C. Chae

Comparison of the virulence of European and North American genotypes of porcine reproductive and respiratory syndrome virus in experimentally infected pigs

The objective of this study was to compare the virulence of Korean types 1 and 2 porcine reproductive and respiratory syndrome virus (PRRSV) isolated from weaned pigs with respiratory disease. Affected pigs were within the same herd and animals infected with type 2 virus had significantly higher mean rectal temperatures than those with type 1 virus between days 2 and 9 post-inoculation (P<0.05). Similarly, mean serum viral titres, expressed as tissue culture infective doses 50% (TCID50)/mL, as well as macroscopic and microscopic pulmonary lesion scores, were significantly higher at multiple time points in pigs infected with type 2 PRRSV compared to those infected with type 1 virus. Mean numbers of PRRSV-positive cells/unit area of lungs and lymph nodes were also significantly higher in type 2 PRRSV infected pigs. This study demonstrates that type 2 PRRSV is more virulent than type 1 PRRSV in this experimental setting as reflected by the pulmonary pathology induced, the extent of virus distribution, and oral shedding of the virus.

Localisation of swine hepatitis E virus in experimentally infected pigs.

The distribution of intravenously inoculated swine hepatitis E virus (HEV) was assessed by in situ hybridisation for a period of 50 days. Evidence of apparent clinical disease was found in only one pig in the HEV infected group. The only gross lesion observed was mildly enlarged mesenteric lymph nodes at 50 days post infection (dpi). Histopathologically, mild lymphoplasmacytic infiltration and focal hepatocellular necrotic lesions were found in HEV-infected pigs. Swine HEV nucleic acids were detected by RT-PCR in the faeces at 3 dpi in 100% of the 18 pigs infected with the virus. Thereafter, the number of positives declined. The most consistent and intense signal was found in the liver of infected animals using in situ hybridisation. The positive cells were hepatocytes, Kupffer cells, bile epithelial cells and interstitial lymphocytes. Swine HEV RNA was localised in the cytoplasm of the hepatocytes, with a slightly granular pattern of staining, but hybridisation signals were not observed in degenerative or vacuolated hepatocytes. HEV was much less frequently detected in extrahepatic tissues such as lymph nodes, tonsil, spleen and small and large intestine. It was concluded that swine HEV had replicated primarily in the hepatocytes and infection resulted in subclinical infection with minimal histopathological changes in the liver.

Pathogenesis of Korean type 1 (European genotype) porcine reproductive and respiratory syndrome virus in experimentally infected pregnant gilts.

The aim of this study was to elucidate the pathogenesis of experimental infection with Korean type 1 porcine reproductive and respiratory syndrome virus (PRRSV) by defining the virus distribution, sites of viral replication, viraemia and gross and microscopical lesions in conventional pigs studied for 28 days after intranasal inoculation. Mean rectal temperature was significantly higher in infected pigs than in negative control pigs at 2 days post inoculation (dpi) (P=0.004), 3 dpi (P<0.001), 4 dpi (P=0.003) and 5 dpi (P=0.034). The log(10)TCID(50)/ml of type 1 PRRSV increased significantly at 0-1 dpi (P=0.024) and 5-7 dpi (P=0.029), but decreased at 10-14 dpi (P=0.026) and 14-21 dpi (P=0.012) in infected pigs. Infected pigs developed multifocal, tan-mottled areas of lung tissue with irregular and indistinct borders. Microscopical lesions, when present, were multifocal, mild to moderate, generally most extensive at 5-7 dpi (P=0.036), and were nearly resolved at 28 dpi. Type 1 PRRSV nucleic acid and antigen were detected exclusively within the cytoplasm of macrophages and type I and II pneumocytes. The score for PRRSV-positive cells increased at 3-7 dpi (P<0.05) and decreased at 10-14 dpi (P=0.034) in infected pigs. Thus, respiratory disease was reproduced in conventional pigs by infection with Korean type 1 PRRSV.

Role of AMP-activated Protein Kinase and Adiponectin during Development of Hepatic Steatosis in High-fat Diet-induced Obesity in Rats

Obesity, an abnormal condition of adipose tissue, has recently been recognized as a major cause of metabolic syndromes, especially non-alcoholic fatty liver disease (NAFLD). The aim of the present study was to examine the possible involvement of adipokines in the development of fatty liver. Sprague-Dawley (SD) rats fed a high-fat (HF) diet for 15 weeks developed increased hepatocellular vacuolation, hepatic triglyceride (TG) content and serum TG, total cholesterol and free fatty acid levels, with increases in adipose tissue mass. The serum concentration of adiponectin decreased slightly in these animals. Western blotting analysis demonstrated a decrease in the levels of AMP-activated protein kinase (AMPK) and phosphorylated-AMPK in the livers of these rats. These results indicate similarities between the diet-induced obesity rat model of NAFLD and human NAFLD, thus making the rat a useful model for the further study of NAFLD, including the interactions between adipokines and hepatic fat metabolism.

Genotypic prevalence of the fimbrial adhesins (F4, F5, F6, F41 and F18) and toxins (LT, STa, STb and STx2e) in Escherichia coli isolated from postweaning pigs with diarrhoea or oedema disease in Korea.

A PCR was used to determine the genotypic prevalence of five fimbrial adhesins (F4, F5, F6, F41 and F18), two heat-stable enterotoxins (STa and STh), the heat-labile enterotoxin (LT), and the shiga toxin 2e (Stx2e) in 230 isolates of Escherichia coli from postweaning pigs with diarrhoea or oedema disease. Ninety-four (40.9 per cent) of the isolates carried genes for at least one of the fimbrial adhesins or toxins. Genes for the F18 fimbrial adhesin were detected in 18.3 per cent, and genes for F4, F6, F5 and F41 were detected in 10.0 per cent, 4.3 per cent, 1.7 per cent and 0.8 per cent of the isolates, respectively. Genes for STa, STb and LT were detected in 25.7 per cent, 15.2 per cent and 8.7 per cent of the isolates, respectively. Genes for Stx2e were detected in 36 (15.6 per cent) of the isolates, and among them 24 also contained the gene for F18ab and four also contained the gene for F18ac.

Detection of Porcine Circovirus 2 in Mammary and Other Tissues from Experimentally Infected Sows

The aim of this study was to determine whether porcine circovirus 2 (PCV2) may infect the mammary gland of sows and be shed in the milk. Six pregnant sows were inoculated intranasally with PCV2 three weeks before their expected farrowing date and two further sows acted as uninfected controls. The animals remained clinically healthy and farrowed normally. Milk samples were collected from all sows on the first, second and third days of lactation. PCV2 DNA was detected in the milk of infected sows from day 1 of lactation but not in the milk of uninfected controls. PCV2 antigen and DNA were detected in the mammary gland and other tissues by immunohistochemistry and in-situ hybridization, respectively. Simultaneous detection of viral protein and DNA provided molecular evidence of PCV2 infection and replication within these tissues.

Necrotising lymphadenitis associated with porcine circovirus type 2 in pigs

PORCINE circovirus type 2 (PCV-2), a single-stranded DNA virus within the family Circoviridae, is now recognised as the aetiological agent of postweaning multisystemic wasting syndrome (PMWS) (Mankertz and others 1997, Allan and Ellis 2000, Chae 2004). In addition to PMWS, PCV-2 has convincingly been linked to porcine dermatitis and nephropathy syndrome, porcine reproductive disorders and porcine respiratory disease complex (Choi and Chae 2001, Kim and others 2003, 2004, Chae 2005). This short communication describes PCV-2-associated necrotising lymphadenitis, which may be a new clinical manifestation of PCV-2 infection in pigs. Seven cases of necrotising lymphadenitis were diagnosed from seven herds in the Republic of Korea. All herds were continuous farrowing units. Two pigs, aged 56 and 62 days, were submitted because of a retardation of growth that was accompanied by diarrhoea. Four pigs, aged 43, 58, 60 and 73 days, were presented with a slight incoordination and pyrexia (40·5 to 41·7°C) and one 65-day-old pig died suddenly without showing clinical signs. Gross necropsy findings in all submitted pigs included homogeneous, white cut surfaces of the inguinal lymph node. Tissue samples were taken from three pigs and processed for histology and virus isolation (Allan and others 1998). Lymph node and kidney samples were frozen and stored at –70°C for subsequent virus isolation. Bacterial culture was performed on samples of the lungs, livers, lymph nodes, kidneys, and small and large intestines from all seven pigs. Immunohistochemistry for PCV-2, classical swine fever virus (CSFV) and porcine reproductive and respiratory virus (PRRSV) was carried out using monoclonal mouse anti-CSFV antibody (WH303), monoclonal mouse anti-PCV-2 antibody and monoclonal mouse anti-PRRSV antibody (SDOW17) as described by Cheon and Chae (1999) and Choi and Chae (2003). In situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nicked end labelling (TUNEL) was also carried out to detect apoptotic markers as described by Choi and Chae (2002). Two 60-day-old colostrum-deprived specific pathogen-free pigs not showing signs of infection with any known pathogenic porcine virus were used as negative controls. Positive controls were included in the immunohistochemistry and TUNEL staining (Cheon and Chae 1999, Choi and Chae 2002, 2003). PCV-2 was isolated from six lymph nodes from six pigs. No other virus was isolated. In addition, no bacteria was isolated from all seven lymph nodes. The predominant microscopic lesions in the lymph nodes were follicular necrosis in the centre of prominent lymphoid follicles. Necrotic foci of one to 10 cells showing pyknosis and karyorrhexis were commonly observed in the centre of prominent follicles and less often in the surrounding lymphoreticular tissues (Fig 1). Necrotic cells were characterised by cell shrinkage, cytoplasm and chromatin condensation, and some contained nuclear fragments. Granulomatous inflammation and intracytoplasmic inclusion bodies were not seen in any of the lymph nodes from the pigs examined. PCV-2 antigens were detected in all lymph nodes with necrotic foci (Fig 2) but were absent in lymph nodes without necrotic foci by immunohistochemistry. Other viruses such as CSFV and PRRSV were not detected in the lesions. Application of the TUNEL reaction on sections of affected lymph nodes revealed intense, specific staining in nuclei and nuclear fragments of lymphocytes (Fig 3). TUNEL-positive cells had dark brown staining. Apoptotic bodies of various sizes exhibited distinct staining and the cytoplasm of apoptotic cells was also often stained. Sections from the negative control pigs were negative for PCV-2 by immunohistochemistry. The microscopic lesions observed in the necrotising lymphadenitis were different from those seen with PMWS. Granulomatous inflammation and intracytoplasmic inclusion bodies are characteristic histopathological lesions of PMWS but these two lesions were not observed in the necrotising lymphadenitis in the present study. Clinical signs of PMWS were not seen in any of the herds examined. Furthermore, an additional 14 pigs, two from each of the seven herds, were also examined to determine the status of the PCV-2 and PMWS. Necrotising lymphadenitis was not seen in any of these pigs, and no microscopic lesions of PMWS or PCV-2 were observed by histopathology or immunohistochemistry, respectively. Therefore, necrotising lymphadenitis was not linked to PMWS in any of the seven herds. The mechanism of apoptosis in these lesions was not determined. Apoptosis has been proposed to account for the

Lack of evidence of porcine circovirus type 1 and type 2 infection in piglets with congenital tremors in Korea.

Chae (2003). Positive and negative control tissues for each virus were included in each in situ hybridisation procedure (Kim and Chae 2001, Choi and Chae 2003). On microscopic examination of the tissues, mild to moderate cerebellar and spinal hypomyelination was detected in 33 of the 36 piglets. Neither PCV-1 nor PCV-2 nucleic acid was detected in any brain or spinal cord samples from the 36 piglets by in situ hybridisation; hybridisation signals of PCV-1 were seen occasionally in liver tissue from one piglet and lymph node from another piglet. PCV-2 DNA was also detected occasionally in the liver and spleen in one piglet, and in the spleen and lymph node in two other piglets with congenital tremor. No piglet had hybridisation signals of both PCV-1 and PCV-2. The granulomatous inflammatory reaction that is typical of lesions associated with PCV-2 infection in pigs with PMWS was not observed in the liver or lymph node from the PCV-1or PCV-2-positive piglets. CSF virus was not detected in any tissue of any of the piglets by in situ hybridisation. All the tests for PCV-1, PCV-2 and other cytopathic viruses were negative by the virus isolation procedure. In the present study of 36 cases of congenital tremor in piglets in Korea, no evidence of PCV-1 or PCV-2 nucleic acid was found in the central nervous system by in situ hybridisation. PCV-1 and PCV-2 were detected in non-neuronal tissues, such as the liver, spleen and lymph node, in two and three piglets, respectively. However, the identification of PCV-2 may not be significant, since PCV-2 can be detected in the lymph nodes of normal pigs by PCR and in situ hybridisation (Chae 2004). The detection of PCV-1 and PCV-2 in day-old piglets was interpreted as a consequence of infection in utero. However, it could not be ruled out that the piglets had been infected with PCV-1 or PCV-2 postnatally. The absence of CSF virus, the involvement of both male and female piglets, the absence of British Saddleback parentage and the lack of prenatal exposure to organophosphates excludes congenital tremor types A1, A3, A4 and A5, respectively. Therefore, the present cases with cerebellar and spinal hypomyelination may be classified as congenital tremor type A2; the two cases with no such histological lesions can be classified as congenital tremor type B. These results support those of researchers in Europe who failed to detect PCV infection in the central nervous system or non-neuronal tissues of piglets with congenital tremor (Kennedy and others 2003). These results are contradictory to those of a previous study, which found that PCV isolates from cases of congenital tremor type A2 can cause congenital tremor when inoculated into susceptible pregnant sows (Hines and Lukert 1994). Furthermore, PCV-2 was detected mainly in the brain and spinal cord from piglets with congenital tremor by Stevenson and others (2001). The apparent lack of consistency regarding the presence or absence of PCV infection in piglets with congenital tremor is difficult to explain. It is possible that a geographical difference exists in the involvement of PCV in congenital tremor; PCV may be associated with congenital tremor in pigs in the USA, but not in European and Asian countries. Further studies are therefore needed to determine the role of PCV in congenital tremor type A2.

Prevalence of porcine circovirus types 2a and b in pigs with and without post-weaning multi-systemic wasting syndrome

The objective of this study was to determine the prevalence of porcine circovirus (PCV) type 2 genotypes in pigs with post-weaning multi-systemic wasting syndrome (PMWS) and in pigs that that did not have the disease. Nested PCR was used to analyse tissue from 540 formalin-fixed, paraffin-embedded lymph nodes. The number of PCV2a-positive pigs that had (χ(trend)(2)=54.584, P<0.001) and did not have (χ(trend)(2)=70.066, P<0.001) PMWS decreased significantly between the years 2000 and 2008. However, over this time-period, there was a significant increase in the number of animals infected with PCV2b that had (χ(trend)(2)=31.356, P<0.001) and did not have (χ(trend)(2)=9.494, P<0.001) PMWS. The findings demonstrate a significant overall increase in PCV2b infection in pigs that both have and do not have PMWS. Further studies will be required to determine the potential relationship between PCV2b infection and the increasing incidence of PMWS in Korea.

Expression of Mucins and Trefoil Factor Family Protein-1 in the Colon of Pigs Naturally Infected with Salmonella typhimurium

The expression patterns of different mucins (MUC1, MUC2, MUC4, MUC5AC, MUC5C and MUC6) and trefoil factor family protein-1 (TFF1) in the colon of healthy pigs and pigs naturally infected with Salmonella typhimurium is reported. Twenty infected pigs approximately 80-160 days of age from 20 different herds were studied. These animals had similar microscopical change in colonic tissue characterized by mucosal erosion or sloughing and acute inflammation. S. typhimurium was cultured from all lesions and the identity of each isolate was confirmed by serotyping. Immunohistochemical studies of colonic tissue revealed reduced expression of MUC4 on the surface of the cryptal epithelium of S. typhimurium-infected pigs compared with non-infected pigs (P<0.001). By contrast, colon from infected animals had increased expression of MUC5AC (P<0.0001) and TFF1 (P=0.0095) relative to controls and there was a significant positive correlation between expression of these two molecules (Spearman coefficient 0.64, P<0.0001). Further studies are needed to evaluate the functional relationship between altered expression of these molecules and inflammation in Salmonella-infected pigs.