Lymphocyte subsets in the small intestine of piglets fed with probiotic and zinc: a qualitative and quantitative micro-anatomical study.

BACKGROUND: Piglet mortality is a real concern to the pig farmers. The major cause is due to the late maturation of the immune system and dietary changes in postweaned piglets. The potential role of probiotic and zinc in the stimulation of the immune system is well established. Hence, the present study was undertaken to evaluate alterations of T and B cells in the small intestine after dietary inclusion of probiotic and zinc in pre and post-weaned piglets. MATERIALS AND METHODS: A total of 18 healthy Large White Yorkshire (LWY) piglets, irrespective of sex obtained from 3 litters at the age-group of 20, 30 and 60 days. They were divided into a control group fed with basal diet and a treatment group fed with probiotic and zinc supplement along with the basal diet, consisting of three animals in each group. The piglets were weaned at 28 days of age. After sacrificing the animals at day 20, 30 and 60 from both the groups, the abdominal cavity was opened and small intestinal tissue samples were collected, processed and stained by indirect immunofluorescence technique. The slides were evaluated under the fluorescent light microscope. The data were statistically analysed. RESULTS: The different T and B cell subsets were recorded in the lining epithelium, core of villus, crypt area of lamina propria and Peyer's patch area. The number of CD4+, CD8+, IgA+ and IgM+ cells was higher in the treated piglets than the control group of animals, irrespective of segments of intestine and age-group. CONCLUSIONS: It can be concluded that the dietary supplementation of probiotic and zinc was found to be good additives as they can stimulate the immune response in piglets, especially during the critical early post-weaning period.

Multidrug resistant staphylococci isolated from pigs with exudative epidermitis in North eastern Region of India.

Exudative epidermatitis or greasy pig disease (GPD) is a contagious disease of pig and endemic worldwide caused by toxigenic strains under genus Staphylococcus. The present study reported an outbreak of GPD in Champhai district of Mizoram adjoining to the southern border of Myanmar. A total of 60 samples were collected from 22 clinically affected animals and processed for isolation and identification of Staphylococcus spp. All the isolates were subjected to antimicrobial sensitivity assay, biofilm production assay and detection of virulence genes, biofilm genes and mec genes followed by cloning and sequencing for phylogenetic analysis. A total of 44 staphylococci belonged to four species (S. sciuri, S. aureus,S. lentus, and S. hyicus) were isolated. Majority of the isolates were multidrug resistant with maximum resistance against ampicillin, penicillin including vancomycin. None of the S. hyicus isolates was methicillin resistant (MRSH) but 66·67% isolates were MRSA. By PCR, mecA gene was detected in S. aureus (n = 2), S. sciuri (n = 4) and S. lentus (n = 3). Biofilm associated gene icaD was detected in S. aureus (n = 3), S. sciuri (n = 5), S. hyicus (n = 4) and S. lentus (n = 6). The exfoliative toxin genes (ehxB, shetA and tsst1) were detected in S. hyicus (n = 3) and S. aureus (n = 1) isolates. All the isolates were closely related with the isolates from pigs of China, Germany, Japan and USA. The pathogens might be transmitted through illegal migration of pigs from Myanmar to India.

Cytokine responses in pigs after natural infection with classical swine fever virus.

Classical swine fever (CSF) is a highly contagious viral infection that affects domestic and wild pig population. The classical swine fever virus (CSFV) targets immune cells which perturb the immune functions causing immunopathological disorders such as immunosuppression, leukopenia and haemorrhage. In the present study, ELISA and quantitative real-time reverse transcription PCR (qRT-PCR) analysis was employed to determine cytokine profiles in pigs naturally infected with CSFV using whole blood assay (WBA) under field conditions. Significantly higher TNF-α, IL-10, and IL-6 expression levels were found in unvaccinated pigs infected with CSFV (group B) compared to vaccinated pigs that recovered after CSF (group A), the difference being statistically significant (p = 0.001). However, the expression of IFN-γ was significantly higher in group A compared to group B (p = 0.001). The findings of this field-supported study will help us to understand the immune biology of CSFV infection in infected pigs. The WBA technique can be used as a reliable, fast and feasible in vitro method to assess porcine cellular immune responses as it imitates the porcine blood conditions. Such studies could be of some value in determining the immune status of the ailing animals infected with CSFV. Keywords: classical swine fever virus; immune response; field conditions; interleukin; IFN-γ; TNF-α.

Endotheliotropic herpesvirus infection in Asian elephants (Elephas maximus) of Assam, India.

BACKGROUND AND AIM: Elephant endotheliotropic herpesvirus (EEHV) is an emerging disease of elephant. Therefore, a study was conducted to know the actual status of the disease in Assam State of India. MATERIALS AND METHODS: A total of 289 Asian elephants of Assam were screened during 2 years of study from April 2017 to March 2019. The clinical symptoms of diseased as well as gross and histopathological changes of dead elephants were recorded for the diagnosis of the disease. Virus involved in the occurrence of the disease was confirmed by polymerase chain reaction (PCR). RESULTS: In the present study, a total of three elephant calves out of 22 were found positive to EEHV1A. On the other hand, three adult asymptomatic elephants were also found positive for EEHV1 on screening 267 captive Asian elephants of Assam. The amplified PCR product showed band size of 520, 600, and 930 bp. The PCR amplified product with size 600 bp had shown the gene sequence for EEHV1U77/HEL. Gross lesions include congested blood vessels of the liver and intestinal mucosa, foci of petechiae in the spleen, and heart and focal ulceration in the dorsal surface of the tongue. Microscopically, the kidneys showed intertubular edema and focal areas of degeneration associated with coagulative necrosis of the tubular epithelium. The liver showed hydropic degeneration and fatty changes of the hepatocytes. There was a massive proliferation of fibroblasts in the interlobular spaces which penetrated the necrosed areas of the hepatic lobules. CONCLUSION: A total of three wild rescued elephant calves and three asymptomatic adults were found positive for EEHV1A during the 2 years of study. The PCR amplified product with size 600 bp had shown the gene sequence for EEHV1U77/HEL.

Unusual rotavirus genotypes in humans and animals with acute diarrhoea in Northeast India.

Rotavirus (RV) infection causes acute infantile diarrhoea in humans and animals and remains a major concern for vaccine development. The close proximity of humans to animals may foster cross-species infection resulting in the emergence of novel/unusual strains by genetic reassortment. In this study, we characterized 500 diarrhoeal samples for group A rotaviruses (RVA) from children (n = 290), piglets (n = 95) and calves (n = 115) in Northeast India during 2012-2013. The data showed that 142/500 (28·4%) faecal samples were positive for RVA with the highest level of infection detected in piglets (57/142, 40·1%) followed by children (51/142, 35·9%) and calves (34/142, 23·9%). Sequence-based G- and P-typing showed G1P[8] (25%) and G1P[7] (35%) were the prevailing genotypes in both humans and animals. Single cases of unusual genotypes, i.e. G9P[8], G5P[8] in humans and G1P[13], G1P[23] and G3P[7] in animals were also identified. Cluster analyses of the sequences showed regional strains were genetically closer to their homologous strains. However, human G5P[8] and porcine G1P[8] strains showed homology to heterologous hosts of their prototype strains. The subsequent global spread of unusual RV strains may result in their establishment over time, presenting challenges to future vaccine evaluation programmes. More studies on emerging genotypes are required to elucidate how RVA strains evolve post-vaccination. This study supports the need for continuous surveillance of RVA infections after detecting from diverse hosts in a common setting.

Classical Swine Fever in Wild Hog: Report of its Prevalence in Northeast India.

Classical swine fever virus (CSFV) is the causative agent of a highly contagious disease, hog cholera in pigs. The disease is endemic in many parts of the world and vaccination is the only way to protect the animals from CSFV infection. Wild hogs belong to the species Sus Scrofa Cristatus under the family Suidae are quite susceptible to CSFV infection. The epidemiological role concerning classical swine fever (CSF) in India is largely unknown. We report here the three isolated cases of CSF in wild hogs from three National parks, namely Kaziranga National Park, Manas National Park and Jaldapara National Park, from north-east part of India. The post-mortem and histopathological findings were clearly indicative for CSFV infection. The presence of CSFV genome was demonstrated in several organs and tissues collected from hogs died due to viral infection. In addition, CSF-specific antibodies were detected in two wild hogs as well as in eighteen feral pigs from the same locations. The phylogenetic analysis of the partial E2 protein gene and 5' untranslated region of CSFV isolates from the wild hog showed identities with genotype 2.2 of the Indian isolates. Occurrence of CSF in wild hogs may pose a potent threat in the epidemiology of the virus in Northeast part of India. To the best of our knowledge, the report presented in the manuscript is the first comprehensive report on CSF in wild hogs form Northeast India. The findings reported would help us to understand the epidemiology and biology of CSFV in wild animals.

Restriction fragment length polymorphism analysis of rotavirus VP7-encoding gene from humans and animals of Northeast India: a relative study of Indian and global isolates.

A restriction fragment length polymorphism (RFLP) assay was developed to examine the genetic relationship between 67 (29 Indian, 38 global) rotavirus isolates of human, bovine and porcine neonates. The assay involved direct digestion of RT-PCR amplified VP7 cDNAs with three restriction enzymes (VspI, HaeIII, NlaIV) independently. Forty-eight RFLP patterns were identified for all 67 strains, and of these 20 patterns were associated with Indian isolates. A correlation between the restriction patterns and G type was apparent through deduction of enzyme restriction sites from known sequences. Major G serotypes (G1, G2, G6, G8) with a few mixed types could be differentiated where there was a positive assortment of intrinsic serotypes from multiple host origin, and certain single or combined enzyme profiles were highly dominant in the population. Significant genetic variations were established between global and Indian isolates and none of the RFLP patterns were shared between them. These data suggest that the Indian wild-type rotavirus population is distinguishable based on the VP7 gene, and co-circulation of distinct strains in different hosts is foremost, indicating the possible likelihood of inter-species transmission.

Comparative efficacy of fluorescent antibody test, immunoperoxidase test and enzyme linked immuno sorbent assay in detection of rotavirus in cell culture.

Rotavirus is prevalent worldwide and has been established as a leading cause of mortality due to severe diarrhoea in neonates. Isolation of the virus is a gold standard method for confirmation of rotavirus infection in the host. Propagation of rotavirus in cell culture is a challenge as in many instances the virus does not produce detectable cytopathic effect. This study was undertaken to evaluate the efficacy of fluorescent antibody test (FAT), immunoperoxidase test (IPT) and sandwich ELISA (S-ELISA) to detect rotavirus antigen propagated in MA 104 cell line. The intensity of fluorescence and colour development for I-FAT and I-IPT was categorized and the ELISA OD values were analyzed. The overall mean of detection were 5.16 ± 0.47, 5.16 ± 0.54 and 5.66 ± 0.33 for I-FAT, I-IPT and S-ELISA, respectively. Significantly less number of samples were positive in the initial one or two passage, which increased up to 100 % from third/fourth passage onwards. The study concluded that I-FAT, I-IPT and S-ELISA were equally effective in detecting propagated rotavirus in cell line, and the former two tests are suitable for in situ demonstration of the virus while the later could be used to assay antigen in cell culture fluid.

Classical swine fever in the pygmy hog.

The pygmy hog is a rare, small and highly endangered mammal belonging to the Suidae family, and it is presently found only in the Assam state of India. While investigating the cause of death of pygmy hogs housed at a conservation centre for captive breeding and research at Basistha, Assam, it was confirmed that they were susceptible to and died as a result of contracting classical swine fever (CSF), caused by CSF virus (CSFV), which is a highly infectious endemic disease of domestic pigs in India. The post-mortem findings and serum CSFV-specific antibody titres, along with the isolation of CSFV from two pygmy hogs, and further confirmation by CSFV genomic E2 and 5' untranslated region (UTR) gene amplification in PCR (polymerase chain reaction), clearly established the cause of death of the pygmy hogs. Further, on phylogenetic analysis, the pygmy hog CSFV 5' UTR sequences were grouped in the genotype 1.1 cluster of Indian CSFVs, and hence the strains causing infection were closely related to CSFV isolates circulating in domestic pigs. Therefore, the occurrence of CSF in this endangered species may pose a potent threat to their existence unless properly controlled, and thus it needs urgent attention. To the authors' knowledge this is the first report on CSF in pygmy hogs.

5'-UTR-based phylogenetic analysis of Classical swine fever virus isolates from India.

Classical swine fever (CSF) caused by Classical swine fever virus (CSFV) is a globally significant disease of pigs. Genetic typing of CSFV isolates can help in understanding the epidemiology of disease and trace down the source of outbreak. 5'-UTR sequence analysis and subsequent genetic classification of nine CSFV field isolates from India indicated that 3 isolates belonged to genotype 2.1 and were closely related to European CSFV strains. The remaining 6 isolates belonged to genotype 1 that contained old and new strains. However, the genotype 2.1 group consisted of recent field isolates only. The study showed circulation of both genotypes 1 and 2.1 in north-eastern part of India.

Characterization and expression of e2 glycoprotein of classical Swine Fever virus in a eukaryotic expression system.

Classical swine fever (CSF) is an economically important Office International des Epizooties (OIE) list A disease of swine characterized by high fever and multiple haemmorhages. The E2 glycoprotein of CSFV is immunogenic and induces neutralizing antibodies against CSFV. In the present study, complete coding region of the E2 gene from Indian virulent field isolate (Mathura) was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and subsequently cloned into a mammalian expression vector; pcDNA3.1(+) at BamHI and XbaI site. The recombinant plasmid; pcDNA.E2.CSFV. was confirmed by restriction enzyme digestion. The pcDNA.E2.CSFV. transfected Vero cell expressed E2 protein which was confirmed by western blotting, immunoperoxidase and indirect immunofluorescent tests. Additionally, flow cytometry analysis also confirmed that 15% of transfected Vero cells expressed the E2 glycoprotein compared to mock or vector alone transfected cells. Further study is under way to evaluate recombinant pcDNA.E2.CSFV. Mathura clone as DNA vaccine against CSFV.

Molecular Characterization of Classical swine fever virus Involved in the Outbreak in Mizoram.

Classical swine fever is the most insidious and devastating disease of pigs and wild boars. The virus is closely related to the other members of the genus Pestivirus. The outbreak recorded in Mizoram, India was strategically important as the state shares porous international boundary with East Asian countries. Both immunodiagnostic and molecular techniques were used to confirm the involvement of Classical swine fever virus (CSFV) in this outbreak. Sandwich ELISA and direct FAT could detect CSFV in the tissue samples. RT-nPCR specifically amplified E2 and 5'NTR product of 271 bp. Phylogenetic analysis showed, that the Mizoram isolate (MZ4/69) was very close to the Chinese strain Shimen-HVRI (93.0%) rather than other Indian isolate (CSF-30-03). Present study provides a valuable sequence based molecular data on Indian isolate of CSFV and will be useful in investigation on transmission of such disease from neighbour countries.

Molecular characterization of shiga like toxin-producing Escherichia coli (STEC) isolates from pigs oedema.

BACKGROUND & OBJECTIVE: An oedema outbreak occurred in a Guwahati pig farm. Escherichia coli isolates from different necropsy samples collected from the dead piglets with oedema were characterized to confirm the virulence. METHODS: Haemolytic E. coli isolates recovered from liver, lung and intestine of pigs with oedema were examined for presence of genes encoding pathogroups such as enteropathogenic Escherichia coli (EPEC), (eae/bfpA), enteroaggregative Escherichia coli (EAggEC), (eagg), enterotoxigive Escherichia coil (ETEC), (elt/est) and shiga like toxin producing Escherichia coli (STEC), (stx1/ stx2) by PCR and molecular typing by randomly amplified polymorphic DNA-PCR (RAPD-PCR). RESULTS: The three haemolytic E. coli recovered from diseased pigs were STEC because of presence of the stx2 and eae genes. Analysis by RAPD-PCR indicated that two of the three isolates were genetically related. INTERPRETATION & CONCLUSION: The isolation of STEC isolates from pigs with oedema was shown. Although the three isolates were untypable, presence of eae and stx2 genes clearly indicated these as prime cause of pig oedema disease. Further, demonstration of STEC in pigs becomes a public health concern, as pigs are potential reservoir of such agents, which may cause human illness.

B and also T lymphocytes migrate via gut lymph to all lymphoid organs and the gut wall, but only IgA+ cells accumulate in the lamina propria of the intestinal mucosa.

In pigs the lymphocytes emigrating from the intestinal wall were collected by cannulating the lymphatics, labeled in vitro using a fluorescent dye and retransfused. The injection of 6.6+/-4.2 x 10(8) cells resulted in a labeling index between 1.5% in intestinal lymph, 0.2% in the spleen and lymph nodes, approximately 0.1% in the intestinal lamina propria and 0.003% in intraepithelial lymphocytes. About 25 % of the injected cells were present in the blood and 1 % was recovered in the lymph. T cells were found in similar proportions in the injected and the recovered cells in the organs (70-80%). The proportion of IgA+ cells among the immigrated cells in the intestinal lamina propria ranged from 5 to 8%, which in absolute numbers was up to 60% of the injected IgA+ cells. T and IgM+ cells did not show a higher accumulation in any organ. These experiments in conventional, unrestrained animals revealed that (1) T cells immigrate into the intestinal lamina propria, (2) preferential migration of IgA+ cells from gut lymph to the intestinal lamina propria is obvious under in vivo conditions and (3) the immigrated IgA+ cells represent a very small population which is difficult to detect when analyzed in relative numbers.

Passive immunization of piglets against enterotoxigenic colibacillosis by vaccinating dams with K88ac pili bearing bacterins.

Day-old-piglets were passively immunized by vaccinating the pregnant sows with K88ac enterotoxigenic Escherichia coli (ETEC) vaccine. High level of ETEC specific antibodies was excreted in colostrum (3733.33 +/- 1152.13) and maintained a detectable level (100.00 +/- 0.00) up to 21 day post partum (DPP). The IgG was the predominant immunoglobulin followed by IgA and IgM. Piglets born of vaccinated dam (group A) and unvaccinated dam (group B) were challenged in 7 day of age. Clinical and faecal scores were significantly (P < 0.01) low in group A than that of group B. Piglets of group A developed mild diarrhoea (33.33%), while all the control piglets developed profuse diarrhoea and 3 of these died before 14 day of challenge infection.

Jejunal and ileal Peyer's patches in pigs differ in their postnatal development.

The postnatal development of the jejunal and ileal Peyer's patches was studied before and after weaning in 1-, 1.5- and 2-month-old pigs. The follicles of the jejunal Peyer's patches grew with age and were two times longer and wider in specified pathogen-free and conventional pigs than in germ-free animals, thus indicating an influence of the living microbial antigens from the gut lumen. In germ-free pigs the size of the ileal Peyer's patch follicles increased between the 1st and 2nd month, whereas in the specified pathogen-free and conventional animals these follicles were comparable in size in all three age groups. In 1- to 1.5-month-old pigs the interfollicular area of jejunal Peyer's patches was wider (0.1 +/- 0.04 mm) than that of the ileal Peyer's patch (0.04 +/- 0.03 mm). Immunohistological studies showed that in germ-free pigs preferentially surface IgM+ but few IgA+ B cells were present in the follicles, domes and dome epithelia. In specified pathogen-free and conventional pigs the B cells expressed different levels of surface or cytoplasmic IgM or IgA. In all groups studied, more T cells were observed in the jejunal than in the ileal Peyer's patch. Here, few T lymphocytes were found because of the small interfollicular areas. Small numbers of Null cells were distributed in the interfollicular regions of all animals. The results show that living microbial antigens have a major influence on the jejunal and ileal Peyer's patches in pigs. The morphological differences between the two types of Peyer's patches are an indication that they develop differently during postnatal life. So far it remains unclear whether these morphological differences reflect a specific function of the pig's ileal Peyer's patch, such as the expansion of the genetically determined B cell repertoire as has been reported for sheep.

Development of bronchus-associated lymphoid tissue in goats.

Development of bronchus-associated lymphoid tissue (BALT) was studied in clinically healthy and diseased Assam local goats. Animals were sacrificed before term as well as at different postnatal periods to screen lung sections for the presence of BALT. In a retrospective study sections prepared from pneumonic lungs were examined for any alteration of BALT. No BALT-like structure was found in neonatal goats. Bronchial lymphoid structures appeared in half of the animals from 1 month of age onward. The frequency of the BALT/4.5 cm2 of lung section ranged from one to two in 1-month-old and three to six in 1-year-old goats. In pneumonic lungs BALT became hyperplastic, and the size was also increased. The incidence of BALT was increased in lungs with fibrotic pneumonia. The number of BALT/section was high (five to eight/4.5-cm2 area) in mesenchymal cell proliferation. The present study shows that BALT did not develop in prenatal periods. But in the presence of potent antigens lymphoid aggregates appeared in the bronchial lamina propria of normal and diseased lungs.

Lymphoid cells in afferent and efferent intestinal lymph: lymphocyte subpopulations and cell migration.

Gut wall emigrating cells have been characterized in the intestinal lymph. The intestinal lymph duct was cannulated in 6-month-old minipigs. Under non-restraining conditions the efferent lymph from the mesenteric lymph nodes was collected in seven normal animals. Lymph coming directly from the gut (afferent lymph) was also collected in 18 pigs after resection of the mesenteric lymph node chains 3 months previously. The intestinal lymph flow was similar in both groups (around 18 ml/h). The lymphoid cell yield was 1.2 +/- 1.0 x 10(6)/h in control animals, while in mesenteric lymph node resected pigs it was around 20 times higher (26.2 +/- 17.6 x 10(6)/h). In the gut-derived lymph 76.5 +/- 8.8% T lymphocytes were observed (CD4+, 48.1 +/- 15.5%; CD8+, 53.6 +/- 12.7%). The percentage of immunoglobulin-positive cells was lower (IgM+, 10.1 +/- 4.5; IgA+, 1.7 +/- 1.1). In 14 mesenteric lymph node resected pigs a mean of 5.6 +/- 3.1 x 10(8) lymphocytes from the gut lymph were labelled in vitro with a fluorescent dye and retransfused. The labelling index of fluorescent cells in the intestinal lymph increased rapidly and remained at a high level until 44 h after cell transfusion. A four-to-ten times lower labelling index was found in the spleen, various lymph nodes and Peyer's patches. Most of the recovered lymphocytes were T cells. This model provides access to the cell pool leaving the gut wall, thus allowing an examination of its role in the gastrointestinal tract and other mucosal-lined organs.