Rapid detection of porcine sapelovirus by reverse transcription recombinase polymerase amplification assay.

BACKGROUND: Porcine Sapelovirus (PSV) infection has been confirmed in pigs worldwide, mostly asymptomatic, but in some cases, it can lead to significant issues in the gastrointestinal, respiratory, neurological, or reproductive systems. PSV is considered an emerging pathogen of porcine species. Recombinase polymerase amplification (RPA) is a simple and fast isothermal technique that uses three enzymes for amplification without the use of any sophisticated equipment. METHODS AND RESULTS: The reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed and optimized for field based detection of PSV. The assay was developed by targeting 5´UTR region of PSV genome and optimized for reaction time, temperature, primer and MgOAc concentration. The analytical sensitivity and specificity of assay was determined. The assay was evaluated on 85 porcine faecal samples collected from field. In addition to conventional format, this assay was also optimized for visual dye-based detection format and lateral flow strips based detection (in combination with probe). The developed assay works at constant temperature of 35 °C for 20 min with forward primer concentration 20pm, reverse primer concentration 10pm and MgOAc concentration of 14mM. This assay is highly sensitive and detects up to 28 copies of viral nucleic acid both in the conventional as well as in fluorescent dye based detection format. Using the newly developed assay 21 samples out of 85 samples were found positive, showing positivity rate of 24.7%. The positivity rate of RT-RPA assay corroborated with the gold standard RT-PCR test. CONCLUSIONS: This study presented the development of an RT-RPA isothermal assay for rapid and accurate detection of PSV. The assay is highly sensitive, specific, works at a low and constant temperature, does not require any high-end instrument and can be a potential diagnostics tool for pen-side testing of PSV in the field conditions. The newly developed RT-RPA assay could successfully detect PSV circulating in swine population of Haryana, India. This is a first report of this kind from the region.

Acaricide resistance status of deltamethrin and coumaphos in Hyalomma anatolicum ticks collected from different districts of Haryana.

To study the acaricide resistance status and possible mechanisms of action in conferring resistance to commonly used acaricides (deltamethrin and coumaphos), Hyalomma anatolicum ticks were collected from 6 dairy farms of Hisar and Charkhi Dadri districts of Haryana. By using standard larval packet test, H. anatolicum tick larvae of Charkhi Dadri isolates were found to be susceptible (100% mortality) to both the acaricides. Level-I resistance against coumaphos was recorded from four isolates, whereas, level-II was observed in only one isolate, collected from Hisar. One isolates (Kaimri) from Hisar also showed level-I resistance against deltamethrin. Biochemically, the ticks having higher values of resistance factor (RF) against coumaphos were found to possess increased enzymatic activity of α-esterase, ß-esterase, glutathione-S-transferase (GST) and mono-oxygenase enzymes, whereas, the monoamine oxidase did not show any constant trend. However, the RF showed a statistical significant correlation with GST only. Native PAGE analysis of H. anatolicum ticks revealed the presence of nine types of esterases (EST-1 h to EST-9 h) by using napthyl acetate as substrate. In the inhibitory assay, esterases were found to be inhibited by PMSF, indicating the presence of serine residue at catalytic triad. The partial cds of carboxylesterase and domain II of sodium channel genes were sequenced to determine any proposed mutations in resistant isolates of H. anatolicum ticks, however, no mutations were observed in either gene, indicating that increased expression of detoxification enzymes as a possible mechanism for resistance development, in the current study.

Emergence of Mycobacterium orygis-Associated Tuberculosis in Wild Ruminants, India.

Tuberculosis caused by Mycobacterium orygis was detected in 2 spotted deer from a wildlife sanctuary in western India and an Indian bison from a national park in central India. Nationwide surveillance is urgently required to clarify the epidemiology of the Mycobacterium tuberculosis complex at the human-livestock-wildlife interface.

Apoptin NLS2 homodimerization strategy for improved antibacterial activity and bio-stability.

The emergence of antibiotic resistance prompts exploration of viable antimicrobial peptides (AMPs) designs. The present study explores the antimicrobial prospects of Apoptin nuclear localization sequence (NLS2)-derived peptide ANLP (PRPRTAKRRIRL). Further, we examined the utility of the NLS dimerization strategy for improvement in antimicrobial activity and sustained bio-stability of AMPs. Initially, the antimicrobial potential of ANLP using antimicrobial peptide databases was analyzed. Then, ANLP along with its two homodimer variants namely ANLP-K1 and ANLP-K2 were synthesized and evaluated for antimicrobial activity against Escherichia coli and Salmonella. Among three AMPs, ANLP-K2 showed efficient antibacterial activity with 12 µM minimum inhibitory concentration (MIC). Slow degradation of ANLP-K1 (26.48%) and ANLP-K2 (13.21%) compared with linear ANLP (52.33%) at 480 min in serum stability assay indicates improved bio-stability of dimeric peptides. The AMPs presented no cytotoxicity in Vero cells. Dye penetration assays confirmed the membrane interacting nature of AMPs. The zeta potential analysis reveals effective charge neutralization of both lipopolysaccharide (LPS) and bacterial cells by dimeric AMPs. The dimeric AMPs on scanning electron microscopy studies showed multiple pore formations on the bacterial surface. Collectively, proposed Lysine scaffold dimerization of Apoptin NLS2 strategy resulted in enhancing antibacterial activity, bio-stability, and could be effective in neutralizing the off-target effect of LPS. In conclusion, these results suggest that nuclear localization sequence with a modified dimeric approach could represent a rich source of template for designing future antimicrobial peptides.

Prevalence of porcine viral respiratory diseases in India.

The pig industry is growing rapidly in India and contributes a major share of growth in the livestock sector. Over the last few years, there is a gradual increase in the adoption of pigs for production by economically weaker sections of the country. However, this production is affected by many respiratory diseases which are responsible for significant economic loss. The occurrence and impact of these diseases are still under-documented. The four important pathogens including porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza A viruses (SIV) and classical swine fever virus (CSFV) are documented here. These diseases are highly devastating in nature and frequent outbreaks have been reported from different parts of the country. The rapid and specific diagnosis, effective prevention and control measures are required for the eradication of these diseases which is urgently required for the growth of the pig industry. This review highlights the prevalence, epidemiology, diagnostics and information gaps on important respiratory viral pathogens of pigs reported from different parts of India. This review also emphasizes the importance of these viral diseases and the urgent need to develop vaccines and effective measures for the eradication of these diseases.

Development of real-time RT-PCR systems for detection and quantitation of bovine enteric viral pathogens.

The enteric viruses in animals are responsible for severe and devastating losses to the livestock owners with a profound negative impact on animal, health, welfare, and productivity. These viruses are usually transmitted via the feco-oral route and primarily infect the digestive tract of the humans, bovines and different mammals as well as birds. Some of the important enteric viruses in ruminants are: Rotavirus A (RVA), Peste des petits virus (PPRV), Norovirus (NV), Bovine corona virus (BoCV) and Bluetongue virus (BTV). In the present study, sensitive, specific and reliable TaqMan probe-based RT-qPCRs were developed and standardized for the rapid detection and quantification of enteric viruses from fecal samples. The assays result in efficient amplification of the RVA, BTV and BoCV RNA with a limit of detection (LoD) of 5, 5 and 4 copies, respectively, which is 1000 times more sensitive than the traditional gel-based RT-PCR. The reproducibility of each assay was satisfactory, thus allowing for a sensitive and accurate measurement of the viral RNA load in clinical samples. In conclusion, real time PCR developed for these viruses are highly specific and sensitive technique for the detection of diarrheic viral pathogens of cattle and buffalo.

VP2 Gene-Based Molecular Evolutionary Patterns of Major Circulating Bluetongue Virus Serotypes Isolated during 2014-2018 from Telangana and Andhra Pradesh States of India.

INTRODUCTION: Bluetongue disease is an economically important viral disease of livestock caused by bluetongue virus (BTV) having multiple serotypes. It belongs to the genus Orbivirus of family Reoviridae and subfamily Sedoreovirinae. The genome of BTV is 10 segmented dsRNA that codes for 7 structural and 4 nonstructural proteins, of which VP2 was reported to be serotype-specific and a major antigenic determinant. OBJECTIVE: It is important to know the circulating serotypes in a particular geographical location for effective control of the disease. The present study unravels the molecular evolution of the circulating BTV serotypes during 2014-2018 in Telangana and Andhra Pradesh states of India. METHODS: Multiple sequence alignment with available BTV serotypes in GenBank and phylogenetic analysis were performed for the partial VP2 sequences of major circulating BTV serotypes during the study period. RESULTS: The multiple sequence alignment of circulating serotypes with respective reference isolates revealed variations in antigenic VP2. The phylogenetic analysis revealed that the major circulating serotypes were grouped into eastern topotypes (BTV-1, BTV-2, BTV-4, and BTV-16) and Western topotypes (BTV-5, BTV-12, and BTV-24). CONCLUSION: Our study strengthens the need for development of an effective vaccine, which can induce the immune response for a range of serotypes within and in between topotypes.

Identification of circulatory microRNA based biomarkers for early pregnancy diagnosis in buffalo.

Introduction: The most crucial factor in improving animal reproduction efficiency is early pregnancy diagnosis. Early diagnosis not only reduces the time interval between two calvings but also aids farmers in identifying open animals, thereby preventing significant milk production losses. Therefore, the objective of this study was to discover circulatory miRNAs that would be useful for early pregnancy diagnosis in buffalo. Material and methods: Blood samples were taken on 0, 6th, 12th, and 18th day after artificial insemination from pregnant animals (n = 30) and non-pregnant animals (n = 20). During these stages of pregnancy, total RNA was extracted, and a small RNA library was subsequently generated and sequenced on the Illumina platform. Subsequently, Real-time PCR was used to validate the findings. Results and discussion: There were 4,022 miRNAs found during the pregnancy, with 15 of those lacking sequences and 4,007 having sequences already in the database. From the beginning of pregnancy until the 18th day, 25 of these miRNAs showed a substantial shift in expression levels in the maternal blood, with a change more than two logs. Furthermore, based on qPCR results, 19 miRNAs were found to be more abundant in pregnant animals than in non-pregnant animals. We used target prediction analysis to learn how maternally expressed miRNAs relate to fetal-maternal communication. In conclusion, miRNA based biomarkers that could be associated with the diagnosis of pregnancy were identified including miR-181a and miR-486 highly upregulated on the 18th day of pregnancy. This study also provides a comprehensive profile of the entire miRNA population in maternal buffalo blood during the early stages of pregnancy.

Development of a multiplexed Luminex assay for simultaneous detection of enteric viruses in cattle.

Viral and bacterial gastroenteritis and diarrhea have long been a problem in livestock with devastating effects on animal health and production causing a heavy financial burden on producers. Therefore, the bead-based multiplex detection assay was created for simultaneous detection of three livestock viral diarrheic agents viz. bovine rotavirus (BRV), bovine coronavirus (BCoV) and bluetongue virus (BTV). The primers and probes for triplex MAGPIX assay for simultaneous detection of three enteric viruses were designed and the assay was optimized for hybridization temperature, primer-probe and bead concentrations. The newly developed MAGPIX assay was used to determine the prevalence of these diarrhea-associated viruses by testing 200 fecal samples collected from Haryana state of India during 2018-2019. The limit of detection of the developed triplex assay was 1 × 105, 1 × 104, and 1 × 105 RNA copies for BRV, BCoV, and BTV, respectively, being lower than the reverse transcription-quantitative polymerase chain reaction (RT-qPCR). However, it was higher than the conventional RT-PCR, showing it to be more sensitive. The newly developed MAGPIX assay was a rapid, cost-effective and high throughput diagnostic tool for identification of three major entero-pathogenic diarrhea associated viruses, either alone or in tandem, with the aim to prevent and control viral diarrhea in animals.

Complete genome sequence analysis of a reference strain of bluetongue virus serotype 16.

The entire genome of the reference strain of bluetongue virus (BTV) serotype 16 (strain RSArrrr/16) was sequenced (a total of 23,518 base pairs). The virus was obtained from the Orbivirus Reference Collection (ORC) at IAH, Pirbright, United Kingdom. The virus strain, which was previously provided by the Onderstepoort Veterinary Research Institute in South Africa, was originally isolated from the Indian subcontinent (Hazara, West Pakistan) in 1960. Previous phylogenetic comparisons show that BTV RNA sequences cluster according to the geographic origins of the virus isolate/lineage, identifying distinct BTV topotypes. Sequence comparisons of segments Seg-1 to Seg-10 show that RSArrrr/16 belongs to the major eastern topotype of BTV (BTV-16e) and can be regarded as a reference strain of BTV-16e for phylogenetic and molecular epidemiology studies. All 10 genome segments of RSArrrr/16 group closely with the vaccine strain of BTV-16 (RSAvvvv/16) that was derived from it, as well as those recently published for a Chinese isolate of BTV-16 (>99% nucleotide identity), suggesting a very recent common ancestry for all three viruses.

Genome sequence of a reassortant strain of bluetongue virus serotype 23 from western India.

The full genome sequence (19,177 bp) of an Indian strain (IND1988/02) of bluetongue virus (BTV) serotype 23 was determined. This virus was isolated from a sheep that had been killed during a severe bluetongue outbreak that occurred in Rahuri, Maharashtra State, western India, in 1988. Phylogenetic analyses of these data demonstrate that most of the genome segments from IND1988/02 belong to the major "eastern" BTV topotype. However, genome segment 5 belongs to the major "western" BTV topotype, demonstrating that IND1988/02 is a reassortant. This may help to explain the increased virulence that was seen during this outbreak in 1988. Genome segment 5 of IND1988/02 shows >99% sequence identity with some other BTV isolates from India (e.g., BTV-3 IND2003/08), providing further evidence of the existence and circulation of reassortant strains on the subcontinent.

The genome sequence of a reassortant bluetongue virus serotype 3 from India.

All 10 genome segments (Seg-1 to 10-a total of 19,188 bp) were sequenced from a strain of bluetongue virus serotype 3 (BTV-3) from India (strain IND2003/08). Sequence comparisons showed that nine of the genome segments from this virus group with other eastern topotype strains. Genome Seg-2 and Seg-6 group with eastern BTV-3 strains from Japan. However, Seg-5 (the NS1 gene) from IND2003/08 belongs to a western lineage, demonstrating that IND2003/08 is a reassortant between eastern and western topotype bluetongue viruses. This confirms that western BTV strains have been imported and are circulating within the subcontinent.

The genome sequence of bluetongue virus type 2 from India: evidence for reassortment between eastern and western topotype field strains.

Bluetongue virus type 2, isolated in India in 1982 (IND1982/01), was obtained from the Orbivirus Reference Collection at IAH Pirbright (http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/btv-2.htm#IND1982/01). Full genome sequencing and phylogenetic analyses show that IND1982/01 is a reassortant virus containing genome segments derived from both eastern and western topotypes. These data will help to identify further reassortment events involving this or other virus lineages in the subcontinent.

The genome sequence of bluetongue virus type 10 from India: evidence for circulation of a western topotype vaccine strain.

Bluetongue virus is the type species of the genus Orbivirus in the family Reoviridae. We report the first complete genome sequence of an isolate (IND2004/01) of bluetongue virus serotype 10 (BTV-10) from Andhra Pradesh, India. This isolate, which is stored in the Orbivirus Reference Collection (ORC) at IAH Pirbright, shows >99% nucleotide identity in all 10 genome segments with a vaccine strain of BTV-10 from the United States.

Complete genome sequence of an isolate of bluetongue virus serotype 2, demonstrating circulation of a Western topotype in southern India.

Bluetongue virus serotype 2 (IND2003/02) was isolated in Tiruneveli City, Tamil Nadu State, India, and is stored in the Orbivirus Reference Collection at the Institute for Animal Health, Pirbright, United Kingdom. The entire genome of this isolate was sequenced, showing that it is composed of a total of 19,203 bp (all 10 genome segments). This is the first report of the entire genome sequence of a western strain of BTV-2 isolated in India, indicating that this virus has been introduced and is circulating in the region. These data will aid in the development of diagnostics and molecular epidemiology studies of BTV-2 in the subcontinent.

Full genome sequence of bluetongue virus serotype 1 from India.

We report the full-genome sequence of an Indian isolate of bluetongue virus serotype 1 (BTV-1), strain IND1992/01. This is the first report of the entire genome sequence (Seg-1 to Seg-10) of an Eastern (e) strain of BTV-1. These sequence data provide a reference for BTV-1e that will help to define the phylogenetic relationships and geographic origins of distinct Indian lineages of BTV-1 as well as their relationships with other BTV strains from around the world. The availability of data for all 10 genome segments of this strain will also help to identify reassortment events involving this and other virus lineages.

Temporal analysis of circulating nucleic acid in early days of pregnancy in buffalo.

Improving reproductive efficiency in livestock relies mainly on the ability to detect pregnancy quickly and accurately. Recently, circulating nucleic acids (CNAs) have been exploited for prenatal diagnosis in humans and animals. In the current investigation, serum samples were collected from pregnant animals (n = 30) and non-pregnant animals (n = 20) on 0th, 6th, 12th, and 18th day post artificial insemination. Total DNA was isolated from these serum samples. Two CNA tags (Bov-B and ART2A) derived from repetitive sections of the bovine genome were amplified using DNA extracted from serum samples. The expression analysis of these CNAs was done using real-time polymerase chain reaction assay, and copy number of each tag was calculated in pregnant and non-pregnant animals. The average number of copies of Art2A increased approximately threefold (P < 0.01) from day zero of pregnancy (7,000 copies) to the day 18 of pregnancy (> 21,000). Similarly, BovB levels in the pregnant group increased significantly (approximately 2.9-fold) from day zero (93,900 copies) till day 18 (> 2, 72,310 copies) (P < 0.01). There was no significant change observed on the 6th and 12th day of pregnancy and on the 18th day in the non-pregnant animals. In conclusion, based on these findings, the defined cut-off value can distinguish between pregnant and non-pregnant animals with a sensitivity of nearly 80% and specificity of nearly 70%. It is possible to employ these two CNA tags as biomarkers for early detection of pregnancy in buffaloes.

High throughput Luminex beads based multiplex assay for identification of six major bacterial pathogens of mastitis in dairy animals.

Introduction: Bovine mastitis is caused by over 150 different microorganisms. Specific identification and quantification of multiple bacteria in a single milk sample becomes essential for rapid intervention. Methods: In the present study a Luminex beads based multiplex assay emphasizing on the precise identification of six major bacterial pathogens of mastitis was developed. Assay was developed in two triplex sets, triplex 1 comprised of Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis while triplex 2 consisted of Staphylococcus aureus, E. coli and Klebsiella pneumoniae. Results: The analytical sensitivity was 10 6 copies per reaction mixture for all the six bacteria. A 100% analytical specificity was observed for simultaneous detection of these bacteria. Clinical milk samples from 100 bovine quarters were tested for validation. Discussion: The analytical sensitivity was similar to the findings reported earlier in real time PCR multiplex assay targeting the DNA of the 11 most common bacterial species or groups in mastitis. The analytical specificity of the optimized assay was 100% similar to reported earlier for simultaneous detection of Mycoplasma spp. and for seven entric viruses of humans.The developed assay indicates a concept proof of a rapid, cost effective high throughput diagnostic tool for identification of major bacteria causing mastitis.

Co-infection of bluetongue virus serotypes 12 and 16 in sheep from Haryana, India.

World Organization for Animal Health has listed bluetongue (BT) under notifiable diseases. The BT is an arboviral infectious disease of domestic and wild ruminants caused by the bluetongue virus (BTV). Southern states of India had remained the point of attention for BT since first presence in 1964 in Maharashtra. Recently, northern states of India have also been reported positive for BTV in small ruminants. The present study reported the dual infection of BTV serotypes, BTV-12 and -16 in sheep population from Sirsa district of Haryana in the year 2016. After detection and serotyping with Seg-2 specific real time polymerase chain reaction (PCR), the Seg-2 and Seg-6 of BTV were PCR amplified and sequenced. On phylogenetic analysis it was detected to be clustered in nucleotype G and nucleotype B specific for BTV-12 and BTV-16, respectively. This was the first report of BTV-16 from Haryana. The results signified the co-infection of two different serotypes in an animal from a single outbreak.

Prevalence of Newcastle Disease Virus in Wild and Migratory Birds in Haryana, India.

Newcastle disease virus (NDV) can infect approximately 250 avian species and causes highly contagious Newcastle disease (ND) in domestic poultry, leading to huge economic losses. There are three different pathotypes of NDV, i.e., lentogenic, mesogenic, and velogenic. Wild resident (wild) and migratory birds are natural reservoirs of NDV and are believed to play a key role in transmitting the virus to domestic poultry. The present study was conducted to determine the prevalence of NDV in wild and migratory birds in the state of Haryana, India, during two migratory seasons (2018-19 and 2019-20). In total 1379 samples (1368 choanal swabs and 11 tissue samples) were collected from live (n = 1368) or dead birds (n = 4) belonging to 53 different avian species. These samples belonged to apparently healthy (n = 1338), sick (n = 30), and dead (n = 4) birds. All samples were tested for NDV by real-time reverse transcription-PCR using M gene specific primers and probe. Of the 1379 samples, 23 samples from wild birds [Columba livia domestica (n = 12, 52.17%), Pavo cristatus (n = 9, 39.13%), and Psittaciformes (n = 2, 8.69%)] were found positive for NDV. Only one of the 23 samples (from P. cristatus) was positive for F gene, indicating it to be a mesogenic/velogenic strain. These results indicate that both lentogenic and velogenic strains of NDV are circulating in wild birds in Haryana and that further studies are needed to characterize NDV strains from wild/migratory birds and domestic poultry to determine the extent of virus transmission among these populations. This study considers the disease transmission risk from domestic pigeons and parrots to commercial poultry and vice versa, and the results emphasize the need for strict biosecurity strategies to protect commercial poultry in the region.

Prevalencia del virus de la enfermedad de Newcastle en aves silvestres y migratorias en Haryana, India. El virus de la enfermedad de Newcastle (NDV) puede infectar aproximadamente a 250 especies de aves y causa la enfermedad de Newcastle (ND) altamente contagiosa en la avicultura comercial, lo que genera enormes pérdidas económicas. Hay tres patotipos diferentes del virus de Newcastle, que incluyen, lentogénico, mesogénico y velogénico. Las aves silvestres residentes (silvestres) y migratorias son reservorios naturales del virus de Newcastle y se cree que desempeñan un papel clave en la transmisión del virus a las aves domésticas comerciales. El presente estudio se realizó para determinar la prevalencia del virus de Newcastle en aves silvestres y migratorias en el estado de Haryana, India, durante dos temporadas migratorias (2018-19 y 2019-20). En total, se recolectaron 1379 muestras (1368 hisopos coanales y 11 muestras de tejido) de aves vivas (n = 1368) o muertas (n = 4) pertenecientes a 53 especies de aves diferentes. Estas muestras pertenecían a aves aparentemente sanas (n = 1338), enfermas (n = 30) y muertas (n = 4). Todas las muestras se analizaron para detectar al virus de Newcastle mediante transcripción reversa y PCR en tiempo real utilizando iniciadores y una sonda específicos del gene M. De las 1379 muestras, 23 muestras de aves silvestres [Columba livia domestica (n = 12, 52.17 %), Pavo cristatus (n = 9, 39.13 %) y Psittaciformes (n = 2, 8.69 %)] resultaron positivas para el virus de Newcastle. Solo una de las 23 muestras (de P. cristatus) fue positiva para el gene F, lo que indica que se trata de una cepa mesogénica/velogénica. Estos resultados indican que tanto las cepas lentogénicas como las velogénicas del virus de Newcastle están circulando en las aves silvestres de Haryana y que se necesitan más estudios para caracterizar las cepas del virus de Newcastle de las aves silvestres/migratorias y de las aves domésticas para determinar el alcance de la transmisión del virus entre estas poblaciones. Este estudio considera el riesgo de transmisión de la enfermedad de las palomasdomésticas y loros a las aves comerciales y viceversa, y los resultados enfatizan la necesidad de estrategias estrictas de bioseguridad para proteger las aves comerciales en la región.