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.

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.

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.

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.

[Leptospirosis with necro-haemorrhagic cholecystitis in a Boxer puppy]. / Leptospirose mit hämorrhagisch-nekrotisierender Cholezystitis bei einem Boxerwelpen.

A Boxer puppy from the island of Rügen, which was properly vaccinated according to its age, was presented with acute gastrointestinal symptoms. The presumptive diagnosis of leptospirosis with acute renal failure, hepatic damage, and jaundice was confirmed by seroconversion (increased titre to 1 : 800 in a non-vaccine serogroup 4 weeks after disease onset). Cholecystitis was diagnosed based on clinical symptoms and sonographic results. After an initial improvement, the puppy's condition deteriorated and cholecystectomy was performed. Histopathological diagnosis indicated a haemorrhagic necrotizing cholecystitis.

Lyssavirus distribution in naturally infected bats from Germany.

In Germany, to date three different lyssavirus species are responsible for bat rabies in indigenous bats: the European Bat Lyssaviruses type 1 and 2 (EBLV-1, EBLV-2) and the Bokeloh Bat Lyssavirus (BBLV) for which Eptesicus serotinus, Myotis daubentonii and Myotis nattereri, respectively, are primary hosts. Lyssavirus maintenance, evolution, and epidemiology are still insufficiently explored. Moreover, the small number of bats infected, the nocturnal habits of bats and the limited experimental data still hamper attempts to understand the distribution, prevalence, and in particular transmission of the virus. In an experimental study in E. serotinus a heterogeneous dissemination of EBLV-1 in tissues was detected. However, it is not clear whether the EBLV-1 distribution is similar in naturally infected animals. In an attempt to further analyze virus dissemination and viral loads within naturally infected hosts we investigated tissues of 57 EBLV-1 positive individuals of E. serotinus from Germany by RT-qPCR and compared the results with those obtained experimentally. Additionally, tissue samples were investigated with immunohistochemistry to detect lyssavirus antigen in defined structures. While in individual animals virus RNA was present only in the brain, in the majority of E. serotinus viral RNA was found in various tissues with highest relative viral loads detected in the brain. Interestingly, viral antigen was confirmed in various tissues in the tongue including deep intralingual glands, nerves, muscle cells and lingual papillae. So, the tongue appears to be a prominent site for virus replication and possibly shedding.

[Foot-and-mouth disease and its differential diagnoses]. / Die Maul- und Klauenseuche und ihre wichtigsten Differenzialdiagnosen.

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals, which leads to the formation of vesicles, erosions und ulcerations in the mouth and hairless parts of the skin, in particular on the feet. Due to its dramatic economic consequences, FMD is considered to be one of the most important diseases of animals. There is a permanent risk of introduction of the virus into Europe due to travel and illegal importation of agricultural products. Cloven-hoofed animals (cattle, sheep, goats, pigs and related game animals) are the typical hosts of the FMD virus. However, some zoo and wild animals belonging to other taxonomical groups, such as giraffes, elephants and camels, are also susceptible. Stomatitis and infections of the feet in livestock occur quite frequently, and often the causes of these conditions remain obscure. Sometimes, a differentiation from FMD is not possible on the basis of clinical signs and gross lesions, necessitating further laboratory investigations. This applies in particular to cases caused by the agents of vesicular stomatitis (VS) and swine vesicular disease (SVD). Additionally, other infectious agents can cause stomatitis, e.g. the viruses of mucosal disease (MD), malignant catarrhal fever (MCF), rinderpest, peste des petits ruminants (PPR), papular stomatitis, orf, blue tongue (BT) and epizootic haemorrhagic disease (EHD). In sheep, a stomatitis of unclear etiology was described as "OMAGOD". Furthermore, bacteria, chemicals and mechanical trauma can cause stomatitis and pododermatitis.

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.

Equid herpesvirus 5-associated dermatitis in a horse--Resembling herpes-associated erythema multiforme.

An equid herpesvirus 5 (EHV-5) infection was detected in lesioned skin from a nine-year-old Holsteiner stallion in the south of Germany. Macroscopically, the animal displayed a non-pruritic, multifocal, pustular dermatitis around both eyes, nostrils and the muzzle, which had been ongoing for one year. Histopathologically, skin lesions were characterized by orthokeratotic to parakeratotic hyperkeratosis, pustular dermatitis, epidermal hyperplasia, apoptotic keratinocytes, a lympho-plasmahistiocytic interface dermatitis with hydropic degeneration of keratinocytes, and perivascular to diffuse, lympho-histiocytic infiltrations. The stratum granulosum and the upper part of the stratum spinosum contained multiple amphophilic, intranuclear inclusion bodies. By in situ hybridization and immunohistochemistry herpesvirus DNA and protein, respectively, were detected within keratinocytes containing inclusion bodies. Sequencing of the PCR-product revealed the presence of EHV-5 DNA. This is the first description of a dermatitis associated with EHV-5 in a horse, indicating that EHV-5 should be considered as an etiology of lymphohistiocytic interface dermatitis with intranuclear inclusion bodies in horses and is similar to herpes-associated erythema multiforme in humans.

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.

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.

Epidemiological and ornithological aspects of outbreaks of highly pathogenic avian influenza virus H5N1 of Asian lineage in wild birds in Germany, 2006 and 2007.

In Germany, two distinct episodes of outbreaks of highly pathogenic avian influenza virus of subtype H5N1 (HPAIV H5N1) in wild birds occurred at the beginning of 2006, and in summer 2007. High local densities of wild bird populations apparently sparked clinically detectable outbreaks. However, these remained restricted in (i) number of birds, (ii) species found to be affected, (iii) time, and (iv) location despite the presence of several hundred thousands of susceptible wild birds and further stressors (food shortage, harsh weather conditions and moulting). Northern and southern subpopulations of several migratory anseriform species can be distinguished with respect to their preference for wintering grounds in Germany. This corroborates viral genetic data by Starick et al. (2008) demonstrating the introduction of two geographically restricted virus subpopulations of Qinghai-like lineage (cluster 2.2.A and 2.2.B) into northern and southern Germany, respectively, in 2006. The incursion of virus emerging in 2007, found to be distinct from the clusters detected in 2006 (Starick et al., 2008), may have been associated with moulting movements. Intensive past-outbreak investigations with negative results of live and dead wild birds and of terrestrial scavengers excluded continued circulation of virus on a larger scale. However, persistence of virus in small pockets of local wild bird populations could not be ruled out resiliently. 1.5% of investigated sera originating from cats sampled at the epicentres of the Ruegen 2006-outbreak contained H5-antibodies. Passive monitoring was found to be highly superior to live bird surveillance when aiming at the detection of HPAIV H5N1 in wild birds (P < 0.0001).

Pleomorphic corneal sarcoma resembling malignant peripheral nerve sheath tumor in a horse.

A 14-year-old Haflinger gelding presented with a protruding mass involving the cornea of the right eye. The mass was resected and submitted for histopathologic and immunohistochemical examination. The preliminary diagnosis was corneal sarcoma, most likely fibrosarcoma. The immunohistochemical results confirmed the mesenchymal origin of the neoplastic cells, which were most consistent with a malignant peripheral nerve sheath tumor. Corneal mesenchymal neoplasms are extremely uncommon tumors in human beings and domestic animals. The cause for this tumor was not determined; infection with bovine papillomavirus was not detected.

Detection of canine oral papillomavirus DNA in conjunctival epithelial hyperplastic lesions of three dogs.

Papillomavirus infections are responsible for plaques and papillomas in various locations on the skin and in mucous membranes. The aim of this report was to describe morphologic features of a viral pigmented conjunctival plaque and 2 conjunctival squamous papillomas in 3 dogs, and to investigate these lesions for the presence of papillomavirus DNA by polymerase chain reaction (PCR), DNA sequence analysis, and in situ hydridization (ISH). Histopathology revealed in all neoplasms various degrees of epithelial hyperplasia, acanthosis, and hyperkeratosis with koilocytosis. In all lesions E6, E7, and L1 gene fragments of canine oral papillomavirus (COPV) DNA were detected by PCR and sequencing analysis. ISH revealed COPV DNA in a highly specific pattern within nuclei of the hyperplastic epithelium. The presence of canine papillomavirus in ocular conjunctival plaques and papillomas suggests these benign lesions may have the potential for malignant transformation. This is the first time that the lambdapapillomavirus COPV has been detected in ocular epithelial hyperplastic lesions.

Clinicopathologic features of a systemic coronavirus-associated disease resembling feline infectious peritonitis in the domestic ferret (Mustela putorius).

From 2002 to 2007, 23 ferrets from Europe and the United States were diagnosed with systemic pyogranulomatous inflammation resembling feline infectious peritonitis (FIP). The average age at the time of diagnosis was 11 months. The disease was progressive in all cases, and average duration of clinical illness was 67 days. Common clinical findings were anorexia, weight loss, diarrhea, and large, palpable intra-abdominal masses; less frequent findings included hind limb paresis, central nervous system signs, vomiting, and dyspnea. Frequent hematologic findings were mild anemia, thrombocytopenia, and hypergammaglobulinemia. Grossly, whitish nodules were found in numerous tissues, most frequently the mesenteric adipose tissue and lymph nodes, visceral peritoneum, liver, kidneys, spleen, and lungs. One ferret had a serous abdominal effusion. Microscopically, pyogranulomatous inflammation involved especially the visceral peritoneum, mesenteric adipose tissue, liver, lungs, kidneys, lymph nodes, spleen, pancreas, adrenal glands, and/or blood vessels. Immunohistochemically, all cases were positive for coronavirus antigen using monoclonal antibody FIPV3-70. Electron microscopic examination of inflammatory lesions identified particles with coronavirus morphology in the cytoplasm of macrophages. Partial sequencing of the coronavirus spike gene obtained from frozen tissue indicates that the virus is related to ferret enteric coronavirus.

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.

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.

Distribution of lesions and antigen of highly pathogenic avian influenza virus A/Swan/Germany/R65/06 (H5N1) in domestic cats after presumptive infection by wild birds.

In early 2006, the highly pathogenic avian influenza virus (HPAIV) H5N1 of the Asian lineage caused the death of wild aquatic birds in Northern Germany. In the mainly affected areas, a trans-species transmission of HPAIV H5N1 to mammals occurred between birds and domestic cats and 1 Stone Marten (Martes foina), respectively. Here, we report lesions and distribution of influenza virus antigen in 3 cats infected naturally with HPAIV H5N1 A/swan/Germany/R65/06. The hemagglutinin partial nucleotide sequences of the viruses were genetically closely related to a H5N1 HPAIV obtained from a dead Whooper Swan (Cygnus cygnus) of the same area. At necropsy, within the patchy dark-red and consolidated lungs, there was granulomatous pneumonia caused by Aelurostrongylus sp. Histologically, the main findings associated with influenza in all cats were bronchointerstitial pneumonia and marked random hepatic necrosis. In addition, all animals displayed lymphoid necrosis in the spleen and Peyer's patches and necrosis of the adrenal cortex. Immunohistochemically, nucleoprotein of HPAIV was present intralesionally in the lungs, liver, adrenal glands, and lymphoid tissues. Oropharyngeal swabs were shown to be suited to detect HPAIV by quantitative real-time polymerase chain reaction (RT-PCR) in these cats, despite the paucity of influenza virus antigen in the upper respiratory tract by means of immunohistochemistry. The results show that outdoor cats in areas affected by HPAIV in wild birds are at risk for lethal infection. In conclusion, hepatic necrosis was, besides bronchointerstitial pneumonia, the primary lesion, suggesting that in naturally infected cats, damage to the liver plays an important role in the pathogenesis of H5N1 influenza.

Pathology of natural infections by H5N1 highly pathogenic avian influenza virus in mute (Cygnus olor) and whooper (Cygnus cygnus) swans.

Mortality in wild aquatic birds due to infection with highly pathogenic avian influenza viruses (HPAIV) is a rare event. During the recent outbreak of highly pathogenic avian influenza in Germany, mortality due to H5N1 HPAIV was observed among mute and whooper swans as part of a rapid spread of this virus. In contrast to earlier reports, swans appeared to be highly susceptible and represented the mainly affected species. We report gross and histopathology and distribution of influenza virus antigen in mute and whooper swans that died after natural infection with H5N1 HPAIV. At necropsy, the most reliable lesions were multifocal hemorrhagic necrosis in the pancreas, pulmonary congestion and edema, and subepicardial hemorrhages. Major histologic lesions were acute pancreatic necrosis, multifocal necrotizing hepatitis, and lymphoplasmacytic encephalitis with neuronal necrosis. Adrenals displayed consistently scattered cortical and medullary necrosis. In spleen and Peyer's patches, mild lymphocyte necrosis was present. Immunohistochemical demonstration of HPAIV nucleoprotein in pancreas, adrenals, liver, and brain was strongly consistent with histologic lesions. In the brain, a large number of neurons and glial cells, especially Purkinje cells, showed immunostaining. Occasionally, ependymal cells of the spinal cord were also positive. In the lungs, influenza virus antigen was identified in a few endothelial cells but not within pneumocytes. The infection of the central nervous system supports the view that the neurotropism of H5N1 HPAIV leads to nervous disturbances with loss of orientation. More investigations are necessary to clarify the mechanisms of the final circulatory failure, lung edema, and rapid death of the swans.