Comparison of bacterial culture, histopathology, and immunohistochemistry for the diagnosis of Johne's disease in culled dairy cows.

Paired samples of formalin-fixed, paraffin-embedded ileum and lymph node from 204 culled dairy cows were investigated for evidence of infection by Mycobacterium avium subsp. paratuberculosis. Of the samples, 151 were from animals that were tissue-culture positive for M. avium subsp. paratuberculosis, and 53 were from animals that were tissue and fecal culture negative. From the culture-positive animals, M. avium subsp. paratuberculosis was isolated from 78 samples of ileum and from 107 samples of lymph node. Ziehl-Neelsen acid-fast and immunoperoxidase stained slides were examined for 15 minutes each. Acid-fast organisms were identified in 7 of 78 (8.97%) and 6 of 106 (5.61%) culture-positive ileum and lymph node samples, respectively. Immunohistochemical (IHC) analysis of the same tissues identified infection in the ileum of 9 of 78 (11.54%) and in the lymph node of 5 of 106 (4.67%) culture-positive tissues. All tissues from culture-negative animals tested negative when using acid-fast and IHC staining. The sensitivity of these 2 tests in detecting M. avium subsp. paratuberculosis in culled dairy cows was not significantly different, and the tests exhibited substantial to almost perfect agreement. Both tests were much less sensitive than bacterial culture, detecting less than 6% of tissues positive compared with culture.

Viremia during infectious salmon anemia virus infection of Atlantic salmon is associated with replicating virus in leucocytes.

In situ hybridization (ISH) was used to investigate the presence of viral mRNA in different fractions of blood cells of Atlantic salmon between 6 and 12 d following experimental infection with infectious salmon anemia virus (ISAV). Using a riboprobe targeting ISAV segment 7 mRNA, hybridization signals were observed only in leucocytes in buffy coat smears from samples collected from 8 to 12 d post-infection (dpi). None of the red blood cell smears from any sample showed ISH signal. These observations allow us to conclude that viremia in ISAV infection is associated with virus replication in leucocytes, and that erythrocytes are not target cells for virus replication.

A retrospective study of sporadic bovine abortions, stillbirths, and neonatal abnormalities in Atlantic Canada, from 1990 to 2001.

In a retrospective study on 265 cases of sporadic bovine abortions, stillbirths, and neonatal deaths in Atlantic Canada (1990 to 2001), an etiological diagnosis was made in 117 cases (44.2%). The cases were divided into 2 groups: 234 abortions, and 31 stillbirths and neonatal deaths. Identified causes of abortion were bacteria (24.4%), fungi (6.8%), viruses (6.0%), protozoa (Neospora spp.) (2.1%), congenital anomalies (0.4%), and miscellaneous conditions (1.3%). In addition, placentitis without demonstrable infectious agents was observed in 17 (7.3%). Of the 31 cases of stillbirth and neonatal death, identified causes were dystocia (22.5%), congenital anomalies (22.5%), meconium aspiration syndrome (16.1%), and miscellaneous conditions (6.5%). No etiological diagnosis was made in 59% of abortions and 32.4% of stillbirths and neonatal deaths. The 3 most common identifiable causes of abortion in this study were bacterial, fungal, and viral infections.

Mechanism of cell death during infectious salmon anemia virus infection is cell type-specific.

Infectious salmon anemia virus (ISAV) is a very important fish virus in the Northern hemisphere and there is continued interest in understanding the mechanisms of its pathogenesis and persistence in fish. In this study, the permissive fish cell lines SHK-1, CHSE-214 and TO were used to determine whether ISAV-induced cytopathic effect (CPE) is due to apoptosis or necrosis. Characteristic apoptotic DNA fragmentation was observed only in ISAV-infected SHK-1 and CHSE-214 cells. Apoptosis in ISAV-infected SHK-1 cells was confirmed by fragment end-labelling assay, suggesting that CPE in these cells is associated with apoptosis. ISAV-infected TO cells did not undergo apoptosis, but showed leakage of high-mobility group 1 (HMGB1) protein from the nucleus, which is characteristic of cells undergoing necrosis; this suggests that CPE in these cells is associated with necrosis. ISAV-infected SHK-1 cells did not show leakage of HMGB1 protein. Infection with two different strains of ISAV showed that induction of apoptosis was correlated with the appearance of CPE in SHK-1 cells. ISAV-induced apoptosis was inhibited by a pan-caspase inhibitor, Z-VAD-fmk, indicating a caspase-activation pathway. The ISAV putative PB2 protein and proteins encoded by RNA segment 7 bound caspase-8 specifically in vitro, suggesting that these viral proteins may have a role in ISAV-induced apoptosis. These findings demonstrate for the first time that the mechanism of cell death during ISAV infection is dependent on the cell type, which may have implications for ISAV pathogenesis and persistence.

Infectious salmon anemia virus RNA in fish cell cultures and in tissue sections of atlantic salmon experimentally infected with infectious salmon anemia virus.

Current understanding of the etiopathogenesis of infectious salmon anemia (ISA) virus (ISAV) infection in fish comes mostly from virus detection in homogenized tissues taken from ISA-suspected mortalities. This study combined in situ hybridization (ISH) and histology to demonstrate viral RNA transcripts in different fish cell lines infected with ISAV and in tissues collected during the clinical phase of ISAV infection in Atlantic salmon. For this, a riboprobe to mRNA transcripts of ISAV RNA segment 8 was shown to detect viral mRNA in ISAV-infected TO, CHSE-214, and SHK-1 cell cultures. Specific hybridization was initially detected exclusively in the nuclei of infected cells, which is consistent with the nuclear transcription of orthomyxoviruses. For use of the riboprobe on fish tissues fixed in paraformaldehyde or formalin, the conditions used to permeabilize tissues before ISH (Proteinase K or Tween 20) were first optimized. Tissues were collected 15-20 days after challenge from 7 fresh mortalities of Atlantic salmon parr (approximately 20 g) showing severe gross and microscopic lesions, consistent with ISAV infection. Reverse transcription-polymerase chain reaction on tissue pools confirmed the presence of ISAV in each of the 7 fish. Of the tissues examined in each fish, the heart and liver consistently showed the strongest hybridization signal and, therefore, the most in situ virus, which was located in the endothelium of small blood vessels and in macrophage-like cells.