Investigation of preinguinal approach for removal of urachal abscess in three Japanese black cattle older than 18 months of age.

This study investigated the diagnostic and surgical management of urachal abscessation involving the urinary bladder in three cattle aged >18 months. While the abdominal floor or paralumbar fossa are typically considered for accessing the urachus and bladder in calves and heifers, the distance is too great to perform the procedure under direct vision in adult cows. Therefore, a novel preinguinal approach was used for access in cows with urachal abscesses after 18 months. Access was successfully achieved in all the three cows through a preinguinal incision to the urachal abscess at the apex of the bladder. However, in cases wherein the abscess and adhesions are severe, removal may be challenging or may necessitate a combined median or paramedian approach.

Hepatocellular carcinoma with lung metastasis showing hemochromatosis in an Egyptian fruit bat (Rousettus aegyptiacus).

After an Egyptian fruit bat (Rousettus aegyptiacus) in a zoo became emaciated and died, a necropsy revealed multiple nodules on the liver and lung surfaces. Microscopy revealed that the liver nodules consisted of neoplastic hepatocytes and showed metastasis in the lung lobes. Most of the neoplastic cells in the liver and lung showed positive labeling for HepPar-1, cytokeratin 19, glypican-3, and Ki-67. Hepatocellular degeneration and necrosis were diffuse in the liver parenchyma. Berlin blue staining revealed large amounts of iron in normal and neoplastic cells. Based on these pieces of evidence, this case was diagnosed as hepatocellular carcinoma with hemochromatosis. This is believed to be the first report of hepatocellular carcinoma in an Egyptian fruit bat that has been immunophenotypically examined in detail by pathological examination.

Surgical management of a dermoid cyst with mandibular gland tissue in a Japanese Black calf.

A 131-day-old male Japanese Black calf presented with a swollen right cheek from birth. Imaging examination revealed a cyst under the right buccal area and debris-containing fluid inside the cyst, and puncture aspiration revealed a mildly cloudy fluid containing hair and tissue fragments. Histological examination of the excised cyst revealed stratified squamous epithelium with skin appendages in the cyst wall, which was diagnosed as a dermoid cyst. In addition, some submandibular gland tissue was found within the cyst wall. After removal of the cyst, there was swelling in the same area, which resolved with steroid administration. Surgical treatment of buccal dermoid cysts should be performed with caution to avoid damage to adjacent salivary gland tissue.

Histopathological findings of the nasopharynx-associated lymphoid tissue of pigs co-infected with porcine circovirus 2 and porcine reproductive and respiratory syndrome virus.

Porcine circovirus 2 (PCV2) causes porcine circovirus-associated disease, and co-infection with porcine reproductive and respiratory syndrome virus (PRRSV) severely affects the pig breeding industry. Both viruses target the macrophages in lymphoid tissues. Various porcine pathogens enter via the nasal cavity, and the nasopharynx-associated lymphoid tissue (NALT) acts as the mucosal immune system. However, the pathological analysis has not progressed. This study aimed to histologically examine the NALT of pigs with suspected PCV2 and PRRSV infections. Six pigs were subjected to necropsy, and their NALT, tonsils, and mesenteric lymph nodes were collected. Macrophages, lymphocytic depletion, multinucleated giant cells, intracytoplasmic inclusion bodies, and neutrophil infiltration increased in the NALT. In situ hybridization revealed positive signals for PCV2 in the NALT of all pigs and PRRSV in the NALT of three pigs. PCV2-positive macrophages were mainly identified in the follicles, whereas PRRSV-positive tissues were found primarily around the crypt and directly below the epithelium. Quantitative PCR revealed 108-1010 copies of PCV2 DNA/µL and 102-104 copies of PRRSV DNA/µL in the NALT. Therefore, both PCV2 and PRRSV were detected in the NALT of pigs. In conclusion, the infection and replication of both viruses in the NALT and tonsils may suppress host immunity and promote co-infection with other pathogens.

Detection of Swine Influenza A and Porcine Reproductive and Respiratory Syndrome Viruses in Nasopharynx-Associated Lymphoid Tissue.

Porcine respiratory disease complex, which is caused by a combination of pathogens, including swine influenza A virus (SIV) and porcine reproductive respiratory syndrome virus (PRRSV), results in significant economic losses in pig production systems. Nasopharynx-associated lymphoid tissue (NALT) plays an important role in the uptake of pathogens and defence of the nasal mucosa in rodents and humans. We characterized NALT M cells in pigs and detected SIV antigen and PRRSV nucleic acid in NALT using histopathological, immunohistochemical and in-situ hybridization analyses. All SIV- and PRRSV-positive cases examined had suppurative nasopharyngitis and pneumonia. M cells were detected by immunohistochemistry and the distribution of M cells showed an increase in the middle section of NALT. SIV antigen was detected in M cells and PRRSV nucleic acid was demonstrated in the cytoplasm of macrophages in NALT. We believe that SIV and PRRSV infection in the upper respiratory tract induces local immunosuppression and these results confirm that swine NALT is a location for virus replication and may be strongly associated with the development of pneumonia in pigs.

Adenomyomatous uterine polyp in a Miniature Pinscher: histologic, immunohistochemical, and clinical aspects.

A 14-y-old Miniature Pinscher bitch was admitted to a veterinary clinic because of inappetence and a distended abdomen; ultrasound examination revealed a fluid-filled uterus with a single 1-cm hyperechoic nodule in its lumen. Ovariohysterectomy was performed. Grossly, the uterine horns were distended irregularly and asymmetrically, and the uterine lumen contained 200-300 mL of brown watery fluid. A single white polypoid 0.9-cm diameter nodule was present at the site of the distended uterine horn and arose from the endometrium with a narrow stalk. Histologically, the polyp consisted of dense, smooth muscle fascicles admixed with glandular components; its surface was covered by simple cuboidal epithelium with areas of squamous metaplasia. The myomatous cells expressed the myogenic markers smooth muscle actin and desmin. We interpreted the mass as an adenomyomatous uterine polyp, which is a rare variant of an endometrial polyp.

Production of granulomas in Mycoplasma bovis infection associated with meningitis-meningoencephalitis, endocarditis, and pneumonia in cattle.

Mycoplasma bovis, the most important primary pathogen in the family Mycoplasmataceae, causes pneumonia, arthritis, otitis media, and mastitis in cattle. Histopathologic pulmonary changes associated with M. bovis infection have been characterized as suppurative-to-caseonecrotic bronchopneumonia; infection in other organs has been reported in only a few studies that examined caseonecrotic endocarditis and suppurative meningitis. Granulomatous lesions associated with M. bovis infection have been reported only rarely. We studied the granulomatous inflammation associated with M. bovis infection in several organs of 21 Japanese Black cattle. M. bovis was detected by isolation and loop-mediated isothermal amplification methods; other bacteria were detected using culture on 5% blood sheep agar and a MALDI-TOF MS Biotyper. Tissues were examined by histopathology and by immunohistochemistry (IHC) using anti-M. bovis, anti-Iba1, anti-iNOS, and anti-CD204 antibodies. All 21 cases, which included 2 cases of meningitis-meningoencephalitis, 8 cases of endocarditis, and 11 cases of bronchopneumonia, had caseonecrotic granulomatous inflammation associated with M. bovis infection. The IHC for macrophages revealed a predominance of iNOS-labeled (M1) macrophages in the inner layer of the caseonecrotic granulomas associated with meningitis-meningoencephalitis, endocarditis, and bronchopneumonia in Japanese Black cattle naturally infected with M. bovis.

Neutrophil and M2-polarized Macrophage Infiltration, Expression of IL-8 and Apoptosis in Mycoplasma hyopneumoniae Pneumonia in Swine.

Mycoplasma hyopneumoniae (Mhp) is the primary pathogen of porcine enzootic pneumonia (PEP). Consolidated lung tissue from the cranioventral lung lobes of 15 pigs with PEP was collected for quantitative polymerase chain reaction, histopathology and immunohistochemistry. Histopathology revealed the co-existence of bronchial-associated lymphoid tissue hyperplasia with intra-alveolar neutrophils and macrophage infiltration in lesions of suppurative bronchopneumonia. Immunolabelling of infiltrated macrophages with CD163/CD204 indicated the presence of M2-polarized macrophages. Mhp antigen was detected on respiratory epithelial cells and in phagocytosed neutrophils. The intensity of Mhp immunolabelling and number of CD163/CD204-positive macrophages were correlated with the Mhp load in lung tissue (r = 0.87, 0.56, P <0.05). IL-8 immunolabelling was mainly found in neutrophils and correlated with Mhp load, Mhp immunolabelling and histological lesion score (r = 0.70, 0.66, 0.64, P <0.05), respectively. Apoptosis was seen in intra-alveolar cells and was correlated with Mhp load (r = 0.62, P <0.05). It is postulated that IL-8 attracts neutrophils to the lesions, while M2-polarized macrophages are a major source of IL-10 and promote a Th2-type immune response.

Mycoplasma bovis May Travel Along the Eustachian Tube to Cause Meningitis in Japanese Black Cattle.

Mycoplasma bovis (M. bovis) is a common inhabitant of the upper and lower respiratory tracts of cattle and is considered to be the main aetiological agent of otitis media in calves. The eustachian tube appears to be the most common portal for pathogens to enter the middle ear. We investigated the transmission route of M. bovis causing otitis media that progressed to meningitis or meningoencephalitis in Japanese Black cattle. M. bovis was detected in 10 cases by a loop-mediated isothermal amplification method or by immunohistochemistry. One case of caseonecrotic granulomatous meningoencephalitis, one case of caseonecrotic granulomatous meningitis, one case of suppurative meningoencephalitis, eight cases of eustachitis, nine cases of tonsillitis and six cases of suppurative bronchopneumonia were identified by histopathological examination. M. bovis antigen was detected in the eustachian tubes of eight cases. In nine cases, M. bovis was also detected in tonsillar epithelial crypts and lumina, in intraluminal inflammatory cells and in the epithelial cells of minor salivary glands located around the eustachian tubes and tonsils. The results suggest that M. bovis can infect and colonize the tonsils and enter the eustachian tubes, causing otitis media, which, in cases of chronic infection, can progress to meningitis.

Immunohistochemical and Morphological Features of Bovine Granulosa Cell Tumours in Relation to Growth Pattern and Folliculogenesis.

Granulosa cell tumours (GCTs) are the most common type of bovine ovarian neoplasm. We describe the morphological and immunohistochemical features of GCTs (n = 26) and a thecoma (n = 1) using antibodies to cytokeratin AE1/AE3, vimentin, desmin, nectin-4, anti-Müllerian hormone (AMH), inhibin α, c-erbB-2 oncoprotein and proliferating cell nuclear antigen. The results indicate that, based on the expression similarity of AE1/AE3 and inhibin α, the macrofollicular growth pattern of GCTs derives from highly differentiated granulosa cells (GCs), the Graafian follicle or early atretic follicle. The trabecular growth pattern of GCTs is inferred to be derived from GCs of the atretic follicle in mature animals. AMH is a useful marker for diagnosing bovine sex cord-stromal tumours because it was expressed in all GCT cases and in the single case of thecoma.

Three neoplasms in a Eurasian otter (Lutra lutra): malignant melanoma, trichoblastoma and mammary gland adenoma.

An eighteen-year-old female Eurasian otter became emaciated and died. Necropsy examination revealed nose and thoracic cutaneous masses, abdominal subcutaneous mass, and multiple nodules in the liver and lungs. Malignant melanoma was found in the nose cutaneous mass and to have metastasized to the liver, lungs, kidneys, adrenal glands, mammary glands and left mandibular lymph node. The neoplastic cells were labeled for vimentin, melanoma, and S100. The cutaneous mass in the thoracic area consisted of spindle shaped neoplastic epithelial cells and was diagnosed as trichoblastoma. Mammary gland adenoma was observed in the abdominal subcutaneous mass. This is the first report of primary three neoplasms of malignant melanoma, trichoblastoma and mammary gland adenoma in a Eurasian otter.

A persistent epidemic of porcine epidemic diarrhoea virus infection by serological survey of commercial pig farms in northern Vietnam.

BACKGROUND: Porcine epidemic diarrhoea (PED) is a highly contagious infectious disease with negative economic impacts on the swine industry. PED outbreaks were reported from 2009 to 2015, but sporadic infection has been observed until now in Vietnam. However, the seroprevalence of PEDV infection has not yet been reported for commercial pig farms in Vietnam. The aim of this study was to assess the seroprevalence of PEDV infection in Vietnamese pig farms to reveal the endemic status of PEDV in northern Vietnam. RESULTS: A serological survey of PEDV infection was carried out using indirect ELISA in commercial pig farms in Hai Duong, Hung Yen and Thai Binh provinces in northern Vietnam in 2019. Twenty sera were randomly collected from each of 10 commercial pig farms, from each province; none of the farms had vaccinated for PEDV. Serological evidence of natural PEDV infection, expressed as a high antibody titre, was observed in the pig farms in all 3 provinces. The OD values were significantly higher (p < 0.001) for pig sera from Thai Binh than from Hai Duong and Hung Yen. No significant differences (p > 0.05) were detected for seropositivity to PEDV based on locality, age, pig breed and farm size. CONCLUSIONS: This study indicates serological evidence of natural PEDV infection with high antibody titre in commercial pig farms. PEDV infection was widespread among the pig population in these 3 provinces and that good management and strict biosecurity are needed at these pig farms.

Pathology of the outbreak of subgenotype 2.5 classical swine fever virus in northern Vietnam.

Classical swine fever (CSF) is an endemic disease in southeastern Asia and is one of the most important swine diseases in Vietnam. This study was conducted to characterize the pathology of natural cases of CSF in northern Vietnam in 2018 and their genetic prevalence. A total of 10 representative pigs were collected from four provinces (Hung Yen, Ha Noi, Quang Ninh and Thai Binh) during five outbreaks and examined pathologically. The gross and histopathological findings showed the disease was expressed as the acute or the subacute to chronic form of CSF, depending on the age of the animals. The most consistently observed lesions associated with infection by the classical swine fever virus (CSFV) included lymphoid depletions in tonsils, lymph node and spleen; histiocytic hyperplasia in spleen; cerebral haemorrhage; perivascular cuffing in the brain; renal erythrodiapedesis; urothelial vacuolation and degeneration and interstitial pneumonia. The immunohistochemical findings showed a ubiquitous CSFV antigen mainly in the monocytes/macrophages and in the epithelial and endothelial cells in various organs. CSFV neurotropism was also found in the small neurons of the cerebrum and the ganglia of the myenteric plexus. Analysis of the full-length envelope protein (E2) genome sequence showed that all strains were genetically clustered into subgenotype 2.5, sharing a nucleotide identity of 94.0%-100.00%. Based on the results of this study, the strain was categorized as a moderately virulent CSFV.

Bursa atrophy at 28 days old caused by variant infectious bursal disease virus has a negative economic impact on broiler farms in Japan.

Infectious bursal disease (IBD), caused by IBD virus (IBDV), is highly contagious, immunosuppressive and causes a negative economic impact on poultry industry. IBDV-vaccinated broiler farms at south Kyushu, Japan had a bursa-to-bodyweight ratio (BB ratio) reduction at 28 days (d) old, followed by high mortality 30 d later. We analysed the influence of the IBDV on atrophy of the bursa of fabricius (BF) and the subsequent mortality after 30 d. Ten broilers were sampled at each timepoint from the farm with high mortality at 21, 25, 28 and 35 d. A second flock from the same farm was sampled at 14, 21, 25, 28, 35 and 42 d. IBDV was detected in BF samples at 25, 28 and 35 d and at 21, 25, 28 and 35 d in the first and second flocks, respectively, using immunohistochemical staining and RT-PCR. IBDV isolates from both flocks were closely related to the China KM523643 strain. Histopathology and TUNEL assay indicated apoptosis, severe lymphoid depletion, vacuoles within follicles, lymphoid follicle atrophy and fibrosis in the BF. We observed 75% of the polyserositis and 10% of the airsacculitis at 30 D in dead broilers. The antigenic variant IBDV infection was appeared to be the main influencing factor on BF atrophy and BB ratio reduction in the broilers. High mortality in the broilers after 30 d could be due to secondary infection. The disease caused by IBDV had a negative economic impact in the farm. RESEARCH HIGHLIGHTS New variant IBDV caused bursa atrophy and reduced BB ratio in 28-day-old broilers. After vIBDV had infected broilers, at 21 days old they became immunosuppressed. High mortality at 30 days old in broilers was due to secondary infection. New vIBDV has a negative economic impact on broiler farms in Japan.

New tropisms of porcine epidemic diarrhoea virus (PEDV) in pigs naturally coinfected by variants bearing large deletions in the spike (S) protein and PEDVs possessing an intact S protein.

We previously reported the coinfection of novel porcine epidemic diarrhoea virus (PEDV) variants bearing large deletions in the S protein and PEDVs possessing an intact S protein (S-intact PEDV) in domestic pigs in Japan. The variants were frequently observed in pig farms with persistent or recurrent infection. To elucidate the role of the variants in persistent infections and their tropism properties, we genetically characterized and immunohistochemically detected PEDVs collected in primary and recurrent outbreaks in two persistently infected farms. Our results revealed coinfection of the PEDV variants bearing a 214-amino acid deletion in the S protein and S-intact PEDVs in the lungs of the naturally infected pigs. New tropisms of PEDV, including epithelial cells and submucosal glands of the airway tract, epithelial cells of the bile duct, and monocytes/macrophages were identified. The findings elucidate the mechanism of PEDV infection, epidemiology and pattern changes in the disease.

Severe Degenerative Changes in Cryptorchid Testes in Japanese Black Cattle.

This is a histopathologic and endocrinologic study of 6 calves diagnosed with cryptorchidism. Cases 1-3 were diagnosed as resembling testicular regression syndrome. In cases 1 and 2, the extracted tissue was a small, firm, gray-white mass, and there was lack of obvious testicular tissue in case 3. Histopathologically, the excised tissue in cases 1-3 was a fibrotic testicular remnant with inflammation, mineralization, hemosiderin-laden macrophages or lipofuscin-laden macrophages, and lack of germ cells and interstitial endocrine cells. These findings were compared with cases 4-6, which were diagnosed as testicular hypoplasia due to cryptorchidism. These cases had small but otherwise grossly unremarkable intra-abdominal testicular tissue and histologically had a few germ cells and sustentacular cells with arrested spermatogenesis and an increase in interstitial endocrine cells. Cases 1-3 had more severe degenerative changes compared with cases 4-6. In case 2, the average diameter of the seminiferous tubules was much smaller than in cases 4-6, and there were few tubule cross sections. Anti-Müllerian hormone (214 pg/ml) was detected in the plasma of case 2. Based on the macroscopic and histopathologic findings as well as endocrinologic profiles, the testicular degeneration in cases 1-3 was considered similar to that of testicular regression syndrome. In this condition, it is thought that a normally developing intra-abdominal testis undergoes degeneration due to heat or a vascular disorder such as torsion.

Development of indirect enzyme-linked immunosorbent assay for detection of porcine epidemic diarrhea virus specific antibodies (IgG) in serum of naturally infected pigs.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) infection is a highly contagious infectious disease causing watery diarrhea, vomiting, dehydration and high mortality rate in newborn piglets. PEDV infection can cause high economic losses in pig industry. In Japan, a PEDV outbreak occurred with high mortality from 2013 to 2015. Even though until now, PEDV infection occurs sporadically. For the control and monitoring of PEDV infection, not only symptomatic pigs, but also asymptomatic pigs should be identified. The objective of this study is to develop and optimize novel indirect ELISA as a simple, rapid, sensitive and specific method for the detection of anti-PEDV antibodies and evaluate the efficacy of the assay as a diagnostic method for PED. RESULTS: One hundred sixty-two serum samples, consisting of 81 neutralization test (NT) positive and 81 NT negative sera, were applied to the assay. Indirect ELISA test based on whole virus antigen (NK94P6 strain) derived from Vero cell culture was evaluated by receiver operating characteristic (ROC) analysis with neutralization test (NT) as a reference method, and cut-off value was determined as 0.320 with sensitivity and specificity of 92.6 and 90.1%, respectively. The area under curve (AUC) was 0.949, indicating excellent accuracy of indirect ELISA test. There was significant positive correlation between indirect ELISA and neutralization test (R = 0.815, P < 0.05). Furthermore, the kappa statics showed the excellent agreement between these two tests (kappa value = 0.815). In addition, the sensitivity and specificity of preserved plates with different periods (1 day, 2 weeks, 1, 2, 3, 4, 5 and 6 months) after drying antigen coated plates were 100% and 80-100%, respectively. CONCLUSIONS: The developed indirect ELISA test in our study would be useful as a reliable test for serological survey and disease control of PEDV infection, and our pre-antigen coated ELISA plates can be preserved at 4 °C until at least 6 months.

Development of a Japanese encephalitis virus genotype V virus-like particle vaccine in silkworms.

To counter the spread of multiple Japanese encephalitis virus (JEV) variants harboured in alternative host species and highly neurotoxic variants with new antigenicity, such as genotype V (Muar), methods for developing more effective and low-cost vaccines against a variety of epidemic JEV strains are required. Here, we successfully synthesized large amounts of a Muar virus-like particle (MVLP) vaccine for JEV in silkworm pupae by using a Bombyx mori nuclear polyhedrosis virus recombinant consisting of JEV codon-optimized envelope (E) DNA. In particular, histopathological examination suggested that MVLP was efficiently synthesized in body fat tissues as well as epithelial cells. Quantitative analysis indicated that one silkworm pupa produced 724.8 µg of E protein in the MVLP vaccine. Electron microscopic examination of purified MVLP vaccine defined a typical MVLP morphological structure. Detailed MVLP antigen assessment by immune-electron microscopy revealed that the majority of MVLPs were covered with approximately 10 nm projections. Boosted immunization with MVLP antigens in mice and rabbits tended to show improved plaque inhibition potency against homologous Muar and heterologous Nakayama, but less potency to Beijing-1 strains. Notably, mixed immune rabbit antisera against Nakayama and Muar VLP antigens led to an increase in the low antibody reaction to Beijing-1. Additionally, a stopgap divalent JEV vaccine consisting of MVLP and Nakayama VLP and its immune mouse serum significantly increased plaque inhibition titre against Muar, Nakayama and Beijing-1 strains. These findings suggested that low-cost MVLP vaccines prepared in silkworm pupae are suitable for providing simultaneous protection of individuals in developing countries against various JEV strains.

Molecular characterization of US-like and Asian non-S INDEL strains of porcine epidemic diarrhea virus (PEDV) that circulated in Japan during 2013-2016 and PEDVs collected from recurrent outbreaks.

BACKGROUND: Since late 2013, porcine epidemic diarrhea virus (PEDV) has reemerged in Japan and caused severe economic losses to the swine industry. Although PEDV vaccines have been used widely, the disease has swept rapidly across the county, and is commonly observed in PED-vaccinated farms, and has recurred in domestic herds. To better understand PEDVs responsible for the reemerging outbreaks in Japan, full-length spike (S), membrane (M), and nucleocapsid (N) genes of 45 PEDVs collected in Japan during 2013-2016, were sequenced and analyzed. RESULTS: Phylogenetic analysis based on S gene sequences revealed that all the recent field PEDVs were genetically distinct from the classical Japanese strains, and were classified into three genotypes: North American (NA), S INDEL, and Asian non-S INDEL. Our data suggested a possibility that multiple parental PEDV strains were introduced into Japan from abroad at the same time or similar times. The newly identified Japanese strains showed the closest relationship to the US strains. Two sublineages of Japanese strains circulating in Japan were similar to two sublineages identified in the US, suggesting common ancestors for these strains. In comparison with two vaccine strains used in Japan, the field strains had various changes in epitope regions, glycosylation sites, and phosphorylation sites. These substitutions, particularly observed in epitope regions of the S (521, 553, 568, and 570), M (5), and N (123, 252, and 255) proteins, may have affected antigenicity and vaccine efficacy, resulting in an unsuccessful PEDV control. Sequence comparisons between PEDVs collected from primary and secondary outbreaks in three herds revealed that the disease has developed to an endemic stage in which PEDV could persist for nearly two years in the herds or local regions, causing subsequent epidemics. CONCLUSIONS: These results elucidate the genetic characteristics, origin, and molecular epidemiology of PEDVs circulating in Japan, as well as the PEDV strains causing recurrent outbreaks. This study provides a better insight into the PEDVs responsible for recent outbreaks in Japan, and could potentially help to develop measures for controlling and preventing the disease.

Enhanced detection of Porcine reproductive and respiratory syndrome virus in fixed tissues by in situ hybridization following tyramide signal amplification.

This study evaluated the sensitivity of biotinyl-tyramide-based in situ hybridization (TISH) method by comparison with chromogenic in situ hybridization (CISH) and immunohistochemical staining (IHC) methods. This study also determined the effect of fixative and fixation time on the detection of Porcine reproductive and respiratory syndrome virus (PRRSV) in paraffin-embedded tissues. Lung samples were fixed in 4% paraformaldehyde (PFA) or 10% neutral buffered formalin (NBF) for various times before paraffin embedding. Of 30 paraffin-embedded lung samples, fixed for 1 day in 4% PFA or 10% NBF, 18 (60%) were positive for PRRSV by nested reverse transcription polymerase chain reaction (nRT-PCR). All 18 lung samples (100%) also were positive for PRRSV by TISH, but only 10 of these 18 specimens (56%) were positive for PRRSV by IHC and CISH. We demonstrated that TISH can detect PRRSV RNA in paraffin-embedded tissues after up to 90 days of fixation. PRRSV nucleic acids and antigens were better preserved in 4% PFA than in 10% NBF. Compared with CISH and IHC testing methods, TISH appeared to be more sensitive for the detection of PRRSV in paraffin-embedded tissues.