Epizootic myocarditis associated with encephalomyocarditis virus in a group of rhesus macaques (Macaca mulatta).

Six cases of fatal myocarditis associated with encephalomyocarditis virus occurred over a 14-month period in a group of outdoor-housed juvenile rhesus macaques. All animals were younger than 3 years of age and died or were euthanized following acute onset of dyspnea or pulmonary effusion (3 of 6) or were found dead without premonitory signs (3 of 6). Gross findings included pulmonary congestion (6 of 6), variable degrees of pleural effusion (4 of 6), multifocal pale tan foci throughout the myocardium (3 of 6), hepatomegaly and hepatic congestion (3 of 6), and pericardial effusion (1 of 6). Histologically, affected myocardium was infiltrated multifocally by lymphoplasmacytic and histiocytic inflammation admixed with necrotic and degenerate myofibers and infrequent mineralization (6 of 6). Pulmonary edema was present in all animals. Encephalomyocarditis virus was confirmed in 6 of 6 hearts by immunohistochemistry, and virus was isolated from one case by polymerase chain reaction. Sequencing of virus isolated from 1 affected animal indicated infection with a novel encephalomyocarditis virus. Encephalomyocarditis virus should be considered as a differential etiology in outbreaks of myocarditis and pulmonary edema in juvenile primates.

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.

Unusual and severe lesions of proventricular dilatation disease in cockatiels (Nymphicus hollandicus) acting as healthy carriers of avian bornavirus (ABV) and subsequently infected with a virulent strain of ABV.

A flock of 14 apparently healthy cockatiels, purchased from a single aviary, was tested for the presence of avian bornavirus (ABV). Twelve birds were found to be intermittently shedding ABV, predominantly genotype 4. Four of the cockatiels known to be shedding ABV4 were subsequently challenged with the tissue culture derived, virulent M24 strain of ABV4. The challenged birds remained in apparent good health until day 92 when one was found dead. The remaining three birds began to exhibit severe neurologic signs, ataxia and convulsions on day 110 and were euthanized. On necropsy, all four birds showed mild proventricular enlargement. In contrast, histopathological examination showed unusually severe and widespread tissue lesions. These included massive lymphocytic infiltration and lymphoid nodule formation within and around the ganglia throughout the gastrointestinal tract. There were similar lesions in the medullary cords of the adrenal gland, heart, spleen, liver, kidney, lungs, pancreas, testes and ovary. Immunohistochemistry demonstrated ABV P antigen not only in the cells of the central and autonomic nervous systems, but also within the mononuclear cells infiltrating the various organs. Two healthy cockatiels, one of which was a known ABV carrier, were inoculated with uninfected tissue culture cells and euthanized on day 150. These birds showed no gross lesions of proventricular dilatation disease but had a mild lymphocytic infiltration in their liver, spleen, and kidneys. Prior infection with ABV did not therefore confer significant immunity on these birds, and may have resulted in increased disease severity following challenge.

Infections with weakly haemolytic Brachyspira species in pigs with miscellaneous chronic diseases.

The prevalence of infections with different Brachyspira species was assessed in 202 pigs with various chronic herd problems using different methods. Twenty-seven pigs (13.4%) were positive for Brachyspira spp. with at least one of the methods used. The highest number of positives was identified with mucosal scraping-PCR (23), followed by PET-PCR (22) and bacteriological-biochemical analysis (15). With the exception of three cases of B. pilosicoli infections, only weakly pathogenic Brachyspira species were identified. The majority was B. murdochii, followed by B. innocens and B. intermedia. Concurrent infections with two or more Brachyspira species were common and accounted for 37.1% of the total. Presence of weakly haemolytic Brachyspira was associated with wasting and diarrhoea in a number of cases. This investigation shows that infections with weakly haemolytic Brachyspira spp. may contribute to colonic pathology in pigs with chronic herd problems and that mixed infections seem to occur more frequently than previously noticed.

A retrospective study of neurological disease in 118 rabbits.

A retrospective pathological study of 118 rabbits presenting with neurological disease was conducted. Diagnoses were categorized on the basis of aetiopathogenesis as inflammatory, vascular, traumatic, metabolic-toxic, neoplastic, degenerative or idiopathic. Central nervous system (CNS) lesions were present in 85 (72.0%) of the rabbits and in most of these cases (70.3%) a causative agent was identified. The majority of animals (n=78, 66.1%) had disease of an inflammatory nature and 71 of these 78 rabbits had one of two zoonotic infectious diseases: encephalitozoonosis (n=69, 58.5%) and herpes simplex virus (HSV) encephalitis (n=2). Infections with zoonotic potential are therefore a major cause of CNS disease in the rabbit.

Detection of Cryptosporidium spp., Entamoeba spp. and Monocercomonas spp. in the gastrointestinal tract of snakes by in-situ hybridization.

This report describes the development of a diagnostic method for protozoal infections of the gastrointestinal tract of captive snakes, based on chromogenic in-situ hybridization with probes designed for the detection of 18S rRNA genes from Cryptosporidium spp., Entamoeba spp., Entamoeba invadens and Monocercomonas spp. The specificity of the probes was confirmed with the help of parasitic cultures and gene sequence analysis. The probes gave clear positive signals. Of 182 snakes examined, seven were positive with the Cryptosporidium probe, 13 with the Entamoeba probe (of which nine were also positive with the E. invadens probe), and 34 with the Monocercomonas probe.

Monitoring of Usutu virus activity and spread by using dead bird surveillance in Austria, 2003-2005.

Usutu virus has been causing avian mortality in Austria since its emergence in 2001. Between 2003 and 2005 a total of 504 dead birds were examined by reverse-transcriptase polymerase chain reaction and immunohistochemistry for the presence of Usutu virus nucleic acid and antigen, respectively. In 2003, 92 birds (out of 177 birds) belonging to five different species were positive, while in 2004, only 11 (of 224) birds, and in 2005, 4 (of 103) birds proved positive, all of which were blackbirds (Turdus merula). Within the surveillance period the virus had spread from its initial area of emergence and circulation, the surroundings of Vienna, to large areas of the federal states of Lower Austria, Burgenland and Styria. However, the absolute numbers of Usutu virus associated avian deaths declined significantly during the course of the years. In addition, the proportion of birds with low amounts of virus in their tissues increased continuously, which may indicate developing herd immunity.

Retrospective study of 82 cases of canine lymphoma in Austria based on the Working Formulation and immunophenotyping.

In human beings the prevalence of different non-Hodgkin's lymphoma (NHL) subtypes varies according to geographical region. The aim of this study was to classify canine lymphomas in Austria and to compare the results with those of similar studies in other countries. Eighty-two NHLs were classified according to their morphology (based on the Working Formulation) and their immunophenotype (determined with anti-T-cell and anti-B-cell antibodies). Forty-two (51.2%) were of B-cell subtype, 24 (29.3%) of T-cell subtype, and 16 (19.5%) remained unclassified, because of either negative labelling (9/16) or immunoreaction with both antibodies (7/16). Diffuse lymphomas predominated (99%) over follicular lymphomas, while intermediate grade lymphomas (61%) outnumbered high-grade lymphomas (23.2%) and low grade lymphomas (13.4%). The most common subtype was the diffuse large cell lymphoma (40.2%), followed by the large cell immunoblastic lymphoma (13.4%) and the diffuse small lymphocytic lymphoma (13.4%). Follicular large cell lymphoma and small noncleaved cell lymphoma were uncommon (1.2%). Generally, these findings accord with those of similar studies in Western Europe, making the existence of specific risk factors in Austria unlikely.

Studies on the aetiology of non-suppurative encephalitis in pigs.

Thirty-eight natural cases of aetiologically unclear non-suppurative encephalitis in pigs were studied retrospectively. Brain samples were examined for the presence of porcine circovirus type 2 (PCV-2), porcine respiratory and reproductive syndrome virus (PRRSV), porcine enteroviruses (PEVS), ovine herpesvirus type 2 (OvHV-2), Borna disease virus (BDV) and suid herpesvirus type 1 (SuHV-1) by molecular biological and immunohistochemical methods. Histological examination of the brains revealed variable degrees of lymphohistiocytic encephalitis or meningoencephalitis, characterised predominantly by perivascular mononuclear infiltrates. Two cases could be attributed to PCV-2 infection by in situ hybridisation: viral nucleic acid was found in the mesencephalon, the cerebellum and the medulla oblongata, mainly in the cytoplasm of macrophages, endothelial cells and some glial cells, which were predominantly found in the meninges and around blood vessels. Real-time PCR detected PCV-2 dna in brain samples from seven other pigs. There was no evidence of PRRSV, BDV, SuHV-1, PEVS or OvHV-2 in any of the brain samples examined.

Broad dissemination of Histomonas meleagridis determined by the detection of nucleic acid in different organs after experimental infection of turkeys and specified pathogen-free chickens using a mono-eukaryotic culture of the parasite.

Histomonas meleagridis, a flagellated protozoan parasite, is the causative agent of histomonosis (syn. histomoniasis, blackhead) in turkeys and chickens. The organs primarily affected by the parasite are the caeca and the liver. Until now, only few reports exist in which the parasite has been diagnosed in tissues other than those mentioned above. Hence, the aim of this study was to perform a systematic investigation of various organs of turkeys and specified pathogen-free chickens following an experimental infection with a mono-eukaryotic culture of Histomonas meleagridis in order to determine the dissemination of the flagellate in infected birds. Molecular methods like PCR and in situ hybridization were used for this purpose. For the first time, the DNA of the parasite could be detected in 13 different organs of infected turkeys by PCR including the proventriculus, duodenum, jejunum, caeca, pancreas, bursa of Fabricius, liver, kidney, spleen, heart, lung, thymus and the brain. Most of these findings were further confirmed by in situ hybridization. In contrast to the turkeys that all died shortly after the infection, all of the chickens survived without displaying any clinical symptoms. Even at necropsy, only mild pathological changes were observed in the caeca. Nevertheless, the parasite could also be detected in various organs of these birds, namely the caeca, bursa of Fabricius, kidney, heart and the brain.

In-situ hybridization for the detection and identification of Histomonas meleagridis in tissues.

For use in an in-situ hybridization method, three probes (HM, TR and BL) were designed to hybridize, respectively, with (1) Histomonas meleagridis, (2) Tetratrichomonas gallinarum, and (3) a broad range of micro-organisms, including Blastocystis spp. Mono-eukaryotic cultures were used to test the specificity of the three oligonucleotides and to optimize the hybridization procedure before applying the probes to archived samples of various tissues and to a culture of Trichomonas gallinae. Specific detection of H. meleagridis was possible with the HM probe, but the other two probes were less specific. The TR probe detected members of the Trichomonadidae (Tetr. gallinarum and Tr. gallinae). Positive signals from a great variety of microorganisms, including fungi and protozoa from different animal species, were obtained with the BL probe. However, neither H. meleagridis nor the two members of the Trichomonadidae mentioned above were detected with this probe, allowing the exclusion of these parasites. Use of the three probes makes possible the accurate detection of H. meleagridis and its distinction from other micro-organisms in tissue samples.

Cryptosporidium infection in domestic geese (Anser anser f. domestica) detected by in-situ hybridization.

An in-situ hybridization (ISH) procedure was developed for the detection of Cryptosporidium sp. in paraffin wax-embedded tissues with a digoxigenin-labelled probe targeting the 18S rRNA. This technique was used in addition to traditional methods, such as haematoxylin and eosin staining, periodic acid-Schiff reaction, transmission electron microscopy and the polymerase chain reaction, to examine the bursa of Fabricius (BF), conjunctiva and other tissues from 20 domestic geese aged 16-36 days for the presence of cryptosporidia. Positive signals were found to a moderate or marked extent in both conjunctival samples (89%) and BF samples (88%) but not in other tissues. Sequencing of the PCR amplification product revealed identity with Cryptosporidium baileyi. The infected geese showed no clinical signs and only scanty histological lesions. These results confirm reports showing that young waterfowl are especially vulnerable to cryptosporidium infection and indicate that the BF and conjunctiva are the preferred sites for the presence of the protozoon. ISH proved a good method for detecting and identifying even small numbers of cryptosporidia in tissue sections.

Canine adenovirus type 2 infection in four puppies with neurological signs.

Four nine- to 11-week-old puppies developed respiratory and neurological signs due to an infection with canine adenovirus type 2 (cav-2); three of these were euthanased. They had moderate, diffuse pneumonia but there were no histological abnormalities in the central nervous system. Adenovirus-specific nucleic acid was detected by pcr in samples of lung and brain and the amplified product was 99.8 per cent homologous with the cav-2 reference strain Toronto a26/61. The positive pcr result was confirmed by in situ hybridisation in samples of lung, liver and spleen, but not in brain, and cav was isolated in cell culture from lung material; pcrs for canine distemper virus and canine herpesvirus-specific nucleic acids were negative, but large amounts of Bordetella bronchiseptica were isolated from lung material.

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.

Keratoconjunctivitis in a group of Icelandic horses with suspected γ-herpesvirus involvement.

REASONS FOR PERFORMING STUDY: The role of equid γ-herpesviruses on ocular surface diseases has been disputed, because the diagnosis is usually based on clinical symptoms and detection of viral DNA from samples obtained from live animals. OBJECTIVES: To describe the clinical course, results of polymerase chain reaction (PCR) analysis, in situ hybridisation, cell culture and pathohistological findings of select cases in a presumed outbreak of herpesvirus infection in a group of 15 Icelandic horses. STUDY DESIGN: Case series. METHODS: Pooled ocular and nasal swabs and peripheral blood mononuclear cells of horses diagnosed clinically with herpesvirus-associated keratoconjunctivitis were analysed for presence of equine herpesviruses (EHV)-2 and EHV-5 nucleic acid using real-time PCR. Necropsy specimens from one horse, subjected to euthanasia due to deterioration of clinical symptoms were examined histopathologically, and analysed for presence of EHV-2 and EHV-5 nucleic acid using real-time PCR. In situ hybridisation and cell culture of select samples were performed. RESULTS: All horses with symptoms of severe keratoconjunctivitis were positive for presence of either EHV-2 and/or EHV-5 nucleic acid using real-time PCR. Assessment of necropsy specimens of the most severely affected case, revealed presence of EHV-2 and/or EHV-5 nucleic acid in several ocular and extraocular anatomical locations. The remaining horses responded favourably to symptomatic treatment. CONCLUSIONS: This case series illustrates a severe outbreak of keratoconjunctivitis in a group of Icelandic horses, with suspected γ-herpesvirus involvement. For the first time equid γ-herpesviruses were detected in intraocular anatomical locations.

Borna disease virus infection of adult and neonatal rats: models for neuropsychiatric disease.

Animal models provide unique opportunities to explore interactions between host and environment. Two models have been established based on Borna disease virus infection that provide new insights into mechanisms by which neurotropic agents and/or immune factors may impact developing or mature CNS circuitry to effect complex disturbances in movement and behavior. Note in press: Since this chapter was submitted, several manuscripts have been published that extend findings reported here and support the relevance of BDV infections of neonatal Lewis rats as models for investigating mechanisms of neurodevelopmental damage in autism. Behavioral abnormalities, including disturbed play behavior and chronic emotional overactivity, have been described by Pletnikov et al. (1999); inhibition of responses to novel stimuli were described by Hornig et al. (1999); loss of Purkinje cells following neonatal BDV infection has been demonstrated by Eisenman et al. (1999), Hornig et al. (1999), and Weissenböck et al. (2000); and alterations in cytokine gene expression have been reported by Hornig et al. (1999), Plata-Salaman et al. (1999) and Sauder et al. (1999).

[The use of in-situ hybridization for the detection of Brachyspira spp. in pigs]. / Einsatz der in situ Hybridisierung zum Nachweis von Brachyspira spp. beim Schwein.

Diagnosis of Brachyspira infections in swine and the differentiation of the involved bacteria is time-consuming and in most cases unsatisfactory. Detecting Brachyspira directly in the damaged Brachyspira of the large intestine could provide a direct correlation between histological lesionsa and bacterial growth. In this study we investigated whether in-situ hybridization (ISH) with a digoxigenin-labeled RNA-probe is a suitable method for detecting Brachyspira in the mucosa of the large intestine. Formalin-fixed and paraffin-embedded tissue sections of the large intestine from 78 pigs, which showed macroscopic and histological findings of Brachyspira-associated colitis, were stained with hematoxylin and eosin and Warthin-Starry silver impregnation and subjected to ISH. We used a RNA-probe with a length of 334bp, complementary to a part of the 23S rRNA of all members of the genus Brachyspira. All sections were treated with this anti-sense probe and with a sense control probe. 64 samples (82%) showed clearly positive ISH signals. Thus ISH is a suitable method for detecting Brachyspira directly within the lesions of the large intestine. The quantity of Brachyspira identified by ISH was always lower than by Warthin-Starry staining. Whether this reflects lower sensitivity of the ISH technique, or the fact that other bacteria with morphological similarities to Brachyspira were also stained by Warthin-Starry is unknown as yet. The present investigations provide a basis of further research developing specific probes to distinguish between pathogenic and non pathogenic Brachyspira species and probes detecting other bacteria with morphological similarity to Brachyspira.

Microglial activation and neuronal apoptosis in Bornavirus infected neonatal Lewis rats.

Lewis rats neonatally infected with Borna disease virus have a behavioral syndrome characterized by hyperactivity, movement disorders, and abnormal social interactions. Virus is widely distributed in brain; however, neuropathology is focused in dentate gyrus, cerebellum, and neocortex where granule cells, Purkinje cells and pyramidal cells are lost through apoptosis. Although a transient immune response is present, its distribution does not correlate with sites of damage. Neuropathology is instead colocalized with microglial proliferation and expression of MHC class I and class II, ICAM, CD4 and CD8 molecules. Targeted pathogenesis in this system appears to be linked to microglial activation and susceptibility of specific neuronal populations to apoptosis rather than viral tropism or virus-specific immune responses.

Induction of protective immunity by aerosol or oral application of candidate vaccines in a dose-controlled pig aerosol infection model.

In order to outline basic concepts for the design of a bacterial aerosol infection model, the development of a pig model with Actinobacillus pleuropneumoniae is described. First, reproducibility of aerosol parameters should be maintained by optimizing generating and sampling conditions. Survival rates of the chosen strain must be predictable. Secondly, inhalation conditions for the recipients have to be standardized to enable the determination of deposition sites and the dose administered. Subsequently, dose-response relationship should be evaluated to find a suitable challenge dose. Furthermore, it seems necessary to establish methods to obtain local specimens for determination of the local immune responses. The present study demonstrates that after aerosol challenge pigs were completely protected after inhalation and partially protected after oral application of A. pleuropneumoniae vaccines and describes techniques to administer bacteria in a dose-dependent, viable way. Using the infection model several stages of the disease from acute pleuropneumonia to chronic infection can be induced for research purposes.

Diagnosis of feline herpesvirus infection by immunohistochemistry, polymerase chain reaction, and in situ hybridization.

An adult domestic shorthair cat had severe chemosis due to purulent and necrotizing blepharitis and conjunctivitis. Purulent rhinitis, necrotizing glossitis, and dermatitis were also diagnosed. The cat was positive for feline immunodeficiency virus and feline leukemia virus. Histologically, intranuclear Cowdry type A inclusions were found within numerous epithelial cells adjacent to the lesions in skin, conjunctiva, and tongue. Electron microscopic examination revealed herpesviral particles within the lesions. Paraffin-embedded skin and tongue tissues were processed in a polymerase chain reaction, using primers to amplify a 306-bp region of the thymidine kinase gene of feline herpesvirus type 1, resulting in a distinct amplification product of the predicted size. The distribution of feline herpesvirus was demonstrated by immunohistochemistry and nonradioactive in situ hybridization. Positive immunostaining was found in nuclei and cytoplasm of numerous epithelial cells within and next to the lesions, whereas in situ hybridization, performed with a digoxigenin-labeled double-stranded DNA probe, revealed hybridization signal only in nuclei of intact epithelial cells. Neither immunohistochemistry nor in situ hybridization showed feline herpesvirus type 1 in tissues of lungs, liver, spleen, intestine, or brain.