Characterization of a PCR-based lymphocyte clonality assay as a complementary tool for the diagnosis of feline lymphoma.

Differentiation between resident mature lymphocyte populations and small cell lymphoma cannot be made by cytological review alone and remains challenging in histopathological review. These cases warrant application of complementary tools like PCR-based immunoglobulin (IG) and T-cell receptor (TCR) clonality testing for confirmation. In this prospective study, diagnostic sensitivity and specificity of different primer sets for routine diagnosis of feline TCR gamma (TCRG) and complete IG heavy chain (IGH) gene rearrangements were assessed. Fine needle aspirates from 20 feline lymphoma cases and lymph node material from 10 cats without hematopoietic neoplasia were subjected to clonality testing. Feline lymphoma cell lines and previously confirmed patient material served as positive control. Detection limits for clonal populations within a polyclonal background was 90% for B-cells and 50% for T-cells. Diagnostic sensitivity and specificity of the clonality assay were 70% and 90%. Overall diagnostic accuracy was 77%, positive predictive value 93% and negative predictive value 60%.

Association between Pneumocystis spp. and co-infections with Bordetella bronchiseptica, Mycoplasma hyopneumoniae and Pasteurella multocida in Austrian pigs with pneumonia.

In this retrospective study, 218 pig lung tissue samples were analyzed to examine a possible association between Pneumocystis spp. using in situ hybridization, Bordetella bronchiseptica (B.b.) using immunohistochemistry (IHC), Mycoplasma hyopneumoniae (M.h.) by quantitative PCR, and Pasteurella multocida (P.m.; IHC). Compared to the bacterial agents (B.b., 5%; M.h., 30%; P.m., 23%), Pneumocystis occurred with a higher prevalence (51%). Co-infections with two or three pathogens were present in 28% of the examined cases. Those of Pneumocystis and M.h. were most commonly seen, followed by Pneumocystis and P.m. and M.h. and P.m. Histologically, interstitial pneumonia was found in both the Pneumocystis positive lungs and lungs with a mild M.h. infection. The B.b. and P.m. positive lungs were mainly associated with suppurative bronchopneumonia and severe M.h. cases with fibrinous or fibrino-haemorrhagic pneumonia. In suckling piglets, the number of samples positive for Pneumocystis predominated, whereas samples from fattening pigs were mainly positive for bacteria or Pneumocystis and bacteria.

Encephalitozoonosis in two inland bearded dragons (Pogona vitticeps).

Microsporidiosis is reported rarely in reptiles. Sporadic multisystemic granulomatous disease of captive bearded dragons (Pogona vitticeps) has been associated with microsporidia showing Encephalitozoon-like morphology. Two such cases are described herein. Both animals displayed clinical signs suggestive of renal failure. Necropsy examination revealed granulomatous lesions in the liver and adrenal area in both animals, and in several other organs in one animal. The lesions were associated with intracellular protozoa consistent with microsporidia. Ultrastructural examination of the organisms revealed morphology similar to Encephalitozoon spp. Immunohistochemistry and chromogenic in-situ hybridization for Encephalitozoon cuniculi were positive in both animals. Nucleotide sequencing of the partial small subunit ribosomal RNA gene and the complete internal transcribed spacer (ITS) region revealed high similarity with published E. cuniculi sequences in both animals. However, the ITS region showed a GTTT-repeat pattern distinct from mammalian E. cuniculi strains. This may be a novel E. cuniculi strain associated with multisystemic granulomatous disease in bearded dragons.

Identification of mixed infections with different genotypes of avian bornaviruses in psittacine birds with proventricular dilatation disease.

Proventricular dilatation disease (PDD) is a fatal, progressive neurological disorder of psittacine birds, which is caused by a single-stranded RNA virus, the avian bornavirus (ABV). The disease pattern includes lymphoplasmacytic inflammation of the central, peripheral and autonomic nervous system. Seven avian bornavirus genotypes have been identified during the last years. So far only monoinfections with a single genotype of ABV have been attributed to PDD cases. However, after a recent survey discovered a case of a double infection with two different ABV genotypes, this seemed to indicate the need for a more systematic search for mixed infections. Brain specimens from 21 psittacine birds affected with PDD were examined. Aim of the investigation was to generate partial ABV sequences of a part of the matrix protein (M) gene and to evaluate whether sequences of more than one ABV genotype were present. RNA was extracted, and subjected to reverse transcriptase PCR with primer pairs generating a partial sequence of the matrix protein (M) gene, followed by a cloning procedure. Ten clones per case were sequenced in order to elucidate whether sequences characteristic for one or more than one genotype were present. In 19 of 21 cases clear M gene sequences could be generated; in two cases nucleic acid amplification failed. Seven birds were infected with ABV 2 and nine with ABV 4, representing the predominant genotypes in Europe. Two cases showed a mixed infection with ABV 2 and ABV 4, and one case a mixed infection with ABV 2 and ABV 6. These results suggest that the molecular cloning method is a useful tool for distinguishing between single and multiple infection events by different ABV genotypes.

In situ hybridisation for the detection of Leishmania species in paraffin wax-embedded canine tissues using a digoxigenin-labelled oligonucleotide probe.

The diagnosis of canine leishmaniosis (CanL) is currently predominantly achieved by cytological or histological identification of amastigotes in biopsy samples, demonstration of specific anti-Leishmania antibodies and PCR-based approaches. All these methods have the advantage of being sensitive and more or less specific; nevertheless, most of them also have disadvantages. A chromogenic in situ hybridisation (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 5.8S rRNA was developed for the detection of all species of Leishmania parasites in routinely paraffin wax-embedded canine tissues. This method was validated in comparison with traditional techniques (histology, PCR), on various tissues from three dogs with histological changes consistent with a florid leishmaniosis. Amastigote forms of Leishmania gave clear signals and were easily identified using ISH. Various tissues from 10 additional dogs with clinical suspicion or/and a positive serological test but without histological presence of amastigotes did not show any ISH signals. Potential cross-reactivity of the probe was ruled out by negative outcome of the ISH against selected protozoa (including the related Trypanosoma cruzi) and fungi. Thus, ISH proved to be a powerful tool for unambiguous detection of Leishmania parasites in paraffin wax-embedded tissues.

Localization of avian bornavirus RNA by in situ hybridization in tissues of psittacine birds with proventricular dilatation disease.

Proventricular dilatation disease (PDD) of psittacine birds is caused by a number of different genotypes of a novel viral species, avian bornavirus (ABV). Here we present an in situ hybridization (ISH) procedure using digoxigenin-labeled RNA probes for localizing viral genomic and mRNA of ABV-2 and ABV-4 in tissues of affected birds. Out of eleven immunohistochemically positive birds ISH signals were only found in seven. Partial sequencing of the viral genome had shown that four of them were infected with ABV-2, two with ABV-4 and one had a mixed infection with ABV-2 and ABV-4. ISH signals were present in the brain, in the vegetative nerve system, glandular epithelia and smooth muscle cells of the intestinal tract and in cardiomyocytes. Hybridization signals for viral genome were more abundant than signals for mRNA. As the probes were not strictly genotype-specific, four of the birds had hybridization signals with both, the ABV-2 and ABV-4 probes. The signals achieved with the homologous probes were more intense and more abundant than those resulting from heterologous probes. Taken together, the results of this study show that ISH can be used as a tool for localizing ABV sequences in tissues of birds with PDD and confirm the causative role of ABVs by showing viral replication in affected tissues.