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.

Immunohistochemical characterization of type II pneumocyte proliferation after challenge with type I porcine reproductive and respiratory syndrome virus.

The aim of this study was to characterize histologically and immunohistochemically the lung lesions developing in growing pigs, 10 and 21 days after experimental challenge with a field strain of porcine reproductive and respiratory syndrome virus (PRRSV). Lung lesions were scored for (1) pneumocyte hypertrophy and hyperplasia, (2) septal mononuclear infiltration, (3) intra-alveolar necrotic debris, (4) intra-alveolar inflammatory cell accumulation and (5) perivascular inflammatory cell accumulation. Immunohistochemistry was performed using antibodies specific for cytokeratin, Ki67, thyroid transcription factor (TTF)-1, the myelomonocytic marker MAC387 and PRRSV. Anti-TTF-1 identified type II pneumocytes and there was marked proliferation of these cells compared with control lung (P <0.05). Anti-cytokeratin labelled type I and II pneumocytes as well as bronchial epithelial cells; however, this labelling was not suitable for cell counting purposes. There was a correlation between lesion severity and the number of cells expressing Ki67 (P <0.05).

Otitis media associated with Cryptosporidium baileyi in a Saker falcon (Falco cherrug).

A 7-week-old male Saker falcon died with a history of severe refractory dyspnoea and respiratory signs. Microscopical lesions included moderate to severe lymphoplasmacytic inflammation of the middle ears, conjunctivae, third eyelids, choanae, salivary glands of the tongue, turbinates, larynx, trachea, syrinx and bronchi. The lesions were associated with variable numbers of Cryptosporidium spp., further confirmed by transmission electron microscopy and in-situ hybridization. Cryptosporidium baileyi was identified by DNA sequence analysis. C. baileyi may therefore be a cause of otitis media in raptors as it is in man. It is most likely that the middle ears of the Saker falcon acquired the infection through the eustachian tubes that originate near the pharynx in the oral cavity. This is the first description of otitis media associated with C. baileyi in a bird or a mammal except man.

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.

Porcine ear necrosis syndrome: a preliminary investigation of putative infectious agents in piglets and mycotoxins in feed.

The aim of this study was to identify the causative factors of porcine ear necrosis syndrome (PENS) in 72 pigs, 5.5-10 weeks in age housed on nine farms. Biopsy samples of ear pinnae were collected from all piglets for bacteriology, histopathology and in situ hybridization for porcine circovirus type 2 (PCV2). At the same time, serum samples were taken for serological analysis and viral PCR, and feed was sampled for mycotoxin analysis. The initial lesion of PENS seemed to be a focal epidermal necrosis. Streptococci were isolated from 44 and staphylococci from 36 pinnae. PCV2 could not be detected by in situ hybridization or qPCR. Seven piglets were positive for porcine reproductive and respiratory syndrome virus, and one for Mycoplasma suis. One piglet had antibodies against Sarcoptes scabiei var. suis. No infectious agents were found in 15 samples. Positive virology and parasitology were often found alongside positive bacteriology. Deoxynivalenol, zearalenone and ergot alkaloids were detected in feed. The findings suggest that PENS is multifactorial in origin and that although infectious agents can be involved in the development of the syndrome they are not the exclusive triggering factor.

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.

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.

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.

Experimental infection of turkeys and chickens with a clonal strain of Tetratrichomonas gallinarum induces a latent infection in the absence of clinical signs and lesions.

The pathogenicity of a mono-eukaryotic culture of Tetratrichomonas gallinarum in specific pathogen free (SPF) chickens and turkeys was studied. Two experiments of identical design were performed: the first with SPF chickens and the second with commercial turkeys. Each experiment included three groups. Groups 1 and 2 each contained 12 infected and three in-contact birds. The birds in these groups were infected on the first day of life, either cloacally (group 1) or orally (group 2). Group 3 consisted of four control birds. Re-isolation of the parasite from cloacal swabs was performed to verify the excretion of T. gallinarum. The infected birds excreted trichomonads from the second day post-infection. Spread of the flagellate from infected to in-contact birds was detected after 5 days post-infection (dpi), based on the re-isolation of the protozoa. No clinical signs or deaths were recorded in chickens or turkeys. Three birds were killed at 4, 8, 14 and 21dpi and various tissues were collected for pathological examination. No gross lesions were noted. Protozoal DNA was demonstrated in the oesophagus, duodenum, jejunum, caecum, liver, lung, bursa of Fabricius and brain by polymerase chain reaction and in-situ hybridization. No antibodies were detected in the serum of infected birds by enzyme linked immunosorbent assay. Microscopical changes were only present in the caecum, where there was sloughing of the epithelium associated with the presence of numerous flagellates on the epithelial surface, within the crypts of Lieberkühn and in the lamina propria. These changes were found in caecal samples from infected and in-contact birds. These studies have demonstrated the rapid transmission of T. gallinarum between both turkeys and chickens and the establishment of a latent infection in both species.

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.

Usutu virus in wild birds in northern Italy.

Usutu virus (USUV) infection was diagnosed in two free-living blackbirds and in three captive owls belonging to two different species in northern Italy in the summers of 2006-2008. Diagnosis was established by immunohistochemistry and RT-PCR. RT-PCR was performed on frozen and on paraffin-embedded tissues (PET), respectively. From the frozen samples a partial sequence of the putative USUV E and NS1 proteins (1229 bp) was determined, whereas partial sequences of the putative NS3 (278 bp) and NS5 (159 bp) proteins were obtained from PET. Additionally, one partial sequence (163 bp) of the putative 3'UTR region was determined from all samples. Sequencing of the amplification products revealed 99.8-100% nucleotide identity of the Italian USUV strains to those from other central European countries.

Zoonotic mosquito-borne flaviviruses: worldwide presence of agents with proven pathogenicity and potential candidates of future emerging diseases.

An update on the mosquito-borne flavivirus species including certain subtypes, as listed in the Eighth Report of the International Committee on Taxonomy of Viruses, is given. Special emphasis is placed on viruses which have been shown to cause diseases in animals, and viruses for which no pathogenicity has been proven yet. Several recent examples (Usutu virus and lineage-2 West Nile virus in central Europe, Zika virus in Micronesia) have shown that sources providing information on such scientifically largely neglected viruses are valuable tools for scientists and public health officials having to deal with such disease emergences. Furthermore the effects of global warming will lead to introduction of competent mosquito vectors into temperate climate zones and will increase efficiency of viral replication in less competent vector species. This, facilitated by rising global travel and trade activities, will facilitate introduction and permanent establishment of mosquito-borne viruses, some of which may become of public health or veterinary concern, into novel environments, e.g. industrialized countries worldwide.

Occurrence, morphological characterization and antigen localization of felid herpesvirus-induced pneumonia in cats: a retrospective study (2000-2006).

The pathological findings in nine cases of pneumonia associated with infection by felid herpesvirus-1 (FeHV-1) are presented. The animals affected were five kittens, two subadult cats and two adult cats. The microscopical changes included fibrinonecrotic pneumonia and severe necrosis of the bronchial and bronchiolar epithelium. FeHV-1 antigen was detected immunohistochemically in pneumocytes, alveolar macrophages and necrotic bronchial and bronchiolar epithelial cells. In the youngest kitten viral antigen was also found in the liver. FeHV-1 infection should therefore be considered as a differential diagnosis in cats with fibrinonecrotic pneumonia and demonstration of viral antigen by immunohistochemistry is a useful diagnostic procedure in such cases.

Comparative evaluation of diagnostic methods for Lawsonia intracellularis infection in pigs, with emphasis on cases lacking characteristic lesions.

In this study the following methods for the diagnosis of Lawsonia intracellularis infection in pigs were compared in relation to a reference method (examination of ileal mucosal scrapings by the polymerase chain reaction [PCR]): Warthin-Starry (WS) staining of tissue sections, immunohistochemistry (IHC), in-situ hybridization (ISH), and PCR examination of faeces and of paraffin wax-embedded samples of ileum. Of 204 pigs examined, 32 were considered on the basis of the PCR to be infected. Gross and histopathological examination, including the use of WS staining, were of limited value. PCR examination of faeces proved to be the most sensitive (sensitivity 70%) of the methods used but, due to the occurrence of false positives, its specificity (95%) was the lowest. IHC (sensitivity 66%, specificity 99%) and ISH (sensitivity 54%, specificity 100%) were clearly superior to examination of WS-stained sections (sensitivity 34%, specificity 100%) for routine diagnosis; although less sensitive than the PCR, they indicated only cases of clinical relevance and, moreover, were capable of distinguishing different stages and levels of infection. Because examination of paraffin wax-embedded tissue by the PCR was shown to be associated with low sensitivity (41%), IHC was regarded as the method of choice for retrospective studies.

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.

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.