Isolation and genetic characterization of swinepox virus from pigs in India

Swinepox virus (SWPV), a member of the genus Suipoxvirus causes generalized pock-like lesions on the body of domestic and wild pigs. Although outbreak has been reported in India since 1987, virus isolation and genetic characterization remained elusive. In September 2013, an outbreak of acute skin infection occurred in piglets in a commercial piggery unit at Rohtak district in Haryana, India. The presence of SWPV in scab samples collected from piglets succumbed to infection was confirmed by virus isolation, PCR amplification of SWPV-specific gene segments and nucleotide sequencing. Phylogenetic analysis of host-range genes of the SWPV revealed that the Indian isolate is genetically closely related to reference isolate SWPV/pig/U.S.A/1999/Nebraska. To the best of our knowledge this is the first report on isolation and genetic characterization of SWPV from pigs in India.     

Effect of formalin fixation on the immunohistochemical detection of PRRS virus antigen in experimentally and naturally infected pigs.

The purpose of this study was to determine the effect of formalin fixation on the immunohistochemical detection of porcine reproductive and respiratory syndrome (PRRS) viral antigen in lungs of experimentally and naturally infected pigs. In separate trials, five 24-day-old pigs and six 10-day-old pigs were housed as separate groups in isolation and inoculated intranasally with 10(5.5) TCID50 of an isolate of PRRS virus (PRRSV; P129). The older and younger pigs were euthanatized at 7 and 10 days post inoculation (dpi), respectively. At necropsy, all pigs had gross and microscopic lung lesions typical of PRRS, and PRRSV was isolated from all pigs. To insure uniform fixation, lungs from each pig were cut into 1-cm-thick slices and immersed into 10% neutral-buffered formalin. After fixation in formalin for 8 hours or 1, 2, 3, 5, 6, 8, 10, and 15 days, 3 lung sections from some or all pigs were processed for histological examination using routine methods. Immunohistochemical staining for PRRSV antigen was positive at the following times (days unless otherwise stated) after fixation (percentage of pigs staining positive for PRRSV in parentheses): 8 hours (100); 1 (100); 2 (100); 3 (80); 5 (33); and 6, 8, 10, and 15 (0-all negative). To further evaluate the effects of formalin fixation on PRRSV immunodetection, 31 field cases of PRRS were selected for immunohistochemistry (IHC). Over a 3-month period, submitted cases were selected from the Purdue University Animal Disease Diagnostic Laboratory, W. Lafayette, Indiana, for IHC if 1) the clinical history included respiratory disease, 2) PRRSV was isolated from lung and/or serum from the submitted pigs or tissues, 3) at least 1 section of lung fixed in 10% neutral-buffered formalin was submitted for IHC, and 4) the duration of fixation could be accurately determined from the case history. Of the 31 PRRSV-infected pig cases meeting the selection criteria, 23 were fixed in formalin for 4 days or less. Twenty-one of these 23 (91%) were positive by IHC. Two of 8 cases fixed for greater than 4 days (25%) were positive by IHC. In practical terms, 1-day shipping of fixed samples to a laboratory followed by routine tissue processing within a laboratory should not adversely affect immunohistochemical detection of PRRS viral antigen. But a delay in shipping or processing of more than 2 days could reduce or prevent the detection of PRRS viral antigen by IHC.     

Group A rotavirus excretion patterns in naturally infected pigs

Cross-sectional and cohort studies were conducted in a piggery to investigate the excretion pattern of group A rotavirus in pigs. The cross-sectional survey revealed that 47 (9 per cent) of 521 pigs sampled were excreting rotavirus in the faeces. No rotavirus antigen was detected in the faeces of pigs either less than one week or over two months old. The prevalence of infection increased with age over the sucking period, and was greatest at five weeks old. Diarrhoea was observed in only eight (17 per cent) of the pigs excreting rotavirus. Sixteen piglets from four litters were selected and faecal samples collected daily from each animal from birth to two months old. All the piglets excreted group A rotavirus and the range of ages at which they first became infected was between 13 and 39 days. The average duration of excretion in individual piglets was 7.4 days. Ten of 13 sucking piglets which excreted rotavirus developed diarrhoea soon after it was first detected.     

Hepatitis E virus infection dynamics and organic distribution in naturally infected pigs in a farrow-to-finish farm

The objective of the present study was to determine the pattern of Hepatitis E virus (HEV) infection in a naturally infected, farrow-to-finish herd. For that purpose, a prospective study was conducted in randomly selected 19 sows and 45 piglets. Blood samples were collected from sows at 1 week post-farrowing and from piglets at 1, 3, 6, 9, 12, 15, 18 and 22 weeks of age. Furthermore 3 or 5 animals were necropsied at each bleeding day (but at 1 week of age), and serum, bile, liver, mesenteric lymph nodes and faeces taken. HEV IgG, IgM and IgA antibodies were determined in serum and viral RNA was analysed in all collected samples by semi-nested RT-PCR. Histopathological examination of mesenteric lymph nodes and liver was also conducted. From 13 analysed sows, 10 (76.9%) were positive to IgG, one to IgA (7.7%) and two to IgM (15.4%) antibodies specific to HEV. In piglets, IgG and IgA maternal antibodies lasted until 9 and 3 weeks of age, respectively. IgG seroconversion occurred by 15 weeks of age while IgM and IgA at 12. On individual basis, IgG was detectable until the end of the study while IgM and IgA antibody duration was of 4-7 weeks. HEV RNA was detected in serum at all analysed ages with the highest prevalence at 15 weeks of age. HEV was detected in faeces and lymph nodes for the first time at 9 weeks of age and peaked at 12 and 15 weeks of age. This peak coincided with the occurrence of hepatitis as well as with HEV detection in bile, liver, mesenteric lymph nodes and faeces, and also with highest IgG and IgM OD values at 15 weeks. Finally, different HEV sequences from this farm were obtained, which they clustered within 3 different groups, together with other Spanish sequences, all of them of genotype 3. Moreover, the present study also indicates that the same pig can be infected with at least two different strains of HEV during its productive life. This is the first study characterizing HEV infection in naturally infected pigs with chronological virus detection and its relationship with tissue lesions throughout the productive life of the animals.     

Immunohistochemical evaluation of inflammatory infiltrate in the skin and lung of lambs naturally infected with sheeppox virus

The present study describes immunophenotypic characteristics of inflammatory infiltrate in the skin and lung of lambs naturally infected with sheeppox virus (SPV). Three lambs revealed typical cutaneous and pulmonary lesions of sheeppox. Histologically, cutaneous and pulmonary lesions consisted of hyperplastic and/or degenerative changes in the epithelium with mononuclear cells, neutrophils, and typical sheeppox cells (SPCs), which had a vacuolated nucleus and marginated chromatin with occasional granular intracytoplasmic inclusions. The inflammatory infiltrate in pox lesions in both skin and lung was characterized by the presence of MHC II+ dendritic cells, CD4+, CD8+, gammadelta+ T cells, IgM+ cells, and CD21+ cells. Loss of expression of MHC I and MHC II antigens was observed in the affected areas of skin and lung. SPCs, stained with anti-SPV antibody, were also positive for CD14 and CD172A, antigens expressed on monocytes and macrophages. CD14 and CD172A negative SPCs were considered to be SPV infected degenerated epithelial cells or fibroblasts.     

Analysis of swinepox virus antigens using monoclonal antibodies.

Seventeen monoclonal antibodies (MAbs) against swinepox virus (SPV) were produced and characterized. These MAbs were classified into eight groups (A through H) on the basis of the molecular weight of the polypeptides which they recognized and the staining patterns of antigens in SPV-infected cells by the indirect immunofluorescent (IF) technique. The MAbs belonging to groups A, B, C and G recognized late antigens in cytoplasmic inclusion bodies with molecular weights of 97 kD, 65 kD, 48 kD and 15 kD, respectively. The MAbs belonging to groups D and H respectively recognized 35 kD and 12 kD late antigens, which first appeared in cytoplasmic inclusion bodies and spread to the cytoplasms and surface membranes of the infected cells. The MAb of group F recognized an 18 kD late antigen with granular distribution in the cytoplasm. The MAbs of group E recognized a 32 kD early antigen. Although all the MAbs belonging to the six groups (A, D through H) were specific for SPV, some of those belonging to groups B and C showed cross-reactivity with members of the other genera of poxviridae. An MAb in group B, SP14, cross-reacted with orf and rabbit fibroma viruses. Two MAbs in group C, SP24 and SP32, cross-reacted with vaccinia, cowpox, ectromelia, and rabbit fibroma viruses. These findings indicate that at least two SPV antigens contain cross-reactive epitopes with different genera of poxviridae.