Isolation and genetic analysis of Porcine circovirus 2 in southern India evidences high circulation of Porcine circovirus 2d genotype.

BACKGROUND: Porcine circovirus 2 is globally noted swine pathogen with multiple genotypes associated with vast clinical and subclinical outcomes. This study aimed to isolate and characterize PCV2 genotypes circulating in southern states of India. METHODS AND RESULTS: A total of 434 field samples comprising of serum (n = 273), tissues (n = 109) and swabs (n = 52) collected from swine during 2019 to 2021 from southern states of India were screened for PCV2 by specific polymerase chain reaction (PCR) assay. Molecular prevalence of PCV2 in southern India was found to be 12.21% (n = 53). All the 53 PCV2 positive samples were further subjected to the PCR assay with designed primers targeting full length amplification of ORF2 gene of PCV2 for molecular characterization. Randomly 32 positive samples by full length PCV2-ORF2 gene PCR were sequenced for genotyping. Signature motif and phylogenetic analysis of 32 PCV2 sequences revealed 62.5% (n = 20) prevalence of PCV2d genotype followed by 21.8% (n = 7) of PCV2h or PCV2-IM1 and 15.6% (n = 5) of PCV2b genotypes. Twenty five PCR positive field samples were subjected for virus isolation in PK15 cells and characterized. Out of 25 samples processed 5 (20%) PCV2 isolates obtained in this study were confirmed by PCR and immune fluorescence assay. Molecular characterization of PK15 adapted five PCV2 isolates confirmed circulation of PCV2d, PCV2h and PCV2b genotypes in pigs under field conditions in southern India. CONCLUSIONS: Isolation and molecular epidemiological study of PCV2 in southern states of India evidences high circulation of PCV2d genotypes in field conditions in comparison to other genotypes.

Population, genetic, and antigenic diversity of the apicomplexan Eimeria tenella and their relevance to vaccine development.

The phylum Apicomplexa includes serious pathogens of humans and animals. Understanding the distribution and population structure of these protozoan parasites is of fundamental importance to explain disease epidemiology and develop sustainable controls. Predicting the likely efficacy and longevity of subunit vaccines in field populations relies on knowledge of relevant preexisting antigenic diversity, population structure, the likelihood of coinfection by genetically distinct strains, and the efficiency of cross-fertilization. All four of these factors have been investigated for Plasmodium species parasites, revealing both clonal and panmictic population structures with exceptional polymorphism associated with immunoprotective antigens such as apical membrane antigen 1 (AMA1). For the coccidian Toxoplasma gondii only genomic diversity and population structure have been defined in depth so far; for the closely related Eimeria species, all four variables are currently unknown. Using Eimeria tenella, a major cause of the enteric disease coccidiosis, which exerts a profound effect on chicken productivity and welfare, we determined population structure, genotype distribution, and likelihood of cross-fertilization during coinfection and also investigated the extent of naturally occurring antigenic diversity for the E. tenella AMA1 homolog. Using genome-wide Sequenom SNP-based haplotyping, targeted sequencing, and single-cell genotyping, we show that in this coccidian the functionality of EtAMA1 appears to outweigh immune evasion. This result is in direct contrast to the situation in Plasmodium and most likely is underpinned by the biology of the direct and acute coccidian life cycle in the definitive host.

Quantifying sperm egg interaction to assess the breeding efficiency through artificial insemination in guinea fowls.

1. Guinea fowl hens were inseminated weekly once with two doses of spermatozoa (75 million and 100 million) in two different diluents, Beltsville poultry semen extender (BPSE), and Instruments for Veterinary Medicine (IMV), each with and without pre-insemination vaginal douching. Per cent fertility, hatchability, dead germ, dead in shells along with data on sperm egg interaction and vaginal microbial counts were recorded. 2. Artificial insemination had significantly improved the per cent fertility and hatchability compared to natural mating. Dose dependent improvement in fertility was noticed with both diluents. 3. There was a beneficial effect of vaginal douching, which was more pronounced at lower insemination doses. 4. For optimum fertility and hatchability in guinea fowl, insemination of 75 million spermatozoa diluted in BPSE once in 4 d and 100 million spermatozoa diluted in BPSE or IMV once in 5 d coupled with vaginal douching is recommended.

Molecular cloning, sequencing and structural studies of granulocyte-macrophage colony-stimulating factor (GM-CSF) from Indian water buffalo (Bubalus bubalis).

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that is essential for growth and development of progenitors of granulocytes and monocytes/macrophages. In this study, we report molecular cloning, sequencing and characterization of GM-CSF from Indian water buffalo, Bubalus bubalis. In addition, we performed sequence and structural analysis for buffalo GM-CSF. Buffalo GM-CSF has been compared with 17 mammalian GM-CSFs using multiple sequence alignment and phylogenetic tree. Three-dimensional model for buffalo GM-CSF and human receptor complex was built using homology modelling to study cross-reactivity between two species. Detailed analysis was performed to study GM-CSF interface and various interactions at the interface.

Influence of age, sex and rearing systems on Toll-like receptor 7 (TLR7) expression pattern in gut, lung and lymphoid tissues of indigenous ducks.

Abstract 1. The objective of the experiment was to determine the influence of age, sex and rearing system on Toll-like receptor 7 (TLR7) gene expression in gut, lung and lymphoid tissues and physiological responses to stress in male and female indigenous ducks of Tamil Nadu, India. 2. A total of 36 ducks (12 males and 24 females) were obtained from local farmers and tissue samples of gut tissues (duodenum, jejunum, ileum and caecum), lymphoid organs (spleen and bursa) and lungs were collected in RNAlater solution followed by RNA extraction. 3. After normalisation to ß-actin (endogenous control) qPCR analysis identified a significant effect of age, sex and rearing system on TLR7 expression in the ducks. 4. A significant up-regulation of TLR7 expression was observed in lungs, duodenum, jejunum, ileum and caecum of sexually mature (45 wk) compared with that of immature ducks (16 wk). Among sexes, male ducks had significantly higher TLR7 expression than female ducks. 5. Age and sex interactions were significant in lungs, duodenum, jejunum and caecum. Ducks reared in an extensive housing system showed significantly higher TLR7 expression in bursa, lungs, duodenum, ileum and caecum compared to intensively reared ducks. There were no effects of age, sex and rearing systems on TLR7 expression in the spleen. 6. The heterophil-to-lymphocyte ratio and serum corticosterone were higher in ducks reared on an intensive system compared with ducks from an extensive rearing system.

Application of vaginal tampon as an alternative to nasal swabs for higher recovery of DNA from sheep and goats for PCR based diagnosis of bovine tuberculosis.

A study was conducted to find the applicability of vaginal tampons as an alternative to regular cotton swabs as a nasal secretion collection tool for the higher recovery of DNA. Nasal secretions were collected from sheep and goats using regular cotton swab and tampon swab. The mean yield and purity of the DNA extracted from tampon were significantly higher than that of the DNA extracted from cotton swab. The tampon swabs resulted higher DNA recovery than the cotton swabs after they were allowed to absorb M. bovis culture. The tampon swab was also found to be more sensitive in detecting M. bovis by PCR. This study concluded that vaginal tampons are having a higher absorption capacity with more DNA yield and can be used as a nasal swab in the diagnosis of bovine tuberculosis.

Molecular evidence of porcine circovirus 3 infection in swine: first report in southern India.

The present study examined 434 field samples including serum (n = 273), swabs from natural orifices (n = 52) and postmortem tissue samples (n = 109) from both suspected and asymptomatic swine from Andhra Pradesh, Karnataka, Kerala, Pondicherry, Tamil Nadu, and Telangana states in southern India. All the samples were processed for molecular screening of PCV3 by specific PCR assay. Overall molecular positivity rate of PCV3 was found to be 0.7% in southern India with one sample positive from each state of Tamil Nadu, Kerala and Telangana. All the three PCR positive PCV3 samples are detected from reproductive failures and were processed and propagated in PK15 cell line for virus isolation. Out of 3 samples processed, one (INDKL9PK76) PCV3 isolate could be obtained in this study and it was confirmed by specific PCR at third and fifth passage levels. Sequencing of PCV3 positive PCR amplicon (INDKL9PK76) revealed 1004 nucleotides and BLAST analysis confirmed partial sequence of the PCV3 genome. The aligned contig sequence was submitted to GenBank under the accession number of MW627201. PCV3 sequence in this study revealed 99% homology with PCV3 isolates from Europe and China. Phylogentic analysis of the PCV3 isolate-INDKL9PK76 sequence along with established PCV3 genotypes revealed clustering within PCV3 genotypes. Characterization of PCV3 (INDKL9PK76) isolate based on deduced amino acid composition of PCV3-capsid protein revealed "A" (alanine) and "R" (arginine) at 24th and 27th residues respectively confirming the incidence of PCV3a genotype. This study evidences PCV3 associated reproductive failure in domestic pigs for the first time in southern India.

Emergence of variant avian infectious bronchitis virus in India.

BACKGROUND: Infectious bronchitis virus (IBV) is the etiological agent of an acute and highly contagious disease. Infectious bronchitis (IB) affects chicken of all ages and poses major economic loses to the poultry industry worldwide. The continuous evolution of the spike protein (S1) of IBV is responsible for the prevalence of many serotypes/genotypes around the world. Multiple lineages of IBV strains have been detected in chicken flocks in India since 2003. AIMS: To detect IBV genotypes prevalent in India. METHODS: Organ samples from 20 IBV-positive flocks with variable clinical signs were used for the amplification of the S1 gene of IBV by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Positive PCR amplicons were sequenced. Sequence analysis showed that 14 field isolates belonged to the GI-1 genetic lineage (Mass 41 serotype), two field isolates belonged to the GI-13 (UK 4/91 variant IBV strain), one field isolate grouped with GIII, GV, and GVI genetic lineage and three belonged to a variant genotype unique to India (GI-24). Phylogenetic analysis also showed a similar type of grouping within the field isolates. Among the fourteen GI-1 isolates, 12 were isolated between 2003 and 2006 and only two were isolated between 2009 and 2011. The two field isolates belonging to GI-13 were isolated in 2007, another one belonging to GIII, GV, and GVI was isolated in 2010 and three field isolates were not close to any reference IBV sequences isolated in 2006 (IND-TN-168-06), 2010 (IND-TN-280-10) and 2011 (IND-TN-290-11). CONCLUSION: A unique variant of IBV is emerging in India (GI-24). Our findings will have important implications for future vaccine intervention.

Detection of virulent Marek's disease virus in poultry in India.

Marek's disease virus (MDV) isolated from poultry flocks in three states of India was monitored for the virus occurrence in the field. The MDV genome was isolated from feather follicles, spleen, and liver of the chicken (173 samples). Twenty two samples were positive for MDV genome in PCR and belonged to the serotype 1. The sequencing of MEQ gene of 11 samples revealed that nucleotide sequences of the isolate Ind-KA-01-06 was similar to the very virulent MDV, strain RB-1B. In situ hybridization studies also confirmed a presence of MDV serotype 1 in the infected liver tissues. Furthermore, the ability of the virus to induce apoptosis detected by flow cytometry showed that the virulent MDV induced apoptosis more efficiently than Turkey herpesvirus (HVT) vaccine virus. The present study showed the presence of virulent/very virulent MDV strains in the Indian poultry flocks.

Relationship between the level of signaling lymphocyte activation molecule mRNA and replication of Peste-des-petits-ruminants virus in peripheral blood mononuclear cells of host animals.

Basal signaling lymphocyte activation molecule (SLAM) expression in the peripheral blood mononuclear cells (PBMCs) of cattle, buffalo, sheep, and goats was correlated with Peste-des-petits-ruminants virus (PPRV) replication assessed by real-time PCR and virus titers. PBMCs from goats had the highest level of SLAM mRNA followed by sheep, cattle, and buffalo. Different breeds of goats had different basal levels of SLAM mRNA. In the PBMCs of studied animals, basal SLAM expression had high correlation with their ability to replicate PPRV. After stimulation of PBMCs from different breeds of goats with concanavalin A (con A), the SLAM expression increased 4- to 16-fold, what resulted in a 1.7- to 3.8-fold increase of viral mRNA and the virus titer increased by 0.4-1.3 log units. These findings revealed that SLAM expression and PPRV replication are highly correlated and different levels of SLAM mRNA could influence the virus replication in different animals.

Non-invasive in vivo imaging of fluorescence-labeled bacterial distributions in aquatic species.

In vivo imaging is becoming an advanced tool for noninvasive distribution of longitudinal small animals. However, the aquatic species have been limited to the optical imaging of noninvasively tracking on pathogen distribution. The purpose of this study was to develop shell-less fish and shrimp models of non-invasive in vivo imaging technique for visualization of pathogens. This experiment was utilized Escherichia coli, Edwardsiella tarda, Vibrio alginolyticus and Vibrio harveyi labeled with fluorescence probes to imaging bacterial distributions by IVIS Lumina LT system. The study was traced the internal distribution of fluorescence probes labeled bacteria in systemic organs by quantified their fluorescence intensities. The ex vivo organ images were showed more obvious fluorescent signal in catfish intestine, liver, heart, kidney and the shrimp showed heart, hepatopancreas, and colon. Hence, the in vivo imaging methods using fluorescent labeled bacterial distribution were suggested to quantify by fluorescence intensity in whole pre-infected subjects. Therefore, it can offer the information about the localization and distribution of pathogens in the preclinical research, after immersion and injections.

Chlorhexidine-calcium phosphate nanoparticles - Polymer mixer based wound healing cream and their applications.

In this work, we developed a wound healing cream composed of two different polymers, namely chitosan and gelatin with chlorhexidine along with calcium phosphate nanoparticles. The physicochemical properties of the prepared cream were investigated based on SEM, EDX, Raman, FTIR and the results indicated that the cream contained gelatin, chitosan, calcium phosphate nanoparticles and chlorhexidine. The maximum swelling ratio studies indicated that the ratio was around of 52±2.2 at pH7.4 and the value was increased in acidic and alkaline pH. The antimicrobial activity was tested against bacteria and the results indicated that, both chlorhexidine and the hybrid cream devoid of chlorhexidine exhibited antimicrobial activity but the chlorhexidine impregnated cream showed three fold higher antimicrobial activity than without chlorhexidine. In vivo wound healing promoting activities of hybrid cream containing 0.4mg/L chlorhexidine were evaluated on surgically induced dermal wounds in mice. The results indicated that the cream with incorporated chlorhexidine significantly enhanced healing compared with the control samples. For the field validations, the veterinary clinical animals were treated with the cream and showed enhanced healing capacity. In conclusion, a simple and efficient method for design of a novel wound healing cream has been developed for veterinary applications.

Cryptic Eimeria genotypes are common across the southern but not northern hemisphere.

The phylum Apicomplexa includes parasites of medical, zoonotic and veterinary significance. Understanding the global distribution and genetic diversity of these protozoa is of fundamental importance for efficient, robust and long-lasting methods of control. Eimeria spp. cause intestinal coccidiosis in all major livestock animals and are the most important parasites of domestic chickens in terms of both economic impact and animal welfare. Despite having significant negative impacts on the efficiency of food production, many fundamental questions relating to the global distribution and genetic variation of Eimeria spp. remain largely unanswered. Here, we provide the broadest map yet of Eimeria occurrence for domestic chickens, confirming that all the known species (Eimeria acervulina, Eimeria brunetti, Eimeria maxima, Eimeria mitis, Eimeria necatrix, Eimeria praecox, Eimeria tenella) are present in all six continents where chickens are found (including 21 countries). Analysis of 248 internal transcribed spacer sequences derived from 17 countries provided evidence of possible allopatric diversity for species such as E. tenella (FST values ⩽0.34) but not E. acervulina and E. mitis, and highlighted a trend towards widespread genetic variance. We found that three genetic variants described previously only in Australia and southern Africa (operational taxonomic units x, y and z) have a wide distribution across the southern, but not the northern hemisphere. While the drivers for such a polarised distribution of these operational taxonomic unit genotypes remains unclear, the occurrence of genetically variant Eimeria may pose a risk to food security and animal welfare in Europe and North America should these parasites spread to the northern hemisphere.

Rapid Detection of Avian Infectious Bronchitis Virus by Reverse Transcriptase-Loop Mediated Isothermal Amplification.

A reverse-transcription loop mediated isothermal amplification (RT-LAMP) was developed for rapid diagnosis of infectious bronchitis (IB) in poultry by targeting the spike protein 2 gene (S2). RT-LAMP primers were designed for IBV-S2 targets and optimized to run at 60 °C for 45 min. As compared with RT-PCR, RT-LAMP was 100 times more sensitive for IBV-S2 gene. RT-LAMP showed specific amplification with IB viral genome but not with other avian respiratory pathogens due to their mismatching with IBV-S2-RT-LAMP primers. RT-LAMP reaction products were visually detected by the addition of propidium iodide stain. Out of 102 field samples tested for detection of IBV, RT-LAMP detected IBV in 12 samples for S2 gene whereas RT-PCR detected IBV in six samples for S2 gene. The sensitivity of the RT-LAMP was 100 % and the specificity was 94 % for S2 gene. Since the developed RT-LAMP to detect IBV is simple, rapid, sensitive and specific, it can be a useful diagnostic tool for detection of IB in poultry in less equipped laboratories and in field conditions.

Complete Genome Sequences of Lineage IV Peste des Petits Ruminants Viruses from the Indian Subcontinent.

The complete genome sequences of two virulent lineage IV peste des petits ruminants viruses (PPRVs) isolated from clinically infected goats in the Indian subcontinent are reported here. This is the first report of a complete genome sequence of a virulent PPRV isolate from India in recent decades.

A simplified objective method for quantification of peste des petits ruminants virus or neutralizing antibody.

A simplified and standardized assay based on haemagglutination of infected culture supernatants was developed to detect peste des petits ruminants (PPR) virus growth in vero cells and to quantify PPR neutralizing antibody. Virus titres estimated by visual reading of cytopathic effects as a criterion were compared with those estimated by haemagglutination of infected supernatants and no statistically significant differences were seen between them. During quantification of PPR antibodies, the titres based on haemagglutination of supernatants on day 5 post-infection had a high sensitivity, specificity and agreement in qualititative comparison with those determined by visual examination of cytopathic effects. In quantitative comparison, the correlation coefficient between serum neutralization titres estimated by visual examination of cytopathic effects or haemagglutination of supernatants was 0.96, when haemagglutination was done on day 5 post-infection. The virus and serum neutralization titres can thus be estimated objectively using the haemagglutination of supernatants as criterion to measure endpoints. The haemagglutination-inhibition titres also correlated well with serum neutralization titres with a coefficient of 0.78. Thus the haemagglutination-inhibition test appears to be a suitable alternative to the serum neutralization test for quantification of PPR neutralizing antibody.

Protectotypic differentiation of avian infectious bronchitis viruses using an in vitro challenge model.

Two vaccine and three virulent strains of infectious bronchitis virus (IBV) were used to infect day-old specific-pathogen-free chickens. Precocious development of oviducts was induced in young female chicks by oestrogen injections. Tracheal and oviduct organ cultures prepared from immunised chickens were challenged in vitro with homologous and heterologous viruses to assess tracheal and oviduct cross-protection. Tracheal cross-protection was seen between serologically related and unrelated viruses. Protection at at the level of the oviducts was lower compared to that seen at the tracheal level. However, the yields of the homologous challenge viruses from immunised oviducts were significantly reduced as compared to yields from normal oviducts. After challenge, tracheal and oviduct ciliary scores were used to group the five strains of IBV into protectotypes. The five viruses could be grouped into three protectotypes and four serotypes. The virulent IBV strains were found to have caused complete stasis of oviduct cilia when examined at three weeks post-infection. Oestrogen treatment of chicks did not significantly alter the lymphocyte proliferation responses to concanavalin A or pokeweed mitogen. The applicability of this model for the study of protection afforded to the oviducts, in terms of protection against virus-induced damage, rather than drop in egg production, is discussed.

Demonstration of alternative and classical complement pathway activity in colostrum from buffalo (Bubalus bubalis).

Buffalo colostrum caused lysis of unsensitized red blood cells (RBC) from sheep, goats, rabbits and chickens. RBC from cattle and buffalo were resistant to lysis. That lysis was due to the presence of natural antibodies to these RBC was ruled out since there was no reduction in haemolytic titres even after adsorption with the respective RBC. The addition of EGTA to the diluent had no effect on the haemolytic activity. These findings indicate the presence of alternative complement pathway (ACP) activity in buffalo colostrum. The haemolytic activity of buffalo complement for unsensitized rabbit RBC was reduced to very low levels by heating at 50 degrees C for 45 min. Treatment with zymosan also inhibited the haemolytic activity, while inulin had no effect. The maximum activity of ACP occurred in the presence of 4 mmol/L Mg(2+) in the diluent. The range of ACP activities in colostrum from buffaloes varied from 4.06 to 8.48 CH50 units/ml. Using a standard system for titrating the classical complement pathway and rabbit red blood cells sensitized with goat haemolysin, the range of complement activity in buffalo colostrum was 4.81-6.77 CH50/ml.

Production and characterisation of monoclonal antibodies to an Indian isolate of peste des petits ruminants virus.

Nine monoclonal antibodies (Mabs) were produced against an Indian isolate of peste des petits ruminants (PPR) virus. These Mabs were directed against the nucleo (N) protein and were of IgG1 isotype. The Mabs produced intranuclear or coarse granular cytoplasmic fluorescence in PPR virus infected Vero cells and did not exhibit any neutralising activity. The Mabs cross-reacted with five other local isolates of PPR virus in slot blot hybridisation, radio immunoprecipitation assay (RIPA) and fixed-cell enzyme linked immunosorbent assay (ELISA). Two of the nine Mabs cross-reacted mildly with the vaccine strain of rinderpest (RP) virus in slot blot hybridisation and fixed-cell ELISA but did not precipitate the N protein of RP virus in RIPA. The N protein specific Mabs will be highly useful in differential diagnosis of PPR from RP.

Partial sequencing of Bm86 gene for studying the phylogeny of an Indian isolate of Rhipicephalus (Boophilus) microplus tick.

Tick gut glycoprotein, designated as Bm86, found on the luminal surface of the plasma membrane of gut epithelial cells of Boophilus microplus, which is a concealed antigen, has been used as vaccine candidate molecule for immunization against ticks. To better understand the molecular diversity of Bm86 gene in ticks, a portion of the cDNA was sequenced from an Indian isolate of B. microplus. Comparison of nucleotide sequence revealed that Indian isolate had 97 % homology (18 polymorphisms) with that of the Australian isolate and 96 % homology (20 polymorphisms) with that of the Cuban vaccine strain. Further, the Indian isolate differed from the Cuban vaccine isolate at 7 amino acid loci, including 5 substitutions (at residues 88, 94, 175, 176 and 177) and 2 deletions (at 183 and 184). However, protein prediction studies did not show any difference in the putative antigenic epitopes of the protein expressed.