B. Kim

Detection and differentiation of porcine epidemic diarrhoea virus and transmissible gastroenteritis virus in clinical samples by multiplex RT-PCR

A multiplex reverse-transcriptase-PCR (RT-PCR) procedure was developed for the simultaneous detection of porcine epidemic diarrhoea virus (PEDV) and transmissible gastroenteritis virus (TGEV) in preweaning pigs with diarrhoea. The membrane gene of PEDV and the nucleocapsid gene of TGEV were chosen as targets. The PCR products of PEDV and TGEV had molecular sizes of 412 and 612 base pairs, respectively. Primers from PEDV did not react with any TGEV tested and vice versa. In addition, the primers did not react with other pig viruses. The multiplex RT-PCR was able to detect 10 tissue culture-infective doses 50 per cent (TCID50)/ml of PEDV or TGEV with each of the primer sets for PEDV and TGEV, respectively. The RNAS of PEDV and TGEV were detected directly in intestinal and faecal samples from pigs infected experimentally with either virus. The results of the assay correlated well with the results of virus isolation. None of the five control specimens was positive. PEDV was detected in 10 intestinal and nine faecal samples, and among the nine positive faecal samples two were culture-negative. TGEV was also detected in 10 intestinal and nine faecal samples, and among the nine positive faecal samples, three were culture-negative.

Reproduction of Postweaning Multisystemic Wasting Syndrome in Pigs by Prenatal Porcine Circovirus 2 Infection and Postnatal Porcine Parvovirus Infection or Immunostimulation

Postweaning multisystemic wasting syndrome (PMWS) was reproduced in prenatally porcine circovirus 2 (PCV2)-infected pigs by either postnatal infection with porcine parvovirus (PPV) or by immunostimulation. Twenty-four randomly selected piglets from 3 sows, which had been experimentally infected during gestation with PCV2, were randomly divided into 3 groups; group 1 (prenatal PCV2 infection, with postnatal PPV infection), group 2 (prenatal PCV2 infection, with postnatal keyhole limpet hemocyanin, emulsified in incomplete Freunds adjuvant [KLH/ICFA] injection), and group 3 (prenatal PCV2 infection only). Twenty-four randomly selected piglets from 3 uninfected sows were randomly divided into 3 groups; group 4 (no prenatal infection, with postnatal PCV2 and PPV infection), group 5 (no prenatal infection, with postnatal PCV2 infection), and group 6 (negative control pigs). Body weight in negative control pigs (group 6) was increased significantly compared with pigs in groups 1, 2, and 4 at 49, 52, 56, 59, and 63 days of age. The granulomatous inflammatory reaction and lymphoid depletion that are typical lesions in pigs with PMWS were observed in the lymph node of piglets in groups 1, 2, and 4 at 63 days of age. Pigs in group 3 had significantly fewer PCV2-positive cells than those from groups 1,2,4, or 5. When the prenatally PCV2-infected pigs were infected with PPV or injected with immunostimulant in the postnatal period, they developed PMWS. Thus, factors that potentiate the progression of prenatal PCV2 infection to PMWS are postnatal infection with PPV or immune stimulation.

Neutrophilic dermatoses associated with myeloid malignancy.

The neutrophilic dermatoses are significantly associated with myeloid malignancies. We now describe the clinical and histological features of 11 patients with these disorders, namely Sweets syndrome in three cases, pyoderma gangrenosum in two, and neutrophilic eccrine hidradenitis in one; there were also five others which could not be categorised as recognised entities. Our observations, as well as those from a review of the literature, support the hypothesis that in the neutrophilic dermatoses associated with myeloid malignancy, a common mechanism may he involved.

Diarrhoea in nursing piglets associated with coccidiosis: prevalence, microscopic lesions and coexisting microorganisms.

A retrospective study was made of natural infections with Isospora suis in nursing piglets, recorded from April 1994 to May 1997, to determine the prevalence, microscopical lesions and other microorganisms associated with coccidiosis. One hundred and five (17.3 per cent) of the 605 nursing piglets submitted from 304 pig farms were diagnosed positive for coccidiosis. The affected piglets were from seven to 20 days old, with a mean age of 11.1 days. Coccidiosis occurred in each year but the incidence peaked in July (15 cases, 14.3 per cent), September (15 cases, 14.3 per cent), October (16 cases, 15.2 per cent) and November (18 cases, 17.1 per cent) and was lowest in May (no cases), August (two cases, 1.9 per cent) and June (four cases, 3.8 per cent). Histopathologically, villous atrophy resulting from the necrosis and sloughing of epithelial cells was a prominent feature of infection with I suis. In 49 5 per cent of the nursing piglets, other enteropathogens were identified, Escherichia coli (47.6 per cent) and transmissible gastroenteritis virus (3.8 per cent) being the most commonly diagnosed. Forty-five of 50 E coli isolates associated with coccidiosis tested negative by polymerase chain reaction for enterotoxigenic virulence factors, such as fimbriae and enterotoxins.

PREVALENCE OF LAWSONIA INTRACELLULARIS IN SELECTED PIG HERDS IN KOREA AS DETERMINED BY PCR

1993). The semi-wild life of the affected deer gave the tumour a long period for growth (as indicated by the long hoof of the affected leg) and for the invasion and destruction of the adjacent bones. However, metastases to the other organs were not found. This is in accordance with the behaviour of malignant human (Enzinger and Weiss 1988) and canine (Cordy 1990, Walder and Gross 1992, Yager and others 1993) schwannomas which very rarely develop metastases. The anti-S-100 pAb has been tested in red deer tissues and showed cross reactivity (Carrasco and others 1998). The remaining antibodies also cross reacted with the red deer tissues used as a control with a distribution pattern similar to that reported for humans beings (Osborn and Weber 1982) and domestic species (Moore and others 1989, Martin de las Mulas and others 1994). Human schwannomas express vimentin and S-100 protein (Enzinger and Weiss 1988, Johnson and others 1988), canine schwannomas also express vimentin and five of seven cases also expressed S-100 protein (Perez and others 1996), and a single case of schwannoma in a pig also expressed both vimentin and S-100 protein (Tanimoto and Ohtsuki 1993). In the present case, almost all the neoplastic cells had diffuse cytoplasmic immunoreactivity to vimentin and numerous tumour cells, mainly in well differentiated areas with a palisade pattern, had nuclear and cytoplasmic immunostaining for S-100 protein whereas highly anaplastic neoplastic cells did not express this protein. The expression of vimentin suggests that the tumour had a mesenchymal origin (Enzinger and Weiss 1988, Moore and others 1989), and the expression of S-100 protein rules out a diagnosis of fibrosarcoma, or muscle tumour, and strongly suggests a diagnosis of schwannoma, which in the present case was also consistent with histopathological patterns of Antoni type A and type B observed in extensive areas of the tumour. The antibodies used therefore cross reacted with normal deer tissues, and could be used as tumour markers in deer, although studies of other neoplasms would be required establish whether they have the same immunophenotype as that reported in domestic species such as the dog (Moore and others 1989, Perez and others 1996) and pig (Tanimoto and Ohtsuki 1993).

Evidence of shedding of porcine circovirus type 2 in milk from experimentally infected sows.

Detection of porcine circovirus type 2 (PCV2) was to evaluate the milk from experimentally infected sows using polymerase chain reaction (PCR) and virus isolation. Six pregnant sows were inoculated intranasally with PCV2 at 93 days of gestation, and milk samples were collected from all sows at 1, 3, 6, 9, 12, 15, 18, 21, 24, and 27 days of lactation. PCV2 was detected in milk as early as day 1 of lactation in all six sows. Thereafter, all infected sows remained positive by PCR for PCV2 in milk until 27 days of lactation. In addition, PCV2 itself was isolated from milk collected from a virus-infected sows. These results suggest that PCV2 may be shed in milk following infection of pregnant sows by the virus.

Differentiation between porcine epidemic diarrhea virus and transmissible gastroenteritis virus in formalin-fixed paraffin-embedded tissues by multiplex RT-nested PCR and comparison with in situ hybridization.

Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) were detected and differentiated in formalin-fixed, paraffin-embedded tissues from experimentally and naturally infected pigs by multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR). The results of this new method were compared with in situ hybridization. A method based on xylene deparaffinization followed by proteinase K digestion yielded RNA of a suitable quality for reliable and consistent multiplex RT-nPCR analyses. PEDV and TGEV cDNAs were detected in jejunal tissues from experimentally and naturally infected pigs by multiplex RT-nPCR. Distinct positive signals for PEDV and TGEV were also detected in the same jejunal tissues by in situ hybridization. The rate of conformity between multiplex RT-nPCR and in situ hybridization was 100% for the detection of PEDV and TGEV in formalin-fixed paraffin-embedded jejunal tissues.

In Situ Hybridization for the Detection and Localization of Swine Chlamydia trachomatis

Gnotobiotic piglets were inoculated intralaryngeally with swine Chlamydia trachomatis strain R33 or orally with swine C. trachmatis strain R27. Archived formalin-fixed, paraffin-embedded tissues from piglets euthanatized 4-7 days postinoculation were examined by in situ hybridization for C. trachomatis nucleic acid using a nonradioactive digoxigenin-labeled DNA probes that targeted specific ribosomal RNA or ompl mRNA molecules of the swine C. trachomatis strains. Positive hybridization signals were detected in bronchial epithelial cells, bronchiolar epithelial cells, pneumocytes, alveolar and interstitial macrophages, and jejunal and ileal enterocytes. Chlamydia-infected cells had a strong signal that was confined to the intracytoplasmic inclusions. Positive hybridization signals were not detected in tissue sections from an uninfected control piglet or in C. psittaci-infected sheep placenta. The morphology of host cells was preserved despite the relatively high temperature required in parts of the incubation procedure. The data indicate that in situ hybridization can be used to detect swine C. trachomatis in formalin-fixed, paraffin-embedded tissue specimens.