Comparative assessment of two in-house-built isothermal assays for visual detection of African swine fever virus.

Owing to the lack of effective vaccines, current control measures and eradication strategies for the African swine fever virus (ASFV) rely on early detection and stringent stamping-out procedures. In the present study, we developed two independent isothermal amplification assays, namely, loop-mediated isothermal amplification (LAMP) and polymerase spiral reaction (PSR), for quick visualization of the ASFV genome in clinical samples. Additionally, a quantitative real-time PCR (qRT-PCR)-based hydrolysis probe assay was developed for comparative assessment of sensitivity with the developed isothermal assays. The analytical sensitivity of the LAMP, PSR, and qRT-PCR was found to be 2.64 ×105 copies/µL, 2.64 ×102 copies/µL, and 2.64 ×101 copies/µL, respectively. A total of 165 clinical samples was tested using the developed visual assays. The relative accuracy, relative specificity, and relative diagnostic sensitivity for LAMP vs PSR were found to be 95.37% vs 102.48%, 97.46% vs 101.36%, and 73.33% vs 113.33%, respectively.

Assessment of reference genes for qRT-PCR normalization to elucidate host response to African swine fever infection.

Viral infection disrupts the normal regulation of the host gene's expression. In order to normalise the expression of dysregulated host genes upon virus infection, analysis of stable reference housekeeping genes using quantitative real-time-PCR (qRT-PCR) is necessary. In the present study, healthy and African swine fever virus (ASFV) infected porcine tissues were assessed for the expression stability of five widely used housekeeping genes (HPRT1, B2M, 18 S rRNA, PGK1 and H3F3A) as reference genes using standard algorithm. Total RNA from each tissue sample (lymph node, spleen, kidney, heart and liver) from healthy and ASFV-infected pigs was extracted and subsequently cDNA was synthesized, and subjected to qRT-PCR. Stability analysis of reference genes expression was performed using the Comparative delta CT, geNorm, BestKeeper and NormFinder algorithm available at RefFinder for the different groups. Direct Cycle threshold (CT) values of samples were used as an input for the web-based tool RefFinder. HPRT1 in spleen, 18 S rRNA in liver and kidney and H3F3A in heart and lymph nodes were found to be stable in the individual healthy tissue group (group A). The majority of the ASFV-infected organs (liver, kidney, heart, lymph node) exhibited H3F3A as stable reference gene with the exception of the ASFV-infected spleen, where HPRT1 was found to be the stable gene (group B). HPRT1 was found to be stable in all combinations of all CT values of both healthy and ASFV-infected porcine tissues (group C). Of five different reference genes investigated for their stability in qPCR analysis, the present study revealed that the 18 S rRNA, H3F3A and HPRT1 genes were optimal reference genes in healthy and ASFV-infected different porcine tissue samples. The study revealed the stable reference genes found in healthy as well as ASF-infected pigs and these reference genes identified through this study will form the baseline data which will be very useful in future investigations on gene expression in ASFV-infected pigs.

Characterization of an African swine fever virus outbreak in India and comparative analysis of immune genes in infected and surviving crossbreed vs. indigenous Doom pigs.

Since 2020, African swine fever (ASF) has affected all pig breeds in Northeast India except Doom pigs, a unique indigenous breed from Assam and the closest relatives of Indian wild pigs. ASF outbreaks result in significant economic losses for pig farmers in the region. Based on sequencing and phylogenetic analysis of the B646L (p72) gene, it has been determined that ASFV genotype II is responsible for outbreaks in this region. Recent studies have shown that MYD88, LDHB, and IFIT1, which are important genes of the immune system, are involved in the pathogenesis of ASFV. The differential expression patterns of these genes in surviving ASFV-infected and healthy Doom breed pigs were compared to healthy controls at different stages of infection. The ability of Doom pigs to withstand common pig diseases, along with their genetic resemblance to wild pigs, make them ideal candidates for studying tolerance to ASFV infection. In the present study, we investigated the natural resistance to ASF in Doom pigs from an endemic area in Northeast India. The results of this study provide important molecular insights into the regulation of ASFV tolerance genes.

Transcriptome signatures of host tissue infected with African swine fever virus reveal differential expression of associated oncogenes.

African swine fever (ASF) has emerged as a threat to swine production worldwide. Evasion of host immunity by ASF virus (ASFV) is well understood. However, the role of ASFV in triggering oncogenesis is still unclear. In the present study, ASFV-infected kidney tissue samples were subjected to Illumina-based transcriptome analysis. A total of 2463 upregulated and 825 downregulated genes were differentially expressed (p < 0.05). A literature review revealed that the majority of the differentially expressed host genes were key molecules in signaling pathways involved in oncogenesis. Bioinformatic analysis indicated the activation of certain oncogenic KEGG pathways, including basal cell carcinoma, breast cancer, transcriptional deregulation in cancer, and hepatocellular carcinoma. Analysis of host-virus interactions revealed that the upregulated oncogenic RELA (p65 transcription factor) protein of Sus scrofa can interact with the A238L (hypothetical protein of unknown function) of ASFV. Differential expression of oncogenes was confirmed by qRT-PCR, using the H3 histone family 3A gene (H3F3A) as an internal control to confirm the RNA-Seq data. The levels of gene expression indicated by qRT-PCR matched closely to those determined through RNA-Seq. These findings open up new possibilities for investigation of the mechanisms underlying ASFV infection and offer insights into the dynamic interaction between viral infection and oncogenic processes. However, as these investigations were conducted on pigs that died from natural ASFV infection, the role of ASFV in oncogenesis still needs to be investigated in controlled experimental studies.

Incidences of Helicobacter infection in pigs and tracing occupational hazard in pig farmers.

Helicobacter species (spp.) is a gram-negative spiral-shaped motile bacterium that causes gastritis in pigs and also colonizes in the human stomach. The present study assessed the prevalence of Helicobacter spp. in pig gastric mucosa and the stool of pig farmers in Assam, India. A total of 403 stomach samples from pig slaughter points, 74 necropsy samples of pigs from pig farms, and 97 stool samples from pig farmers were collected. Among the pig stomach samples, 43 (20.09%) of those with gastritis showed the presence of Gram-negative, spiral-shaped organisms, while only 3.04% of stomach samples without lesions had these organisms. Scanning Electron Microscopy (SEM) of urease-positive stomach samples revealed tightly coiled Helicobacter bacteria in the mucus lining. Histopathological examination showed chronic gastritis with hemorrhagic necrosis, leucocytic infiltration, and lymphoid aggregates. PCR confirmed the presence of Helicobacter suis in 19.63% of pig stomach samples and 2.08% of pig farmer stool samples. Additionally, 3.12% of the stool samples from pig farmers were positive for Helicobacter pylori. Phylogenetic analysis revealed distinct clusters of Helicobacter suis with other Helicobacter spp. These findings highlight the prevalence of Helicobacter in both pig gastric mucosa and pig farmer stool. The findings highlight the need for improved sanitation and hygiene practices among pig farmers to minimize the risk of Helicobacter infection in humans.

Natural co-infection of pigs with African swine fever virus and porcine reproductive and respiratory syndrome virus in India.

Porcine reproductive and respiratory syndrome (PRRS) and African swine fever (ASF) are economically important diseases of pigs throughout the world. During an outbreak, all age groups of animals except piglets < 1 month of age were affected with symptoms of high fever, cutaneous hemorrhages, vomition with blood, diarrhea, poor appetite, ataxia, and death. The outbreak was confirmed by the detection of the N gene of the porcine reproductive and respiratory syndrome virus (PRRSV) and the VP72 gene of the African swine fever virus (ASFV) by PCR in representative blood samples from affected pigs followed by Sanger sequencing. Mixed infection was also confirmed by simultaneous detection of both the viruses using multiplex PCR. Phylogenetic analysis of both the viruses revealed that the outbreak was related to ASFV and PRRSV strains from China which were also closely related to the PRRSV and ASFV strains from the recent outbreak from India. The study confirmed the involvement of genotype II of ASFV and genotype 2 of PRRSV in the present outbreak. Interestingly, PRRSV associated with the present outbreak was characterized as a highly pathogenic PRRSV. Therefore, the present study indicates the possibility of future waves or further outbreaks of these diseases (PRRS and ASF) in this region. This is the first report of ASFV and PRRSV co-infection in pigs from India.

Clinicopathological and ultrastructural study of African swine fever outbreak in North-East India.

The present investigation focuses on examining the clinical, histopathological, and ultrastructural changes that occurred in pig, during an outbreak of African swine fever (ASF) in 2022 in Assam, India. The disease initially manifested as a per-acute case with high mortality but without any evident clinical signs. Subsequently, some animals exhibited an acute form of the disease characterized by high fever (104-106 °F), anorexia, vomiting, respiratory distress, and bleeding from the anal and nasal orifices. During acute African swine fever virus (ASFV) infections, elevated levels of pro-inflammatory IL-1α, IL-1ß, IL-6, TNF, CCL2, CCL5, and CXCL10 were detected in the palatine tonsil, lymph nodes, spleen, and kidney using qPCR assay. These molecular changes were associated with haemorrhages, edemas, and lymphoid depletion. Postmortem examinations revealed prominent features such as splenomegaly with haemorrhages, haemorrhagic lymphadenitis, severe petechial haemorrhage in the kidney, pneumonia in the lungs, and necrotic palatine tonsil. Histopathological analysis demonstrated lymphocyte depletion in lymphoid organs, multi-organ haemorrhages, and interstitial pneumonia in the lungs. Scanning electron microscopy (SEM) further confirmed lymphocyte depletion in lymphoid organs through lymphocyte apoptosis and kidney damage with distorted tubules due to red blood cell destruction. Transmission electron microscopy reaffirmed lymphocyte apoptosis by observing chromatin condensation and nucleus margination in lymphocytes of lymphoid organs. These findings provide comprehensive insights into the clinical, histopathological, and ultrastructural aspects of ASF outbreak in pigs. Understanding the pathological changes associated with ASF can contribute to improved diagnosis, prevention, and control measures for this highly contagious and economically devastating viral disease.

Meta-Analysis of the Prevalence of Porcine Zoonotic Bacterial Pathogens in India: A 13-Year (2010-2023) Study.

The presence of bacterial pathogens such as Brucella spp., Clostridium spp., E. coli, Listeria monocytogenes, Salmonella spp., Staphylococcus spp., and Streptococcus suis not only hampers pig production but also carries significant zoonotic implications. The present study aims to conduct a comprehensive meta-analysis spanning over 13 years (2010-2023) to ascertain the prevalence of these zoonotic bacterial pathogens in Indian pig populations. The study seeks to synthesize data from diverse geographic regions within India and underscores the relevance of the One Health framework. A systematic search of electronic databases was meticulously performed. Inclusion criteria encompassed studies detailing zoonotic bacterial pathogen prevalence in pigs within India during the specified timeframe. Pertinent information including authors, publication year, geographical location, sampling techniques, sample sizes, and pathogen-positive case counts were meticulously extracted. The meta-analysis of zoonotic bacterial pathogens in Indian pig populations (2010-2023) unveiled varying prevalence rates: 9% Brucella spp., 22% Clostridium spp., 19% E. coli, 12% Listeria monocytogenes, 10% Salmonella spp. and Streptococcus suis, and 24% Staphylococcus spp. The application of random effects further revealed additional variability: 6% Brucella spp., 23% Clostridium spp., 24% E. coli, 14% Listeria monocytogenes, 10% Salmonella spp. and Streptococcus suis, and 35% Staphylococcus spp. Notably, the observed heterogeneity (I2) varied significantly from 87% to 99%. The meta-analysis findings underscore the pervasive nature of these diseases throughout India's pig populations, accentuating the substantial impact of these pathogens on pig health and the potential for zoonotic transmission. The present study reinforces the importance of the adoption of a comprehensive One Health approach that acknowledges the intricate interplay between animal, human and environmental health.

Whole genome sequencing-based cataloguing of antibiotic resistant genes in piggery waste borne samples.

The growing use of antibiotics in livestock is one of the main causes of the rapid global spread of antimicrobial resistance (AMR). However, extensive research on AMR in animals is currently absent. In this article, we provide the bacterial antibiotic resistance genes (ARGs) from piggery waste samples in West Bengal, India, based on whole genome sequencing (WGS). According to the study, there are alarmingly high levels of Enterobacteriaceae in piggery waste, especially slaughterhouse waste, that are resistant to beta-lactam, aminoglycoside, sulphonamide, and tetracycline. We found several plasmids carrying multidrug-resistant Enterobacteriaceae including resistant to last-resort medications like colistin and carbapenems. Our findings will serve as a guide for developing AMR management policies for livestock in India and aid in understanding the current AMR profiles of pigs. To grasp the actual situation with AMR in the pig sector, large scale sample screening must be done.

Characterization of piggery farm waste-borne bacterial transposable elements associated with antimicrobial resistance phenotypes.

Even though there is a link between antibiotic resistance and the presence of transposable elements few research has looked at the prevalence and distribution of transposable elements/ integrons in piggery farm samples. Present study identified the presence of six transposable elements namely Tn6763 (Accession number: OQ565300), Tn6764, (Accession number: OQ565299), Tn6765 (Accession number: OQ409902), Tn2003 (Accession number: OQ503494), Tn6072 (Accession number: OQ565298) and Tn6020 (Accession number: OQ503493) in piggery farm waste from India which are belongs to Enterobacteriaceae family. In a conjugative experiment, Klebsiella isolates carrying Tn6020 having the resistant phenotypes for nalidixic acid was used as donor cells while Escherichia coli DH5α Cells carrying chloramphenicol resistant plasmid was employed as recipient cells. Transconjugant bacterial colonies were shown to carry the Tn6020 transposable elements with both nalidixic acid (donor cell origin) and chloramphenicol (recipient cell origin) resistant antibiotic phenotypes. Given the presence of transposable elements in 21.4% of resistant Enterobacteriaceae strains, preventative measures are vital for avoiding the spread of mobile genetic resistance determinants in the piggery sector and to monitor their emergence.

Japanese Encephalitis Virus Genotype III Strains Detection and Genome Sequencing from Indian Pig and Mosquito Vector.

Japanese encephalitis viruses (JEVs) are globally prevalent as deadly pathogens in humans and animals, including pig, horse and cattle. Japanese encephalitis (JE) still remains an important cause of epidemic encephalitis worldwide and exists in a zoonotic transmission cycle. Assam is one of the highly endemic states for JE in India. In the present study, to understand the epidemiological status of JE circulating in pigs and mosquito, particularly in Assam, India, molecular detection of JEV and the genome sequencing of JEV isolates from pigs and mosquitoes was conducted. The genome analysis of two JEV isolates from pigs and mosquitoes revealed 7 and 20 numbers of unique points of polymorphism of nucleotide during alignment of the sequences with other available sequences, respectively. Phylogenetic analysis revealed that the isolates of the present investigation belong to genotype III and are closely related with the strains of neighboring country China. This study highlights the transboundary nature of the JEV genotype III circulation, which maintained the same genotype through mosquito-swine transmission cycles.

Development of CD163 receptor-based enzyme-linked immunosorbent assay for diagnosis of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) is an important economical disease in the global swine industry. The accurate detection of the PRRS virus (PRRSV) antigen is essential for the disease control and prevention programme. In this study, an indirect enzyme-linked immunosorbent test (PRRSVCD163-iELISA) was developed for the detection of the PRRSV antigen in samples of post-mortem swine tissue using the recombinant pig CD163 receptor protein as the capture ligand. The test was found to be specific for PRRSV, with no cross-reactions with other prevalent pig viral pathogens. The assay was validated by testing 217 post-mortem porcine tissue samples and the results were found to be satisfactory with a relative accuracy of 88.88%. Our assay is also quite precise, with intra- and inter-assay CVs of 6% and 10%, respectively. These findings imply that the PRRSVCD163-iELISA developed is capable of detecting the PRRSV antigen in swine post-mortem tissue samples. This research showed that porcine CD163, the PRRSV cellular receptor, can be exploited to build a diagnostic technique for the detection of PRRSV antigen. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03376-z.

Porcine Circovirus type 2 infected myocardial tissue transcriptome signature.

The goal of this study was to compare the global gene expression profile in cardiac tissues of pig infected with porcine circovirus 2 (PCV2) to that of healthy cells. Since PCV2 infection causes severe cardiovascular lesions, the myocardial tissue model was chosen for this study. In High-throughput transcriptome analysis, DESeq2 and CLC genomics workbench analyses revealed a total of 196 significantly differentially expressed genes (DEGs) (p-value < 0.05). Furthermore, 194 transcripts were upregulated, while only two were downregulated (HSPA6 and DNAJA1), with fold changes ranging from 16.293 to -10.002. Among the KEGG canonical pathways targeted by the DEGs in the functional analysis, adrenergic signalling in cardiomyocytes, Cardiac Muscle Contraction, Hypertrophic Cardiomyopathy (HCM), and Dilated Cardiomyopathy (DCM) tends to be enriched. The differentially expressed highly connected (DEHC) biomarker genes in pathogenicity of PCV2 infection, such as LDB3, MYOZ2, CASQ2, TNNT2, MLC2V, MYBPC3, ACTC1, TCAP, TNNI3, TRDN, CSRP3, MYL3, RYR2, LMOD2, MYH7, etc., were identified using protein-protein interaction (PPI) network analysis. The study might provide detailed information on the dysregulated genes and biological pathways in infected myocardial tissues that may be essential for PCV2-related heart pathology.

Selection and validation of suitable reference gene for qPCR gene expression analysis in lamb testis cells under Sheep pox virus infection.

Virus infection alters host gene expression, therefore ideal and stable reference housekeeping genes are required to normalise the expression of other expressed host genes in quantitative real-time PCR (qRT-PCR). The suitable reference gene may vary in response to different viral infections in different hosts or cells. In the present study, we cultured primary lamb testis cells (LTC) and assessed the expression stability of seven widely used housekeeping genes (B2M, HMBS, HPRT1, HSP-90, POLR2A, 18s_RNA, GAPDH) as reference genes in Sheeppox virus (SPPV) infected and control (uninfected-0h) LTC at 0.5h, 4.0h, 8.0h, and 12.0h post-infection) using NormFinder, Bestkeeper, geNorm, and the comparative ΔCT method in RefFinder based on their expression levels. Analysis revealed that HSP90, 18s_RNA, HPRT, POLR2A, and B2M were the most stable genes from the panel in the individual analysis group in 0h, 0.5h, 4.0h, 8.0h, and 12.0h, respectively. Furthermore, B2M was shown to be the most stable reference gene in the combined control with the respective and overall infected groups, except the control group of 4.0hpi of SPPV infection. In this study, we selected the most suitable reference genes in LTC for particular time points of SPPV infection. The identified most suitable housekeeping gene can be used during normalization of expression of other targeted genes at aspecific time point of SPPV infection.

Hepatic transcriptome analysis reveals altered lipid metabolism and consequent health indices in chicken supplemented with dietary Bifidobacterium bifidum and mannan-oligosaccharides.

This study investigated the role of dietary prebiotic mannan-oligosaccharides (MOS), and probiotic Bifidobacterium bifidum (BFD) in lipid metabolism, deposition, and consequent health indices in broiler chicken. The supplementation of 0.2% MOS along with either 106 or 107 CFU BFD/g feed resulted in downregulation of Acetyl-CoA carboxylase, fatty acid synthase, sterolregulatory element binding protein-1, and apolipoprotein B100; and up-regulation of peroxisome proliferator activated receptor-α AMP-activated protein kinase α-1, and stearoyl CoA (∆9) desaturase-1 hepatic expression in broiler chicken. The birds supplemented with 0.2% MOS along with either 106 or 107 CFU BFD/g feed depicted lower body fat percentage, palmitic acid, stearic acid, and saturated fatty acid contents, whereas, higher palmitoleic acid, oleic acid, and MUFA contents were observed. The ∆9-desaturase indices of chicken meat have shown higher values; and elongase index (only thigh) and thioesterase index have shown lower values in birds supplemented with 0.2% MOS along with either 106 or 107 CFU BFD/g feed. The meat health indices such as Polyunsaturated fatty acids (PUFA)/Saturated fatty acids (SFA) ratio, Mono-saturated fatty acids (MUFA)/SFA ratio, unsaturated fatty acids (UFA)/SFA ratio, hypocholesterolemic/hypercholesterolemic fatty acid ratio, saturation index, atherogenic index, thrombogenic index, and hypercholesterolemic fatty acid content were positively improved in birds supplemented with 0.2% MOS along with either 106 or 107 CFU BFD/g feed. Similarly, the birds supplemented with 0.2% MOS along with either 106 or 107 CFU BFD/g feed have shown lower serum triglyceride and total cholesterol levels along with higher high density levels and improved serum health indices cardiac risk ratio, atherogenic coefficient, and, atherogenic index of plasma.

Genome-wide expression analysis reveal host genes involved in immediate-early infections of different sheeppox virus strains.

This study explored the transcriptome of lamb testis cells infected with sheeppox virus (SPPV) wild strain (WS) and vaccine strain (VS) at an immediate-early time. Most of the differentially expressed genes (DEGs) and differentially expressed highly connected (DEHC) gene network were found to be involved in SPPV-VS infection compared to SPPV-WS. Further, the signaling pathways were mostly involved in SPPV-VS infection than SPPV-WS. SPPV modulates the expression of several important host proteins such as CD40, FAS, ITGß1, ITGα1, Pak1, Pak2, CD14, ILK leading to viral attachment and entry; immune-related DEGs such as MAPK, JNK, ERK, NFKB, IKB, PI3K, STAT which provide optimal cellular condition for early viral protein expression; and FOXO3, ATF, CDKNA1, TCF, SRF, BDNF which help in inducing apoptosis and MPTP, BAD and Tp53 inhibits apoptosis or cell death at the immediate-early time. The results captured the specific genes and enabled to understand distinct pathogenic mechanisms employed by VS and WS of SPPV.

Dietary Lactobacillus acidophilus and Mannan-Oligosaccharides Alter the Lipid Metabolism and Health Indices in Broiler Chickens.

The effects of dietary Lactobacillus acidophilus (LBA) and mannan-oligosaccharide (MOS) supplementation on lipid metabolism and consequent lipid profile and health indices in broiler chicken were investigated in this study. Supplementation of 0.2% MOS along with either 106 or 107 LBA/g feed in broiler chicken downregulated hepatic expression of genes involved in lipogenesis, and upregulated expression of lipolytic genes. It caused decline of lipogenesis and increase of lipid oxidation which resulted in lower carcass fat content. None of the genes studied influenced fatty acid profile of chicken meat except the expression of stearoyl CoA (Δ9) desaturase-1 (SCD-1) whose upregulation increased monounsaturated fatty acid (MUFA) content at the cost of saturated fatty acid (SFA) content. The lipid metabolism indices of chicken meat such as ∆9 desaturase index (DI) increased in birds supplemented with 0.2% MOS along with either 106 or 107 CFU LBA/g feed, whereas no effect was observed on ∆5 + ∆6 DI. The supplementation of 0.2% MOS along with either 106 or 107 CFU LBA/g feed in birds improved the health indices of chicken meat due to upregulation of SCD-1 expression. The supplementation of 0.2% MOS along with either 106 or 107 CFU LBA/g feed in broiler chicken produced hypocholesterolemic and hypolipidemic effects with improved serum cardio-protective indices.

VARV B22R homologue as phylogenetic marker gene for Capripoxvirus classification and divergence time dating.

Sheeppox disease is associated with significant losses in sheep production world over. The sheep pox virus, the goatpox virus, and the lumpy skin disease virus cannot be distinguished by conventional serological tests. Identification of these pathogens needs molecular methods. In this study, seven genes viz. EEV maturation protein-F12L, Virion protein-D3R, RNA polymerase subunit-A5R, Virion core protein-A10L, EEV glycoprotein-A33R, VARV B22R homologue, and Kelch like protein-A55R that cover the start, middle, and end of the genome were selected. These genes were amplified from Roumanian-Fanar vaccine strain and Jaipur virulent strain, cloned, and sequenced. On analysis with the available database sequences, VARV B22R homologue was identified as a marker for phylogenetic reconstruction for classifying the sheeppox viruses of the ungulates. Further, divergence time dating with VARV B22R gene accurately predicted the sheeppox disease outbreak involving Jaipur virulent strain.