteurella multocida. The present study describes the real-time PCR based approach for screening of genes associated with biofilm formation. Out of 10 isolates screened for biofilm formation, five of them produced biofilm on Congo red agar of which all are resistant to major antibiotics especially cotrimoxazole, nalidixic acid, enrofloxacin and tetracyclines. All the isolates show presence of genes associated with biofilm formation indicating that other factors influencing the biofilm production. We suggest that the future studies may be targeted to unravel the other factors that influence the biofilm production.

Lumpy skin disease (LSD), a notifiable disease listed by the World Organization for Animal Health and a fast fast-moving transboundary viral disease infecting cattle and buffaloes, was reported in India in 2019 and has since rapidly spread across the country. This study reports the first complete genome sequence and analysis of a pathogenic LSD virus (LSDV) from India (LSDV/208/PVNRTVU/2020) obtained by direct sequencing of a suspected clinical sample using Illumina and Nanopore sequencing technologies. The complete genome sequence of LSDV/208/PVNRTVU/2020 is 150445 bp long, codes for 156 putative genes and carries identical 2254 bp inverted terminal repeats at either ends. The unique features reported in the LSDV isolates from the recent outbreaks in Asia, namely, the insertions of 12 nucleotides in the viral G-protein coupled receptor (GPCR) and 27 nucleotides leading to duplication of 9 aminoacids in the extracellular enveloped virus-specific (EEV) genes were also conserved in LSDV/208/PVNRTVU/2020. Phylogenetic analysis of the complete genome sequence of LSDV/208/PVNRTVU/2020 revealed its close relation with Kenyan strains and clustered away from vaccine strains. Further analysis showed evidence of strong purifying selection without any recombination events. The data presented in this study could be useful for designing effective strategies such as developing rapid diagnostics and vaccines to control LSD.

Viral pathogens are the primary cause of canine gastroenteritis. However, few structured comprehensive studies on the viral etiology of canine gastroenteritis have been conducted. In this study, 475 rectal swabs collected over three years (2018-2021) from clinical canine gastroenteritis cases were screened for the presence of six major enteric viruses - canine parvovirus 2 (CPV-2), canine distemper virus (CDV), canine adenovirus 2 (CAdV-2), canine coronavirus (CCoV), canine astrovirus (CaAstV), and canine rotavirus (CRV) - by real-time PCR. The most frequently detected virus was CPV-2, which was present in 64.8% of the samples (subtype 2a, 21.1%; 2b, 77.4%; 2c, 1.5%), followed by CDV (8%), CaAstV (7.2%), CCoV (5.9%), and CAdV-2 (4.6%). Two to four of these viruses in different combinations were found in 16.8% of the samples, and CRV was not detected. The complete genome sequences of Indian isolates of CDV, CCoV, and CaAstV were determined for the first time, and phylogenetic analysis was performed. This study highlights the need for routine prophylactic vaccination with the appropriate vaccines. Notably, 70.3% of animals vaccinated with DHPPiL were found to be positive for at least one virus. Hence, regular molecular analysis of the prevalent viruses is crucial for addressing vaccination failures.

Sexed semen can be used to produce offspring of the desired sex, and to produce replacement heifers from genetically superior cows. In the Indian dairy industry, this revolutionary technique is in its budding stage. The objective of this study was to assess quantitative and qualitative parameters of a) bovine cryopreserved sexed (for X sperm cells) (XS) spermatozoa produced through two different major commercial semen sorting techniques, and b) unsexed (US) spermatozoa of Bos indicus and Bos taurus breeds. By real-time PCR, it was found that the percentage of X chromosome bearing spermatozoa ranged from 62.88%±0.56 to 71.21±0.93 in US semen samples of four different cattle breeds: Holstein Friesian (HF), Jersey, Gir and Sahiwal. The corresponding values in semen sorted through flow cytometry technique ranged from 99.02% ±3.36 to 99.99%±4.56 in the above four cattle breeds; while the percentage of XS semen obtained through the decapitation of Y chromosome following sorting by flow cytometry ranged from 95.92% ±1.83 to 97.99%±2.74. Qualitatively, US sperm samples were found significantly (P

Against the backdrop of the second wave of COVID-19 pandemic in India that started in March 2021, we have monitored the spike (S) protein mutations in all the reported (GISAID portal) whole-genome sequences of SARS-CoV-2 circulating in India from 1 January 2021 to 31 August 2021. In the 43,102 SARS-CoV-2 genomic sequences analysed, we have identified 24,260 amino acid mutations in the S protein, based on which 265 Pango lineages could be categorized. The dominant lineage in most of the 28 states of India and its 8 union territories was B.1.617.2 (the delta variant). However, the states Madhya Pradesh, JammuKashmir, and Punjab had B.1.1.7 (alpha variant) as the major lineage, while the Himachal Pradesh state reported B.1.36 as the dominating lineage. A detailed analysis of various domains of S protein was carried out for detecting mutations having a prevalence of1%; 70, 18, 7, 3, 9, 4, and 1 (N = 112) such mutations were observed in the N-terminal domain, receptor binding domain, C -terminal domain, fusion peptide region, heptapeptide repeat (HR)-1 domains, signal peptide domain, and transmembrane region, respectively. However, no mutations were recorded in the HR-2 and cytoplasmic domains of the S protein. Interestingly, 13.39% (N = 15) of these mutations were reported to increase the infectivity and pathogenicity of the virus; 2% (N = 3) were known to be vaccine breakthrough mutations, and 0.89% (N = 1) were known to escape neutralizing antibodies. The biological significance of 82% (N = 92) of the reported mutations is yet unknown. As SARS-CoV-2 variants are emerging rapidly, it is critical to continuously monitor local viral mutations to understand national trends of virus circulation. This can tremendously help in designing better preventive regimens in the country, and avoid vaccine breakthrough infections.

Background and Aim: Bluetongue (BT) is a major disease of sheep and goats and is endemic to India. It is known to cause significant economic losses to the sheep industry. The current study aimed to determine the type-specific seroprevalence of BT in sheep population of India during 2018-2019. Materials and Methods: Blood samples (n=405) were collected from 6 months to 1 year old sheep from six districts (Nalgonda, Karimnagar, Khammam, Mahabubnagar, Warangal, and Ranga Reddy) of Telangana state, India. Group- and type-specific seroprevalence (against BT virus [BTV] serotypes BTV-1, 2, 4, 5, 9, 10, 12, 16, 21, 23, and 24) was studied by competitive enzyme-linked immunosorbent assay and serum neutralization test, respectively. Results: Results showed an overall seroprevalence of 14.81% (n=60) with the highest seroprevalence of 50% in Khammam district. Seroprevalence of BTV-1, 2, 4, 5, 9, 10, 12, 16, 21, 23, and 24 was noted as 16.66%, 11.66%, 31.66%, 11.66%, 05%, 6.66%, 16.66%, 8.33%, 13.33%, 6.66%, and 16.66%, respectively. The majority of the sera neutralized more than 1 serotype, indicating superinfection or circulation of multiple serotypes in the sampled flocks. This mixed seroprevalence was observed in 43.33% of the sera with number of BTV serotype-specific antibodies ranging from two to eight in individual animals. Conclusion: Regular monitoring of circulating serotypes, especially in young herds, elucidates pattern of dominating serotypes in a particular area during a season. This knowledge can be applied to design appropriate vaccination strategies by including particular serotypes of virus as part of a multivalent vaccine for a particular period, in a particular area.

Against the backdrop of the second wave of COVID-19 pandemic in India that started in March 2021, we have monitored the spike (S) protein mutations in all the reported (GISAID portal) whole genome sequences of SARS CoV-2 circulating in India from 1 January 2021 to 31 August 2021. In the 43,102 SARS-CoV-2 genomic sequences analysed, we have identified 24, 260 mutations in the S protein, based on which 265 pango lineages could be categorised. The dominant lineage in most of the 28 states of India and its 8 union territories was B.1.617.2 (the delta variant). However, the states Madhya Pradesh, Jammu & Kashmir, and Punjab had B.1.1.7 (alpha variant) as the major lineage, while the Himachal Pradesh state reported B.1.36 as the dominating lineage. A detailed analysis of various domains of S protein was carried out for detecting mutations having a prevalence of >1%; 70, 18, 7, 3, 9, 4, and 1 (N=112) such mutations were observed in the N -terminal domain, receptor binding domain, C -terminal domain, fusion peptide region, heptapeptide repeat (HR)-1 domains, signal peptide domain, and transmembrane region, respectively. However, no mutations were recorded in the HR-2, and cytoplasmic domains of the S protein. Interestingly, 13.39% (N=15) of these mutations were reported to increase the infectivity and pathogenicity of the virus; 2%(N=3) were known to be vaccine breakthrough mutations; and 0.89%(N=1) were known to escape neutralising antibodies. Biological significance of 82% (N=92) of the reported mutations is yet unknown. As SARS-CoV-2 variants are emerging rapidly, it is critical to continuously monitor local viral mutations to understand national trends of virus circulation. This can tremendously help in designing better preventive regimens in the country, and avoid vaccine breakthrough infections.

Canine bufavirus (CaBuV), a novel protoparvovirus of dogs was reported only in Italy and China, till date. It was detected from dogs with enteric and respiratory symptoms and is distantly related to the human bufavirus. To explore the enteric prevalence of CaBuV in India, 186 diarrheic faecal samples were collected in Telangana State between 2019 and 2020. Among the samples, 4.3% (8/186) were positive for CaBuV by PCR. Co-infection with canine parvovirus CPV-2 was seen in 75% (6/8) of CaBuV positive samples. The near complete genome (4292 bp) of CaBuV was amplified and reconstructed for one isolate 407/PVNRTVU/2020. Sequence alignment indicated 93.42–98.81% homology with the other available CaBuV sequences; 70.88-73.39%, and 54.4-54.8% identity with human bufavirus, and CPV-2, respectively. Phylogenetic analysis showed that CaBuV 407/PVNRTVU/2020 was most closely related to CaBuV Chinese strains, together separated as Asian lineage. All Bufaviruses clustered together in one clade; however, the bat and sea otter parvoviruses also showed close relation with Bufaviruses. This first report of prevalence of CaBuV in India provides a good reference, emphasizes the need for further epidemiological surveillance of CaBuV in India and its role in canine enteritis.

Canine bufavirus (CBuV), a novel protoparvovirus of dogs that is associated with enteric and respiratory symptoms, has been reported only in Italy and China. The enteric prevalence of CBuV in India was investigated, and the nearly complete genome sequence (4292 bp) was amplified and reconstructed for one strain. A nucleotide sequence alignment indicated 93.42-98.81% identity to the other available CBuV sequences and 70.88-73.39% and 54.4-54.8% identity to human bufavirus and canine parvovirus 2 (CPV-2), respectively. The current strain is most closely related to Chinese CBuV strains, which together form an Asian lineage. This first report of the prevalence of CBuV in India emphasizes the need for further epidemiological surveillance.

Veterinary clinicians and academicians of India have limited locally generated data on the efficacy of the antimicrobial Ceftiofur hydrochloride in dairy cattle. The present study was undertaken to study the efficacy of Ceftiofur in dry cow therapy of cross bred Indian dairy cattle. Animals in the treatment group (N=254) received 500 mg of ceftiofur into all quarters on the day of drying while the animals in the control group (N=127) were left untreated. It was found that 98.03%, and 96.06% of treatment, and control group animals, respectively were exhibiting intramammary infections on the day of drying. Efficacy of Ceftiofur in terms of cure rates was 70.6% by day 28 postpartum with control group showing a spontaneous cure rate of 16.39%. The proportions of new intramammary infections by day 28 postpartum were higher in control group (73.77%) in contrast to treatment group (9.23%). Our study suggests an important role of ceftiofur as an effective antimicrobial agent in dry cow therapy.

Bluetongue (BT) is an infectious, non-contagious, insect-transmitted viral disease of sheep, other domestic, and wild ruminants caused by bluetongue virus (BTV), a prototype species of the genus Orbivirus of the family Reoviridae. The study was designed to determine the hematological and biochemical alterations and acute phase protein response in BTV sero-negative sheep experimentally infected with bluetongue virus serotype-10 and 24. The BTV-infected group comprising of 18 animals, 6 each inoculated with 6 ml of clarified virus containing 1 × 106/ml TCID50 of BTV-10 or 24 by intradermal route, 6 animals inoculated with equal volumes of BTV-10 and 24 and 4 animals with 6 ml of mock infected culture fluid served as uninfected control group. The blood and serum samples were analyzed at 0, 1, 3, 7, 11, and 16 days post-infection (DPI). Hematological findings in this study showed a significant decrease in the total number of white blood cells in BTV-10 infected group, a significant increase in circulating neutrophils decrease in lymphocytes in all the infected groups, in PCV in BTV-24 and co-infected groups, and RBC count in only BTV-24-infected group at 11 DPI. The results of serum biochemical analyses of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) exhibited increased levels in BTV-24 and co-infected animals. A significant rise in the serum level of creatine kinase (CK) was observed from 3 DPI in the infected groups. Serum levels of total proteins decreased significantly at 11 DPI in BTV-24 infected animals. Significant increase in CRP and fibrinogen levels was observed at 7 and 11 DPI in the infected groups. The significantly decreased levels of TLC, hemoglobin, PCV, CK, and acute phase proteins such as fibrinogen and CRP indicated the inflammatory changes associated with the virus replication and the consequent vascular damage and inflammatory changes in the tissues. Further, the variations in the hemato-biochemical and acute phase response observed between the two serotypes BTV-10 and 24 probes to the difference in virulence and pathogenesis of the serotypes in infected sheep.

Canine adenovirus (CAV) circulates as two distinct serotypes, CAV-1 and CAV-2, which are antigenically related but differ in their clinical manifestations. CAV is one of the important viral agents in the etiology of canine gastroenteritis. Here, we report the molecular surveillance and genetic characterization of CAV from clinical cases of canine gastroenteritis. A total of 302 fecal/rectal swabs were collected from dogs presented with gastroenteritis at various clinics in and around Hyderabad, India during 2018–19. These samples were tested for CAV using polymerase chain reaction with primers designed for the CAV E3 gene and the virus was isolated from positive samples. CAV-2 nucleic acid was present in 4.9% of the test samples. The partial sequence analyses of the E3 gene of the CAV-2 isolates revealed a frameshift mutation by insertion of nucleotide “G” at 1077 position of E3 gene, which resulted in an extension of the polypeptide chain by eleven amino acids. As a result, isolates from the current study formed a novel group, and the virus that was previously subdivided into two groups worldwide is now categorized under three. The study identifies a novel group of CAV-2 circulating in India providing an updated information regarding CAV-2.

Enteritis caused by CPV-2 antigenic variants (CPV-2a, 2b, and 2c) is frequently reported in dogs worldwide leading to significant morbidity and mortality. Here, we describe about a simple, single-step, ARMS-PCR strategy targeting the mutant 426 amino acid of VP2 to differentiate CPV-2 antigenic types. A total of 150 fecal samples were subjected to ARMS-PCR of which 18 were typed as CPV-2a, 79 were typed as CPV-2b, and 6 were typed as CPV-2c. The ARMS-PCR results were validated by randomly sequencing partial VP2 gene of 14 samples. Phylogenetic analysis of partial VP2 gene sequencing of each of the CPV-2 variants revealed that CPV-2a and CPV-2b isolates formed a separate clade of Indian lineage, while CPV-2c shared common evolutionary origin with Asian lineage. The developed technique is first of its kind, one-step, rapid, sequencing independent method for typing of CPV-2 antigenic variants.

We report clinical profile of hundred and nine patients with SARS CoV-2 infection, and whole genome sequences (WGS) of seven virus isolates from the first reported cases in India, with various international travel histories. Comorbidities such as diabetes, hypertension, and cardiovascular disease were frequently associated with severity of the disease. WBC and neutrophil counts showed an increase, while lymphocyte counts decreased in patients with severe infection suggesting a possible neutrophil mediated organ damage, while immune activity may be diminished with decrease in lymphocytes leading to disease severity. Increase in SGOT, SGPT and blood urea suggests the functional deficiencies of liver, heart, and kidney in patients who succumbed to the disease when compared to the group of recovered patients. The WGS analysis showed that these isolates were classified into two clades: I/A3i, and A2a (four according to GISAID: O, L, GR, and GH). Further, WGS phylogeny and travel history together indicate possible transmission from Middle East and Europe. Three S protein variants: Wuhan reference, D614G, and Y28H were identified predicted to possess different binding affinities to host ACE2.

Background: Canine distemper virus (CDV) is one of the important causes for canine gastroenteritis. Its genome codes for six structural proteins N, P, M, F, H and L, of which H is important for viral pathogenesis. Availability of CDV molecular epidemiological data is sparse. Our study reports for the first time, isolation of enteric CDV from India and its molecular epidemiology. Methods: We isolated the circulating wild type CDV from cases of canine gastroenteritis recorded during 2019 in India. Partial H gene of the isolates was amplified and phylogeny was reconstructed with other isolates from the NCBI database using MUSCLE from MEGA7. Results: The multiple sequence alignment of the partial H gene of circulating CDV with reference isolates revealed 88-95% nucleotide identity, and 83-91% amino acid identity along with the presence of Nde1 restriction site at position 1571-1576 that is typical of wild type CDV isolates. Interestingly, phylogenetic analysis revealed that the circulating CDV were grouped with three different lineages i.e., South America-2, Asia-4, and Asia-5/India-1 and distantly related with the vaccine strains. Our study strengthens the need for the development of a relevant vaccine comprising of circulating viral strains for effective vaccination strategies.

Bluetongue (BT) is one of the important viral diseases of domestic and wild ruminants, especially small ruminants such as sheep. Out of the 29 BTV serotypes prevalent in the world, at least 24 of the serotypes are reported in India, either by virus isolation or serology. To better understand the seroprevalence of BTV, we conducted a comprehensive study in the main reservoir hosts of BTV, i.e., cattle and buffaloes of different age groups in Andhra Pradesh and Telangana states of India where the disease is majorly prevalent. A total of 321 blood samples collected from cattle and buffaloes during 2017-2018 were tested for group-specific BTV seroprevalence by c-ELISA, followed by type specific seroprevalence (against BTV-1, 2, 4, 5, 9, 12, 16, and 24) by serum neutralization test. Of the 311 BTV seropositive samples, 112, 98, 102, 127, 2, 113, 160, and 5 samples neutralized BTV-1, 2, 4, 5, 9, 12, 16, and 24, respectively. Twenty-nine samples could not neutralize any of the tested BTV serotypes. Majority of the sera neutralized more than one serotype, up to a maximum of six serotypes. Major finding of the study is detection of BTV serotypes not included in the commercial pentavalent inactivated vaccine. Regular surveillance of circulating serotypes, especially in sentinel reservoir hosts throughout the country can help in designing better multivalent vaccines with suitable vaccine strains, for specific geographic regions.

Bluetongue virus (BTV) is neurotropic in nature, especially in ruminant fetuses and in-utero infection results in abortion and congenital brain malformations. The aim of the present study was to compare the neuropathogenicity of major Indian BTV serotypes 1, 2, 10, 16 and 23 by gross and histopathological lesions and virus distribution in experimentally infected neonatal BALB/c mice. Each BTV serotype (20 μl of inoculum containing 1 × 105 tissue culture infectious dose [TCID]50/ml of virus) was inoculated intracerebrally into 3-day-old mice, while a control group was inoculated with mock-infected cell culture medium. Infection with BTV serotypes 1, 2 and 23 led to 65-70% mortality at 7-9 days post infection (dpi) and caused severe necrotizing encephalitis with neurodegenerative changes in neurons, swelling and proliferation of vascular endothelial cells in the cerebral cortex, cerebellum, midbrain and brainstem. In contrast, infection with BTV serotypes 10 and 16 led to 25-30% mortality at 9-11 dpi and caused mild neuropathological lesions. BTV antigen was detected by immunohistochemistry, direct fluorescence antibody technique and confocal microscopy in the cytoplasm of neuronal cells of the hippocampus, grey matter of the cerebral cortex and vascular endothelial cells in the midbrain and brainstem of BTV-1, -2, -10, -16 and -23 infected groups from 3 to 20 dpi. BTV nucleic acid was detected in the infected brain tissues from as early as 24 h up to 20 dpi by VP7 gene segment-based one-step reverse transcriptase polymerase chain reaction. This study of the relative neurovirulence of BTV serotypes is likely to help design suitable vaccination and control strategies for the disease.

India is enzootic for bluetongue (BT), a predominant disease of small ruminants. The most important task in the control of disease is rapid and sensitive detection of virus. The present study was undertaken to standardize immunofluorescence (IFT) and immunoperoxidase tests (IPT) employing BTV serogroup specific VP7 monoclonal antibodies (MAbs), polyclonal homologous, and polyclonal heterologous antisera against specific serotypes of BTV for detection of BTV antigen. Serial tenfold dilutions of BTV-9 were tested for limit of detection (LoD) of IFT, IPT, and molecular assays by using MAbs against VP7, homologous anti-BTV-9 serum, and heterologous anti-BTV-16 serum. LoD of IFT was found to be 101 TCID50/mL using MAbs against VP7, anti BTV-9 serum, and anti BTV-16 serum. LoD of IPT was found to be 101 TCID50/mL, 102 TCID50/mL, and 102 TCID50/mL using MAbs against VP7, anti BTV-9 serum and anti BTV-16 serum, respectively. LoD of RT-PCR was 101 TCID50/mL and that of real time PCR was 100 TCID50/mL. This standardized assay was then applied for BTV detection in BTV suspected field samples collected from BT outbreaks followed by confirmation with virus isolation and NS3 group specific PCR. The current study shows that IFT and IPT are specific tests for group specific BTV identification. For IFT, monoclonal and polyclonal (homologous and heterologous) source of antibodies had similar sensitivity in the ability of BTV detection; whereas the most sensitive mode of detection by IPT was with MAbs.

Bluetongue (BT) is a viral disease of ruminants and is caused by different serotypes of bluetongue virus (BTV), which is transmitted by several species of Culicoides midges. The disease is endemic in tropical areas, and incursions have been observed in some of the temperate areas. Twenty-seven recognized serotypes of BTV have been reported so far. Some serotype viruses have been shown to circulate in certain geographical areas. BTV-24 has been reported from Africa, the Mediterranean and the Americas, whereas it is exotic to Australasia. Here, we report isolation of BTV-24 from India and show that it has high sequence homology in genome segment 2 with other Western isolates of BTV-24. Entry of this serotype into Australasian region is a cause of concern.

Aim: The current study was designed to understand the infection kinetics and antibody responses of major circulating serotypes of bluetongue virus (BTV) in India, i.e., BTV-4 and BTV-16 through experimental infection and superinfection of Deccani sheep, a popular breed of sheep found in the southern states of India. Materials and Methods: Experimental infection with 106 TCID50/ml BTV-4 was followed by superinfection with BTV-16 and vice versa. Along with observing for clinical signs and immunological responses in the experimentally infected sheep, the effect of infection of one specific serotype on the outcome of superinfection with a different serotype was also studied. Results: Certain interesting findings have been made in the course of experimental infection, such as prominent signs of infection in BTV-4 infection, mild or no clinical signs in BTV-16-infected and superinfected animals, and non-seroconversion of one of the BTV-16-superinfected animals. In addition, BTV was isolated from infected sheep in all the experimental conditions except BTV-16 superinfection. Furthermore, it was observed that immune response in the form of type-specific antibodies was slower with BTV-16 superinfection. Conclusion: Superinfection of a sheep with more than one serotype of BTV is a common phenomenon in BT endemic countries like India. Such situation was replicated in an experimental infection in the current study, and the findings to our knowledge are first of a kind and are likely to aid in unfolding the newer aspects of BTV pathogenesis and virulence.

Aim: The present study was designed to standardize real-time polymerase chain reaction (PCR) for detecting the bluetongue virus from blood samples of sheep collected during outbreaks of bluetongue disease in the year 2014 in Andhra Pradesh and Telangana states of India. Materials and Methods: A 10-fold serial dilution of Plasmid PUC59 with bluetongue virus (BTV) NS3 insert was used to plot the standard curve. BHK-21 and KC cells were used for in vitro propagation of virus BTV-9 at a TCID50/ml of 105 ml and RNA was isolated by the Trizol method. Both reverse transcription -PCR and real-time PCR using TaqMan probe were carried out with RNA extracted from virus-spiked culture medium and blood to compare the sensitivity by means of finding out the limit of detection (LoD). The results were verified by inoculating the detected and undetected dilutions onto cell cultures with further cytological (cytopathic effect) and molecular confirmation (by BTV-NS1 group-specific PCR). The standardized technique was then applied to field samples (blood) for detecting BTV. Results: The slope of the standard curve obtained was -3.23, and the efficiency was 103%. The LoD with RT-PCR was 8.269Ex103 number of copies of plasmid, whereas it was 13 with real-time PCR for plasmid dilutions. Similarly, LoD was determined for virus-spiked culture medium, and blood with both the types of PCR and the values were 103 TCID 50/ml and 104 TCID 50/ml with RT-PCR and 10° TCID 50/ml and 102 TCID 50/ml with real-time PCR, respectively. The standardized technique was applied to blood samples collected from BTV suspected animals; 10 among 20 samples were found positive with Cq values ranging from 27 to 39. The Cq value exhibiting samples were further processed in cell cultures and were confirmed to be BT positive. Likewise, Cq undetected samples on processing in cell cultures turned out to be BTV negative. Conclusion: Real-time PCR was found to be a very sensitive as well as reliable method to detect BTV present in different types of samples, including blood samples collected from BTV-infected sheep, compared to RT-PCR. The LoD of BTV is likely influenced by sample type, possibly by the interference by the other components present in the sample.

Bluetongue (BT) is a Culicoides-borne disease caused by several serotypes of bluetongue virus (BTV). Similar to other insect-borne viral diseases, distribution of BT is limited to distribution of Culicoides species competent to transmit BTV. In the tropics, vector activity is almost year long, and hence, the disease is endemic, with the circulation of several serotypes of BTV, whereas in temperate areas, seasonal incursions of a limited number of serotypes of BTV from neighbouring tropical areas are observed. Although BTV is endemic in all the three major tropical regions (parts of Africa, America and Asia) of the world, the distribution of serotypes is not alike. Apart from serological diversity, geography-based diversity of BTV genome has been observed, and this is the basis for proposal of topotypes. However, evolution of these topotypes is not well understood. In this study, we report the isolation and characterization of several BTV-4 isolates from India. These isolates are distinct from BTV-4 isolates from other geographical regions. Analysis of available BTV seg-2 sequences indicated that the Australasian BTV-4 diverged from African viruses around 3,500 years ago, whereas the American viruses diverged relatively recently (1,684 CE). Unlike Australasia and America, BTV-4 strains of the Mediterranean area evolved through several independent incursions. We speculate that independent evolution of BTV in different geographical areas over long periods of time might have led to the diversity observed in the current virus population.

Bluetongue (BT) is an emerging and re-emerging vector-borne viral disease of domestic and wild ruminants, caused by viruses classified within the species bluetongue virus (BTV), genus Orbivirus, family Reoviridae. There are 27 recognized serotypes of BTV (with two more recently discovered ‘putative’ serotypes) as well as multiple geographic variants (topotypes) and many different strains and genotypes, most of which are transmitted between their vertebrate hosts by certain ‘vector-competent’ biting midges of the genus Culicoides. Bluetongue is an economically important transboundary disease that is listed by the World Organisation for Animal Health, reflecting the ability of BTV to (a) infect all ruminants, including important domesticated species; (b) cause severe disease, with high fatality rates in sheep and certain species of deer; and (c) cause large economic losses due to fatalities, reduced productivity and reproductive performance, animal movement and trade restrictions and surveillance and control strategies (including vaccination). The plurality of BTV serotypes and strains, the involvement of multiple host and vector species and the potential for (re)introduction of exotic BTV strains make the control and eradication of BT very complex and difficult to achieve. In this chapter, we review the current understanding of BTV biology, bluetongue epidemiology, pathogenesis and pathology, laboratory techniques to diagnose the disease and identify the virus, experimental animal models to study the disease and to evaluate vaccines and methods for the control and/or eradication of bluetongue.

Bluetongue (BT) is an insectborne endemic disease in India. Although infections are observed in domestic and wild ruminants, the clinical disease and mortality are observed only in sheep, especially in the southern states of the country. The difference in disease patterns in different parts of the country could be due to varied climatic conditions, sheep population density and susceptibility of the sheep breeds to BT. Over the five decades after the first report of BT in 1964, most of the known serotypes of bluetongue virus (BTV) have been reported from India either by virus isolation or by detection of serotype-specific antibodies. There have been no structured longitudinal studies to identify the circulating serotypes throughout the country. At least ten serotypes were isolated between 1967 and 2000 (BTV-1-4, 6, 9, 16-18, 23). Since 2001, the All-India Network Programme on Bluetongue and other laboratories have isolated eight different serotypes (BTV-1-3, 9, 10, 12, 16, 21). Genetic analysis of these viruses has revealed that some of them vary substantially from reference viruses, and some show high sequence identity with modified live virus vaccines used in different parts of the world. These observations have highlighted the need to develop diagnostic capabilities, especially as BT outbreaks are still declared based on clinical signs. Although virus isolation and serotyping are the gold standards, rapid methods based on the detection of viral nucleic acid may be more suitable for India. The epidemiological investigations also have implications for vaccine design. Although only a handful serotypes may be involved in causing outbreaks every year, the combination of serotypes may change from year to year. For effective control of BT in India, it may be pertinent to introduce sentinel and vector traps systems for identification of the circulating serotypes and to evaluate herd immunity against different serotypes, so that relevant strains can be included in vaccine formulations.

Objectives Lipocalin-2 (Lcn2) is an innate immune protein expressed by a variety of cells and is highly upregulated during several pathological conditions including immune-complex (IC) mediated inflammatory/autoimmune disorders. However, the function of Lcn2 during IC-mediated inflammation is largely unknown. Therefore our objective was to investigate the role of Lcn2 in IC-mediated diseases.

Tumor stromal alternatively activated macrophages are important determinants of antitumor T lymphocyte responses, intratumoral neovascularization, and metastatic dissemination. Our recent efforts to investigate the mechanism of macrophage migration inhibitory factor (MIF) in antagonizing antimelanoma immune responses reveal that macrophage-derived MIF participates in macrophage alternative activation in melanoma-bearing mice. Both peripheral and tumor-associated macrophages (TAMs) isolated from melanoma bearing MIF-deficient mice display elevated proinflammatory cytokine expression and reduced anti-inflammatory, immunosuppressive, and proangiogenic gene products compared with macrophages from tumor-bearing MIF wild-type mice. Moreover, TAMs and myeloid-derived suppressor cells from MIF-deficient mice exhibit reduced T lymphocyte immunosuppressive activities compared with those from their wild-type littermates. Corresponding with reduced tumor immunosuppression and neo-angiogenic potential by TAMs, MIF deficiency confers protection against transplantable s.c. melanoma outgrowth and melanoma lung metastatic colonization. Finally, we report for the first time, to our knowledge, that our previously discovered MIF small molecule antagonist, 4-iodo-6-phenylpyrimidine, recapitulates MIF deficiency in vitro and in vivo, and attenuates tumor-polarized macrophage alternative activation, immunosuppression, neoangiogenesis, and melanoma tumor outgrowth. These studies describe an important functional contribution by MIF to TAM alternative activation and provide justification for immunotherapeutic targeting of MIF in melanoma patients.

Bluetongue is endemic in India and has been reported from most Indian states. Of late, the clinical disease is most frequently seen in the states of Andhra Pradesh, Telangana (erstwhile Andhra Pradesh state), Tamil Nadu and Karnataka. Our analysis of diagnostic samples from bluetongue outbreaks during 2010–2011 from the state of Karnataka identified bluetongue virus (BTV) serotype 5 (BTV-5) for the first time in India. One of the diagnostic samples (CH1) and subsequent virus isolate (IND2010/02) contained both BTV-2 and BTV-5. Segment 2 (seg-2) sequence data (400 bp: nucleotides 2538–2921) for IND2010/02-BTV5, showed 94.3% nucleotide identity to BTV-5 from South Africa (Accession no. AJ585126), confirming the virus serotype and also indicating that Seg-2 was derived from a Western topotype, which is in contrast to serotype 2, that belongs to an Eastern topotype. BTV-5 has been recently reported from Africa, China, French islands and the Americas. Although the exact source of the Indian BTV-5 isolate is still to be confirmed, recent identification of additional exotic serotypes in India is of real concern and might add to the severity of the disease seen in these outbreaks.

Southern Indian isolate IND1994/01 of bluetongue virus serotype 2 (BTV-2), from the Orbivirus Reference Collection at the Pirbright Institute ( http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/btv-2.htm#IND1994/01 ), was sequenced. Its genome segment 6 (Seg-6) [encoding VP5(OCP2)] is identical to that of the Indian BTV-1 isolate (IND2003/05), while Seg-5 and Seg-9 are closely related to isolates from South Africa and the United States, respectively.

Since 1998 there have been significant changes in the global distribution of bluetongue virus (BTV). Ten previously exotic BTV serotypes have been detected in Europe, causing severe disease outbreaks in naïve ruminant populations. Previously exotic BTV serotypes were also identified in the USA, Israel, Australia and India. BTV is transmitted by biting midges (Culicoides spp.) and changes in the distribution of vector species, climate change, increased international travel and trade are thought to have contributed to these events. Thirteen BTV serotypes have been isolated in India since first reports of the disease in the country during 1964. Efficient methods for preparation of viral dsRNA and cDNA synthesis, have facilitated full-genome sequencing of BTV strains from the region. These studies introduce a new approach for BTV characterization, based on full-genome sequencing and phylogenetic analyses, facilitating the identification of BTV serotype, topotype and reassortant strains. Phylogenetic analyses show that most of the equivalent genome-segments of Indian BTV strains are closely related, clustering within a major eastern BTV ‘topotype’. However, genome-segment 5 (Seg-5) encoding NS1, from multiple post 1982 Indian isolates, originated from a western BTV topotype. All ten genome-segments of BTV-2 isolates (IND2003/01, IND2003/02 and IND2003/03) are closely related (>99% identity) to a South African BTV-2 vaccine-strain (western topotype). Similarly BTV-10 isolates (IND2003/06; IND2005/04) show >99% identity in all genome segments, to the prototype BTV-10 (CA-8) strain from the USA. These data suggest repeated introductions of western BTV field and/or vaccine-strains into India, potentially linked to animal or vector-insect movements, or unauthorised use of ‘live’ South African or American BTV-vaccines in the country. The data presented will help improve nucleic acid based diagnostics for Indian serotypes/topotypes, as part of control strategies.

Summary Bluetongue (BT) is an arthropod-borne viral disease mostly of sheep. Bluetongue virus (BTV) is a segmented double-stranded RNA virus belonging to the genus Orbivirus of family Reoviridae and is transmitted by midges belonging to Culicoides spp. The disease is endemic in the tropics and subtropics, and the incidence is high in southern India. Twenty-six serotypes of BTV have been reported worldwide. Although most of the serotypes have been reported in India, information regarding currently circulating serotypes is essential to develop control programs. Both serological assays and nucleic acid-based assays have been used for typing BTV. Segment 2, which codes for the outer capsid protein VP2, is the target for PCR-based typing; however, the VP2 sequence diversity among viruses belonging to the same serotype but isolated from different geographical areas makes it essential to develop geographical based reagents. In this study, reverse transcription PCR was developed based on sequences of Indian isolates of BTV (serotypes 1, 2, 9, 10, 12, 16, 21 and 23), and this was applied to type 52 isolates obtained during the last decade. It was found that multiple serotypes circulate, with involvement of more than one serotype infecting individual animals and herds over a period in a given area. Detection of circulating serotypes and estimation of herd immunity against different serotypes of BTV may provide important information for predicting the distribution of these serotypes and inclusion of serotypes in vaccines.

A bacterial species typically includes heterogeneous collections of genetically diverse isolates. How genetic diversity within bacterial populations influences the clinical outcome of infection remains mostly indeterminate. In part, this is due to a lack of technologies that can enable contemporaneous systems-level interrogation of host-pathogen interaction using multiple, genetically diverse bacterial strains. This chapter presents a prototype microfluidic cell array (MCA) that allows simultaneous elucidation of molecular events during infection of human cells in a semi-automated fashion. It shows that infection of human cells with up to sixteen genetically diverse bacterial isolates can be studied simultaneously. The versatility of MCAs is enhanced by incorporation of a gradient generator that allows interrogation of host-pathogen interaction under four different concentrations of any given environmental variable at the same time. Availability of high throughput MCAs should foster studies that can determine how differences in bacterial gene pools and concentration-dependent environmental variables affect the outcome of host-pathogen interaction.