Chanhee Chae

Porcine postweaning multisystemic wasting syndrome in Korean pig: detection of porcine circovirus 2 infection by immunohistochemistry and polymerase chain reaction.

This report describes the first diagnosis of porcine circovirus (PCV) infection in weaned pigs with postweaning multisystemic wasting syndrome in Korea by immunohistochemistry and polymerase chain reaction. The most unique lesions were multifocal granulomatous inflammation affecting lymph nodes, liver, and spleen, characterized by infiltrates of epithelioid macrophages and multinucleated giant cells. Circoviral antigen was detected in formalin-fixed sections and was usually present in large, round, dendritic cells in the white pulp of spleen and remnants of follicles in lymph nodes. Lymphoid follicles in the tonsils also contained PCV antigen. A 530–bp DNA fragment of circovirus was successfully amplified from all tested lymph nodes, liver, and spleen.

Differentiation of porcine circovirus (PCV)-1 and PCV-2 in boar semen using a multiplex nested polymerase chain reaction

A multiplex nested polymerase chain reaction (PCR) was developed for the detection of and differentiation between porcine circovirus (PCV)-1 and PCV-2 in boar semen. Eighteen (30%) and 30 (50%) out of 60 whole semen samples were found to be positive for PCV using multiplex conventional PCR and multiplex nested PCR, respectively. Of the 30 positive samples obtained using multiplex nested PCR, two were found to be positive for PCV-1 only, eight for PCV-2 only, and 20 for PCV-1 and PCV-2. When the separated fractions of PCV-contaminated semen were analyzed using multiplex nested PCR, PCV DNA was found to be present mainly in the seminal fluid and nonsperm cell fractions. When compared with the virus isolation method commonly used to detect viruses, this PCR assay was found to be more sensitive and rapid and, as such, may prove to be a good alternative method for the detection of and differentiation between PCV-1 and PCV-2 in boar semen.

Commercial porcine circovirus type 2 vaccines: efficacy and clinical application.

Porcine circovirus type 2 (PCV2) is the one of the most economically important pathogens of pigs. After postweaning multisystemic wasting syndrome (PMWS) was first identified and reported in western Canada in 1991, it took 13years for the first commercial PCV2 vaccine to be used under special licence in France and Germany in 2004. Along with PMWS, PCV2 is also associated with a number of diseases and syndromes, collectively referred to as porcine circovirus-associated disease (PCVAD). Currently, five commercial vaccines are available on the international market. Commercial PCV2 vaccines were initially developed to control PMWS, but they are now also used against other PCVAD. This review focuses on (1) the types of commercial vaccines; (2) the criteria of vaccine efficacy; (3) the clinical, virological, immunological and pathological efficacy of the vaccines; and (4) the use of PCV2 vaccines against different clinical manifestations of PCVAD.

Prevalence of porcine circovirus type 2 in aborted fetuses and stillborn piglets

A retrospective study of natural cases of abortion, recorded between October 2000 and September 2002, was made to determine the prevalence of abortions associated with porcine circovirus type 2 (PCV-2). The virus was detected by PCR, immunohistochemistry and in situ hybridisation. A total of 46 (13.1 per cent) of 350 aborted fetuses and stillborn piglets were positive for PCV-2 by PCR, and the virus was detected in fetuses at all stages of gestation. Viral antigen was detected in macrophages from the aborted fetuses and stillborn piglets by immunohistochemistry, and viral DNA was detected by in situ hybridisation.

Localization of swine hepatitis E virus in liver and extrahepatic tissues from naturally infected pigs by in situ hybridization

BACKGROUND/AIMS The objective of this study was to detect and localize the swine hepatitis E virus (HEV) in the liver and extrahepatic tissues from 20 pigs naturally infected with swine HEV. METHODS cDNA probe 289 base pairs for swine HEV were generated by reverse transcription-polymerase chain reaction. In situ hybridization with a nonradioactive digoxigenin-labeled cDNA probe was used for the detection of swine HEV in formalin-fixed, paraffin-embedded tissues. RESULTS When liver tissues from the pigs naturally infected with swine HEV were hybridized with the nonradioactive digoxigenin-labeled cDNA probe, a strong signal was seen in hepatocytes and bile duct. Positive hybridization signals were also detected in small and large intestine, lymph node, tonsil, spleen, and kidney. CONCLUSIONS Swine HEV was detected primarily in the hepatocytes. Swine HEV may also replicate in tissues other than the liver. In situ hybridization described in the present study has greatly facilitated the application of in situ hybridization procedures to the clinical setting, thus allowing for the diagnosis of swine HEV infection while preserving the morphology of the tissue.

Optimized protocols for the detection of porcine circovirus 2 DNA from formalin-fixed paraffin-embedded tissues using nested polymerase chain reaction and comparison of nested PCR with in situ hybridization

Optimized DNA extraction method and nested polymerase chain reaction (PCR) were developed for the detection of porcine circovirus 2 (PCV2) from formalin-fixed, paraffin-embedded tissues. Conventional PCR, nested PCR, and in situ hybridization methods were also compared for the detection of PCV2 in archival tissues. A method based on xylene deparaffinization followed by proteinase K digestion yielded DNA of sufficient quality for PCR analyses reliably and consistently. Twenty-six (70%) of the 37 tissues examined gave positive results with conventional PCR, whereas all the 37 tissues gave positive results using the nested PCR. A distinct positive signal for PCV2 was detected in spleen and lymph node from all the 37 pigs by in situ hybridization. The nested PCR and in situ hybridization could be applied successfully to archival tissues for the detection of porcine circovirus 2 DNA.

Comparison of virus isolation, reverse transcription-polymerase chain reaction, immunohistochemistry, and in situ hybridization for the detection of porcine reproductive and respiratory syndrome virus from naturally aborted fetuses and stillborn piglets.

Virus isolation, reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and in situ hybridization methods were compared for the detection of porcine reproductive and respiratory syndrome virus (PRRSV). Seven aborted fetuses and 6 stillborn piglets naturally infected with PRRSV were used in the study. Viral antigen and viral nucleic acid were detected in macrophages and dendritic cells in the spleen, tonsil, lymph nodes, and thymus; in macrophages of liver, heart, and lung; and in endothelial cells and myocytes of the heart. Viral antigen and viral nucleic acid were most consistently detected in the spleen. Of the 13 samples, 6 were positive for PRRSV by all 4 techniques. Four (31%) samples were positive for PRRSV by RT-PCR, in situ hybridization, and virus isolation. Two (15%) samples were positive for PRRSV by virus isolation, RT-PCR, and in situ hybridization. One (8%) was positive for PRRSV by virus isolation and RT-PCR. The RT-PCR identified the presence of PRRSV more frequently than the other methods. However, when only formalin-fixed tissues are submitted, immunohistochemistry and in situ hybridization would be useful methods for the detection of PRRSV antigen and nucleic acid.

Expression of interleukin-10 and interleukin-12 in piglets experimentally infected with porcine reproductive and respiratory syndrome virus (PRRSV).

The expression of mRNA encoding interleukin-10 (IL-10) and IL-12 was studied, by the reverse transcription-polymerase chain reaction and by in-situ hybridization with a non-radioactive digoxigenin-labelled cDNA probe, in formalin-fixed, paraffin wax-embedded lung tissue from piglets inoculated intranasally with a Korean isolate (North American genotype) of porcine reproductive and respiratory syndrome virus (PRRSV). IL-12p35-positive cells were detected in the lung at 1 day post-inoculation (dpi), their number increasing at 5 dpi, and rapidly decreasing thereafter. In contrast, IL-10- and IL-12p40-positive cells were detected in the lung at 1 dpi, their number increasing at 3 dpi, and rapidly decreasing thereafter. Hybridization signals for IL-10, IL-12p35 and IL-12p40 were always associated with inflammation. Expression of these cytokines was minimal in non-lesional lung of PRRSV-infected piglets and in normal lung from control piglets. In-situ hybridization in serial sections of lung tissues indicated close co-localization of PRRSV and these cytokines in interstitial pneumonia. The results suggest that the expression of IL-10 and IL-12 plays a role in pulmonary defence mechanisms against PRRSV infection.

Prevalence of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) gene in isolates in weaned pigs with diarrhea and/or edema disease.

A total of 476 Escherichia coli isolated from weaned pigs with diarrhea and/or edema disease were screened for the presence of the enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1) gene by polymerase chain reaction (PCR). E. coli strains that carried EAST1 genes were also tested by PCR for the presence of genes for five fimbriae (F4, F5, F6, F18 and F41), two heat-stable (STa and STb) and one heat-labile (LT) enterotoxin, and Shiga toxin 2e (Stx2e). One hundred and forty nine (31.3%) of the 476 E. coli isolates carried the gene for EAST1. Of these 149 isolates, 66 (44.3%) carried the east1 gene only and 83 (55.7%) carried genes for the fimbrial adhesins or enterotoxins. E. coli which carried east1 gene also possessed genes for STa or F4 frequently. EAST1 may represent an additional determinant in the pathogenesis of E. coli diarrhea in weaned pigs.

Detection of nucleic acids of porcine reproductive and respiratory syndrome virus in the lungs of naturally infected piglets as determined by in-situ hybridization

Summary Replication of porcine reproductive and respiratory syndrome virus (PRRSV) was studied in formalin-fixed paraffin wax-embedded lung tissues from seven naturally infected piglets by in-situ hybridization with a non-radioactive digoxigenin-labelled probe. A 433 base pair cDNA probe for the viral RNA encoding the nucleocapsid proteins of a Korean PRRSV isolate was generated by the polymerase chain reaction. All seven piglets infected with PRRSV showed a distinct, positive signal, scattered throughout the alveolar septa and spaces. Positive cells typically exhibited dark brown staining deposits in the cytoplasm without background staining. In-situ hybridization demonstrated that PRRSV replicated primarily in interstitial and alveolar macrophages, and occasionally in type 2 pneumocytes. The bronchial or bronchiolar epithelium did not exhibit a hybridization signal for PRRSV nucleic acids. The anterior and middle lobes of the lung were more reliable than the caudal or accessory lobes for the detection of PRRSV nucleic acids. The in-situ hybridization technique used was rapid, specific and sensitive, and may prove useful for the diagnosis of PRRSV infection in routinely fixed and processed tissues.