Molecular and genomic characterization of a novel equine molluscum contagiosum-like virus.

Cases of pox-like lesions in horses and donkeys have been associated with poxviruses belonging to different genera of the family Poxviridae. These include the orthopoxviruses vaccinia virus (VACV), horsepoxvirus (HPXV) and cowpoxvirus (CPXV), as well as a potentially novel parapoxvirus and molluscum contagiosum virus (MOCV). However, with the exception of VACV, HPXV and CPXV, the genomic characterization of the causative agents remains largely elusive with only single short genome fragments available. Here we present the first full-length genome sequence of an equine molluscum contagiosum-like virus (EMCLV) directly determined from skin biopsies of a horse with generalized papular dermatitis. Histopathological analysis of the lesions revealed severe epidermal hyperplasia with numerous eosinophilic inclusion bodies within keratinocytes. Virions were detected in the lesions in embedded tissue by transmission electron microscopy. The genome sequence determined by next- and third-generation sequencing comprises 166 843 nt with inverted terminal repeats (ITRs) of 3473 nt. Overall, 20 of the predicted 159 ORFs have no equivalents in other poxviruses. Intriguingly, two of these ORFs were identified to encode homologues of mammalian proteins involved in immune signalling pathways, namely secreted and transmembrane protein 1 (SECTM1) and insulin growth factor-like family receptor 1 (IGFLR1), that were not described in any virus family so far. Phylogenetic analysis with all relevant representatives of the Poxviridae suggests that EMCLV should be nominated as a new species within the genus Molluscipoxvirus.

Mesenteric lymphangitis and sepsis due to RTX toxin-producing Actinobacillus spp in 2 foals with hypothyroidism-dysmaturity syndrome.

Actinobacillus suis-like organisms (ASLOs) have been isolated from the genital, respiratory, and digestive tracts of healthy adult horses, horses with respiratory disease, and septic foals. Two foals with congenital hypothyroidism-dysmaturity syndrome from separate farms developed ASLO infection. At necropsy, both had contracted carpal flexor tendons, thyroid hyperplasia, and thrombotic and necrotizing mesenteric lymphangitis and lymphadenitis; one foal also had mandibular prognathism. Numerous ASLOs were isolated from tissues from both foals, including intestine. Biochemical testing and mass spectrometric analysis of the two Actinobacillus isolates did not allow unequivocal identification. Comparative genetic analysis was done on these and similar isolates, including phylogeny based on 16S rRNA, rpoB and recN genes, as well as RTX (repeat in toxin) toxin typing of apxIA-apxIVA and aqxA genes. One isolate was identified as Actinobacillus suis sensu stricto, based on the presence of apxIA and apxIIA but not aqxA, whereas the other isolate had aqxA but neither apxIA nor apxIIA, consistent with A equuli ssp haemolyticus. Based on genotypic analysis of the isolates included for comparison, 3 of 3 equine ASLOs and 2 of 5 A equuli isolates were reclassified as A equuli subsp haemolyticus, emphasizing the importance of toxin genotyping in accurate classification of actinobacilli.

Clinical course and pathology in rats (Rattus norvegicus) after experimental cowpox virus infection by percutaneous and intranasal application.

Recently, several cases of human cowpox virus (CPXV) infections were reported in France and Germany, which had been acquired through close contact with infected pet rats. The animals exhibited respiratory signs or skin lesions and died shortly after purchase. After natural infection of white rats with CPXV in the USSR in 1978, a peracute pulmonary form, a milder dermal form, and a mixed form exhibiting features of both have been described. To the best of the authors' knowledge, 3 experimental cowpox virus infection studies using rats have been performed to date; however, neither results of histomorphological examinations nor immunohistochemical analyses have yet been reported in rats after experimental infections. To investigate the impact of the infection route on the clinical course, the development of lesions, and tropism, rats were infected intradermally, intranasally, or by a combination of both routes. The authors found a correlation between clinical manifestation, pathology, and infection routes. Intradermal and contact exposure yielded a mild dermal form, characterized by the development of vesiculopustular dermatitis. In contrast, intranasally infected animals died peracutely, showing severe dyspnea. Occasionally, a combination of the dermal and the respiratory form occurred after intranasal infection. Immunohistochemically, CPXV antigen was detected in the epithelial and mesenchymal cells of the upper respiratory tract and affected skin lesions and rarely in mesenchymal cells of lymph nodes. This is the first histomorphological and immunohistochemical analysis of CPXV in rats after experimental infection.

Neurotropism in blackcaps (Sylvia atricapilla) and red-billed queleas (Quelea quelea) after highly pathogenic avian influenza virus H5N1 infection.

The epidemiologic role of passerine birds in the spread of highly pathogenic avian influenza virus (HPAIV) remains controversial. However, confirmed natural infections with HPAIV in Passeriformes, their close contact to poultry and humans, and their role as a human food source indicate a need for increased research on passerines. To date, there are only a few studies on viral shedding and pathomorphologic changes in songbirds infected with HPAIV. To investigate susceptibility, clinical outcome, virus spread, and pathomorphology, the authors inoculated oculo-oronasally 22 red-billed queleas (Quelea quelea) and 11 blackcaps (Sylvia atricapilla) with A/Cygnus cygnus/Germany/R65/2006 (H5N1) using 2 different doses of either 10(4) EID50 (50% egg infective dose) or 10(6) EID50 per animal. They monitored all birds for clinical signs and oropharyngeal and cloacal virus shedding. They also performed immunohistochemistry and obtained molecular virologic data by real-time reverse transcription polymerase chain reaction in tissue samples. In contrast to blackcaps, where 100% of the infected individuals died, queleas were much less susceptible, with a mortality of 82% and 18%, depending on the doses applied. In both species, the virus was shed within 3 to 6 days postinfection, mainly via the respiratory tract. Viral antigen was detected in 100% of the succumbed birds, particularly in the central nervous system. In blackcaps, the heart, lungs, and pancreas were mainly infected. In contrast, the pancreas was predominantly affected in queleas, whereas the heart and the lower respiratory tract were of minor relevance. The authors hypothesize that neurotropism should be considered a main factor for the fatal course of disease in Passeriformes after infection with HPAIV.

Imaging Mass Spectrometry and Proteome Analysis of Marek's Disease Virus-Induced Tumors.

The highly oncogenic alphaherpesvirus Marek's disease virus (MDV) causes immense economic losses in the poultry industry. MDV induces a variety of symptoms in infected chickens, including neurological disorders and immunosuppression. Most notably, MDV induces transformation of lymphocytes, leading to T cell lymphomas in visceral organs with a mortality of up to 100%. While several factors involved in MDV tumorigenesis have been identified, the transformation process and tumor composition remain poorly understood. Here we developed an imaging mass spectrometry (IMS) approach that allows sensitive visualization of MDV-induced lymphoma with a specific mass profile and precise differentiation from the surrounding tissue. To identify potential tumor markers in tumors derived from a very virulent wild-type virus and a telomerase RNA-deficient mutant, we performed laser capture microdissection (LCM) and thereby obtained tumor samples with no or minimal contamination from surrounding nontumor tissue. The proteomes of the LCM samples were subsequently analyzed by quantitative mass spectrometry based on stable isotope labeling. Several proteins, like interferon gamma-inducible protein 30 and a 70-kDa heat shock protein, were identified that are differentially expressed in tumor tissue compared to surrounding tissue and naive T cells. Taken together, our results demonstrate for the first time that MDV-induced tumors can be visualized using IMS, and we identified potential MDV tumor markers by analyzing the proteomes of virus-induced tumors.IMPORTANCE Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that infects chickens and causes the most frequent clinically diagnosed cancer in the animal kingdom. Not only is MDV an important pathogen that threatens the poultry industry but it is also used as a natural virus-host model for herpesvirus-induced tumor formation. In order to visualize MDV-induced lymphoma and to identify potential biomarkers in an unbiased approach, we performed imaging mass spectrometry (IMS) and noncontact laser capture microdissection. This study provides a first description of the visualization of MDV-induced tumors by IMS that could be applied also for diagnostic purposes. In addition, we identified and validated potential biomarkers for MDV-induced tumors that could provide the basis for future research on pathogenesis and tumorigenesis of this malignancy.

Classical swine fever virus strain "C" protects the offspring by oral immunisation of pregnant sows.

The aim of this study was to evaluate if oral immunisation of wild sows protects the fetuses from transplacental infection. Two experiments were carried out with gilts vaccinated orally with C-strain virus approximately 5 weeks after insemination. They were challenged at mid-gestation with highly virulent classical swine fever virus (CSFV) or moderately virulent field virus. The results revealed that oral vaccination has no negative impact on the pregnancy, and all vaccinated sows developed neutralising antibodies. After infection no symptoms were detected in the six vaccinated-infected sows. Challenge virus could neither be found in blood, nasal and fecal swabs or saliva nor in organs sampled at necropsy. Likewise, all fetuses originating from vaccinated sows were virologically and serologically negative. In contrast, the controls developed a short viremia and as a result of the transplacental infection all fetuses were CSFV positive. In addition, 22 serologically positive wild sows of an endemically infected area, where oral vaccination had also been carried out, and their offspring were free from CSFV or viral RNA. Our results confirm that oral immunisation of pregnant wild sows with C-strain vaccine may protect the fetuses against CSF.

[Gingival fibromatosis (hereditary hyperplastic gingivitis) in a wild European red fox (Vulpes vulpes)]. / Gingivafibromatose (hereditäre hyperplastische Gingivitis) bei einem europäischen Wildrotfuchs (Vulpes vulpes).

This report describes a case of gingival fibromatosis in an otherwise healthy and well nourished wild European red fox (Vulpes vulpes), which was shot by a hunter and submitted to the state laboratory in the context of the rabies monitoring program of the federal state of Brandenburg, Germany. At necropsy, a severe papillomatous proliferation of the complete gingival tissue of the upper and lower jaw was present. This gingival proliferation had already resulted in malocclusion, loosening and loss of several incisival, premolar and molar teeth. Histologically, the primary lesion was a massive increase in the amount of collagen rich and relatively avascular connective tissue within the gingival lamina propria mucosae. A papillomavirus infection was excluded by electron microscopy, immunohistochemistry and molecular biological methods. The gingival lesions in the red fox are identical to those seen in hereditary hyperplastic gingivitis in farmed silver foxes and hereditary gingival fibromatosis in man. It is presumed that, in analogy to the genetic diseases in silver foxes and man, a still unidentified genetic defect is responsible for the development of the disease in the red fox, too.

Persistence of viral RNA in rabbits which overcome an experimental RHDV infection detected by a highly sensitive multiplex real-time RT-PCR.

An internally controlled multiplex real-time RT-PCR using TaqMan probes and external standards for absolute RNA quantification was developed as a new diagnostic tool for the detection of rabbit haemorrhagic disease virus (RHDV). The test revealed a specificity of 100%, an analytical sensitivity of 10 copies/well and a linearity over a range from 10(1) to 10(10) copies. The viral loads in organs, leukocytes, sera and excretions of seropositive, convalescent rabbits which were overcoming an experimental infection with RHDV were determined using the validated assay. As a result, viral RNA was demonstrated and quantified for at least 15 weeks. Thus, a persistence of viral RNA after experimental infection of rabbits could be shown for the first time. In contrast, neither antigen nor infectious virus could be detected by antigen-ELISA, immunohistochemistry or experimental transmission. Therefore, further experiments are necessary to prove that the persistence of RNA is linked with the persistence of infectious virus particles.

Encephalitis in a stone marten (Martes foina) after natural infection with highly pathogenic avian influenza virus subtype H5N1.

Recent outbreaks of disease in different avian species, caused by the highly pathogenic avian influenza virus (HPAIV), have involved infection by subtype H5N1 of the virus. This virus has also crossed species barriers and infected felines and humans. Here, we report the natural infection of a stone marten (Martes foina) from an area with numerous confirmed cases of H5N1 HPAIV infection in wild birds. Histopathological examination of tissues from this animal revealed a diffuse nonsuppurative panencephalitis with perivascular cuffing, multifocal gliosis and neuronal necrosis. Additionally, focal necrosis of pancreatic acinar cells was observed. Immunohistochemically, lesions in these organs were associated with avian influenza virus antigen in neurons, glial cells and pancreatic acinar cells. Thus, the microscopical lesions and viral antigen distribution in this stone marten differs from that recently described for cats naturally and experimentally infected with the same virus subtype. This is the first report of natural infection of a mustelid with HPAIV H5N1.

Transplacental transmission of Neospora caninum in moose (Alces alces).

A 5-years-old moose (Alces alces) cow kept in a zoo in the German Federal State of Brandenburg aborted a female foetus of 44cm crown rump length (CRL). Pathohistological analysis revealed several Neospora (N.) caninum infected cells and cysts, as well as multifocal gliosis, necrosis, haemorrhages, dystrophic mineralisation and haemosiderosis in the brain, predominantly in cerebrum and brainstem. In addition, mild lymphocytic meningitis was present. Together with the fresh foetus, a mummified foetus of 16cm CRL was expelled. Neither focal necrosis, nor inflammation was detected in the brain of the mummified foetus. By two polymerase chain reactions (PCR) targeting the pNc5 gene of N. caninum (i.e. an end point PCR and a real-time PCR), by two serological methods (immunofluorescence test and immunoblot), by histological and immunohistochemical analyses, transplacental N. caninum infection was confirmed in the fresh foetus and interpreted as possible cause of abortion. Infection with other agents causing abortion including Bovine Herpesvirus 1 (BHV1), Bluetongue Virus (BTV), Bovine Virus Diarrhoea Virus (BVDV), Brucella spp., Chlamydia spp., Coxiella burnetii and Toxoplasma gondii were excluded. Our findings show that control measures may be necessary to protect captive moose against accidental N. caninum infection. Further studies are needed to explore the importance of neosporosis in wild and captive moose.

Choroid plexus carcinoma in a goat.

A 15-year-old female goat suddenly developed right-sided head tilting with anorexia and depression. Post-mortem examination of the brain revealed a large, unilateral, well-demarcated, intraventricular neoplasm which was diagnosed as a choroid plexus carcinoma. The neoplasm, which occupied about 75% of the left lateral ventricle, led to unilateral obstructive hydrocephalus and invaded the white and grey matter of the left piriform lobe, with focal subarachnoid spread and meningeal implantation. Histopathological examination revealed loss of branching papillary architecture, invasive growth, a high mitotic index and marked necrosis in the undifferentiated areas of the tumour. Neoplastic cells expressed vimentin and, multifocally, broad spectrum cytokeratins, but were negative for GFAP, NSE and Sl00 antigen. This is the first report of a choroid plexus carcinoma in a goat.

Experimental Infection of Calves with Escherichia coli O104:H4 outbreak strain.

In 2011, a severe outbreak of hemolytic-uremic syndrome was caused by an unusual, highly virulent enterohemorrhagic E. coli (EHEC) O104:H4 strain, which possessed EHEC virulence traits in the genetic background of human-adapted enteroaggregative E. coli. To determine magnitude of fecal shedding and site of colonization of EHEC O104:H4 in a livestock host, 30 (ten/strain) weaned calves were inoculated with 10(10) CFU of EHEC O104:H4, EHEC O157:H7 (positive control) or E. coli strain 123 (negative control) and necropsied (4 or 28 d.p.i.). E. coli O157:H7 was recovered until 28 d.p.i. and O104:H4 until 24 d.p.i. At 4 d.p.i., EHEC O104:H4 was isolated from intestinal content and detected associated with the intestinal mucosa. These results are the first evidence that cattle, the most important EHEC reservoir, can also carry unusual EHEC strains at least transiently, questioning our current understanding of the molecular basis of host adaptation of this important E. coli pathovar.

Detection of canine oral papillomavirus-DNA in canine oral squamous cell carcinomas and p53 overexpressing skin papillomas of the dog using the polymerase chain reaction and non-radioactive in situ hybridization.

Nineteen cutaneous and mucocutaneous papillomas, as well as 29 oral and 25 non-oral squamous cell carcinomas of dogs were analyzed immunohistologically for the presence of papillomavirus (PV)-antigens. Canine oral papillomavirus (COPV)-DNA was detected in formalin-fixed, paraffin-embedded tissues by polymerase chain reaction (PCR) and non-radioactive in situ hybridization (ISH). Furthermore, the expression of the tumor suppressor protein p53 was investigated. PV-antigens were detectable in more than 50% of the oral and cutaneous papillomas, while no PV-antigens could be demonstrated in venereal papillomas. One squamous cell carcinoma was PV-antigen positive. Only two cutaneous papillomas of the head showed a strong p53-specific immunostaining, while overexpressed p53 was detectable in approximately 35% of all squamous cell carcinomas. It was possible to amplify fragments of the E6, E7 and L1 gene by polymerase chain reaction (PCR) from five of eight oral and from five of eight cutaneous papillomas as well as from three oral squamous cell carcinomas. Nine of 10 papillomas showed a strong nucleus-associated hybridization signal typical for COPV-DNA. In three squamous cell carcinomas COPV-DNA was located in nests of the epithelial tumor cells surrounding 'horn pearls' or disseminated in the carcinoma tissue. These observations support the view that COPV may also induce non-oral papillomas in the dog and confirm the opinion that a progression of viral papillomas into carcinomas in dogs may occur.

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.

Immunohistochemical detection of P53 overexpression in paraffin wax-embedded squamous cell carcinomas of cattle, horses, cats and dogs.

One hundred and six squamous cell carcinomas (SCCs) of cattle, horses, cats and dogs were analysed immunohistochemically for overexpression of p53 protein. The monoclonal antibody pAb 240, which recognizes only mutant p53, was used. Of 41 bovine ocular SCCs, 26 (63.4%) showed p53 nuclear reactivity. All of six (100%) equine ocular SCCs and seven of nine (77.7%) SCCs of the equine penis or vulva gave positive reactions. In nine of 11 (81.8%) feline SCCs of the ear and in seven of 14 (50%) feline SCCs of other locations, p53 immunoreactivity was detected. Only seven of 25 (29.5%) canine cutaneous SCCs gave a positive reaction. Thus p53 antigen could be detected immunohistochemically in formalin-fixed and paraffin wax-embedded tissues of SCCs of domestic animals. The results support the view that, as in man, p53 overexpression plays an important role in the development of most SCCs of the animal species studied. This was in particular true for feline SCCs of the ear and for bovine and equine ocular SCCs, which are assumed to be related to ultraviolet radiation.

Heterotopic hairs in the tongue of two dogs.

The occurrence of hairs in the median sulcus of the tongue in two dogs, a 7-year-old Rottweiler and a 10-year-old Cocker spaniel, is reported as an incidental observation at necropsy. The gross pathology of both cases and the histological and scanning electron microscopical findings in one case indicated a heterotopic development of the hairs, with secondary inflammation. The term pili heterotopici mediani linguae is suggested.

Subacute liver necrosis after experimental infection with rabbit haemorrhagic disease virus (RHDV).

Rabbit haemorrhagic disease (RHD) is usually peracute to acute, while subacute to chronic disease is rare. This paper describes gross and histopathological findings in four out of 20 rabbits aged 14 weeks, experimentally infected with one of two German field isolates of RHD virus. Eight rabbits survived the infection for 10 days and were killed after four of them, infected with 100 to 10 000 haemagglutination units, had started to develop progressive jaundice. Histopathologically, icteric livers showed severe subacute centrilobular bridging necrosis with calcification, and proliferation of periportal hepatocytes and bile ducts. Positive-strand RHDV RNA was detected by in-situ hybridization, mainly in periportal macrophages. Loss of the normal hepatic architecture, reparation (fibrosis) and hepatocellular regeneration, together with moderate inflammatory reaction, are signs of liver cirrhosis. These signs, observed in young rabbits given small doses of RHD virus, are interpreted as an unusual outcome of experimental inoculation.

Detection of European porcine reproductive and respiratory syndrome virus in porcine alveolar macrophages by two-colour immunofluorescence and in-situ hybridization-immunohistochemistry double labelling.

Two groups of five pigs aged 6 weeks were each infected oronasally with one of two different European isolates of porcine reproductive and respiratory syndrome virus (PRRSV). The animals were killed sequentially at 4, 7, 14 or 21 days post-inoculation for examination. The methods used consisted of histopathology, and mono- and double-labelling techniques based on in-situ hybridization, immunofluorescence and immunohistochemistry. Porcine alveolar macrophages (PAMs) contained large amounts of PRRSV antigen and PRRSV RNA, as shown by double labelling with (1) either PRRSV immunofluorescence or PRRSV-specific in-situ hybridization with digoxigenin-labelled riboprobes, and (2) immunolabelling with Mac 387 antibody for calprotectin. Expression of PRRSV-RNA was not detectable in cytokeratin-positive hypertrophic and proliferating pneumocytes or in cells of alveolar ducts or bronchiolar epithelium. The use of two-colour immunofluorescence with confocal laser scanning microscopy and double labelling with in-situ hybridization-immunohistochemistry showed that PAMs were the only pulmonary target cells. This contradicts earlier reports that epithelial pulmonary cells may also be infected by PRRSV.

Evaluation of formalin-fixed paraffin-embedded tissues from vaccine site-associated sarcomas of cats for papillomavirus DNA and antigen.

OBJECTIVE: To determine whether vaccine site-associated sarcomas (VSS) from cats contain papillomavirus antigen or DNA. SAMPLE POPULATION: 50 formalin-fixed paraffin-embedded tissue blocks of VSS from cats. PROCEDURE: Sections from each tissue block were evaluated for papillomavirus antigen by use of an avidin-biotin-complex immunohistochemical staining method, using rabbit anti-bovine papillomavirus type-1 antibody. The DNA was extracted from sections of each tissue block, and polymerase chain reaction assays were performed, using primers designed to amplify regions of the E5 gene of bovine papillomavirus and consensus primers designed to amplify a region of the L1 gene of animal papillomaviruses. Sections from 20 of the tissue blocks were evaluated by use of nonradioactive in situ hybridization for bovine papillomavirus DNA. RESULTS: Papillomavirus antigen and DNA were not detected in any of the VSS. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that papillomaviruses likely do not have any direct involvement in the pathogenesis of VSS in cats.

[Halicephalobus (Syn. Micronema) deletrix as a cause of granulomatous gingivitis and osteomyelitis in a horse]. / Halicephalobus (Syn. Micronema) deletrix als Ursache einer granulomatösen Gingivitis und Osteomyelitis bei einem Pferd.

Therapy resistant swellings of the maxillary region and unilateral nasal discharge in older horses are mainly thought to be consequences of neoplasias of the oral cavity, especially of the gingiva and the teeth, or to develop from tumours of the nasal cavity. We report an unilateral swelling of the left nasal and buccal region in a 13-year-old gelding, which was accompanied by an aggressive destruction of involved osseous tissue due to a severe proliferative granulomatous inflammation. The granuloma was caused by the nematode Halicephalobus (syn. Micronema) deletrix. This nematode infection is known for over 30 years, even though the here reported form is uncommon and rarely diagnosed. However, this report shows that even in cases of unilateral maxillary swellings in horses a granulomatous inflammation due to nematodiasis should be considered as an additional differential diagnosis.