Infectivity and pathobiology of H7N1 and H5N8 high pathogenicity avian influenza viruses for pigeons (Columba livia var. domestica).

Avian influenza (AI) is one of the most important viral diseases in poultry, wildlife and humans. Available data indicate that pigeons play a minimum role in the epidemiology of AI. However, a degree of variation exists in the susceptibility of pigeons to highly pathogenic AI viruses (HPAIVs), especially since the emergence of the goose/Guangdong H5 lineage. Here, the pathogenesis of H5N8 HPAIV in comparison with a H7N1 HPAIV and the role of pigeons in the epidemiology of these viruses were evaluated. Local and urban pigeons (Columba livia var. domestica) were intranasally inoculated with 105 ELD50 of A/goose/Spain/IA17CR02699/2017 (H5N8) or A/Chicken/Italy/5093/1999 (H7N1) and monitored during 14 days. Several pigeons inoculated with H5N8 or H7N1 seroconverted. However, clinical signs, mortality, microscopic lesions and viral antigen were only detected in a local pigeon inoculated with H5N8 HPAIV. This pigeon presented prostration and neurological signs that correlated with the presence of large areas of necrosis and widespread AIV antigen in the central nervous system, indicating that the fatal outcome was associated with neurological dysfunction. Viral RNA in swabs was detected in some pigeons inoculated with H7N1 and H5N8, but it was inconsistent, short-term and at low titres. The present study demonstrates that the majority of pigeons were resistant to H5N8 and H7N1 HPAIVs, despite several pigeons developing asymptomatic infections. The limited viral shedding indicates a minimum role of pigeons as amplifiers of HPAIVs, regardless of the viral lineage, and suggests that this species may represent a low risk for environmental contamination. RESEARCH HIGHLIGHTS H7N1 and H5N8 HPAIVs can produce subclinical infections in pigeons. The mortality caused by H5N8 HPAIV in one pigeon was associated with neurological dysfunction. Pigeons represent a low risk for environmental contamination by HPAIVs.

Experimental infection of domestic geese (Anser anser var. domesticus) with H5N8 Gs/GD and H7N1 highly pathogenic avian influenza viruses.

Prior to the emergence of the Asian-origin H5 Goose/Guangdong/1/96 (Gs/GD) lineage, highly pathogenic avian influenza viruses (HPAIV) had rarely caused high mortalities in domestic geese. In 2016/2017 European epidemics, H5N8 Gs/GD clade 2.3.4.4 Group B produced an unprecedented number of outbreaks in waterfowl holdings. In this study, the pathogenesis of H5N8 HPAIV in comparison with H7N1 HPAIV, and the role of domestic geese in the epidemiology of these viruses, were evaluated. Local and commercial geese (Anser anser var. domesticus) were intranasally inoculated with 105 ELD50 of A/goose/Spain/IA17CR02699/2017 (H5N8) or A/Chicken/Italy/5093/1999 (H7N1) and monitored daily during 15 days. H5N8 was highly virulent to domestic geese, reaching 100% mortality by 10 days post-infection. Systemic microscopic necrotizing lesions associated with widespread AIV-antigen were detected by IHC techniques, the central nervous system being the most severely affected. High viral loads, measured by qRT-PCR, were present in all samples collected: oral and cloacal swabs, plasma tissues, and moderate levels in pool water. Domestic geese were also susceptible to H7N1 infection, as demonstrated by seroconversion and detection of viral RNA in tissues and plasma in some geese, but all lacked clinical signs. Viral shedding was confirmed in only some geese and was restricted to the oral route, but levels were high and still detected at the end of the study. Overall, H7N1 presents a lower lethality and shedding than H5N8 in geese; however, the viral shedding indicates that these species could play a role in the epidemiology of Gs/GD and other lineages of HPAIVs. RESEARCH HIGHLIGHTS H5N8 Gs/GD clade 2.3.4.4 Group B is highly virulent to domestic geese. The severity of H5N8 is associated with multisystemic replication. H7N1 can infect domestic geese but is avirulent to this species. Domestic geese could play a role in the epidemiology of Gs/GD HPAIVs.

Epidemiological and pathological investigation of fowl aviadenovirus serotypes 8b and 11 isolated from chickens with inclusion body hepatitis in Spain (2011-2013).

Inclusion body hepatitis caused by different fowl aviadenovirus (FAdV) serotypes has been described in several countries in recent years. In Spain, from the spring of 2011 to 2013, an increased number of outbreaks in broiler and broiler breeder flocks from different regions occurred. The objectives of the present work were to carry out the molecular characterization of FAdV strains from Spanish inclusion body hepatitis cases and to study the pathogenicity and viral dynamics of these strains in specific pathogen-free (SPF) chickens. A total of 52 inclusion body hepatitis clinical cases, including 45 from broiler farms and seven from broiler breeder farms, were analysed by conventional polymerase chain reaction and sequencing targeting the FAdV hexon gene. From these, 37 strains were classified as FAdV type 8b, while the remaining 15 were classified as FAdV types 11 (n = 10), 2 (n = 4) and 8a (n = 1). In addition, two different FAdVs belonging to the genotypes 8b and 11 were used for experimental infection. Specific pathogen-free five-day-old birds were inoculated intramuscularly with a high (106.5 tissue culture infective dose (TCID)50/ml) or low (104 TCID50/ml) dose of the above-mentioned FAdVs. No mortality was observed in any of the experimental groups, and only one bird showed evident clinical signs. However, macroscopic and microscopic hepatic lesions, as well as viral DNA, were detected in birds from all infection groups. Inclusion bodies and viral DNA were also detected in the pancreas and in the small and the large intestine in some birds. Long-lasting shedding and transmission to contact birds were confirmed in all infected groups.

Coronavirus Infection in Ferrets: Antigen Distribution and Inflammatory Response.

Multisystemic granulomatous lesions are the most common finding in ferrets infected by ferret systemic coronavirus (FRSCV). To characterize the inflammatory response developed against this virus, lesions from 4 naturally infected ferrets were examined. Lesions were classified into the 4 known types of granulomas (granulomas without necrosis [G], granulomas with necrosis [G-N], granulomas with neutrophils [G-NL], and diffuse granulomatous inflammation [DG]). The cellular composition of the lesions was characterized on the basis of cellular morphology and immunohistochemistry using markers for T and B-lymphocytes, plasma cells, macrophages, and neutrophils. The extent and distribution of viral antigen expression was also assessed. In G lesions, macrophages were mainly located in the center of the granuloma, with a moderate number of T-lymphocytes scattered among the macrophages, plasma cells, and B-lymphocytes. G-N lesions exhibited a necrotic center surrounded by abundant macrophages, some T-lymphocytes, plasma cells, and a few B-lymphocytes. In G-NL lesions, there was a central area dominated by neutrophils with low numbers of macrophages, plasma cells, and lymphocytes. DG presented similar cell proportions, but distributed evenly throughout the lesions. FRSCV was expressed in G, G-NL, G-N, and DG, with decreasing numbers of immunoreactive cells. This study reveals the important role of macrophages in the inflammatory response of ferrets against the virus and the variable proportions of leukocytes among different types of lesions, indicating their variable age. The results also confirm the similarities of the disease in ferrets to feline infectious peritonitis.

Six-Year Follow-up of Slaughterhouse Surveillance (2008-2013): The Catalan Slaughterhouse Support Network (SESC).

Meat inspection has the ultimate objective of declaring the meat and offal obtained from carcasses of slaughtered animals fit or unfit for human consumption. This safeguards the health of consumers by ensuring that the food coming from these establishments poses no risk to public health. Concomitantly, it contributes to animal disease surveillance. The Catalan Public Health Protection Agency (Generalitat de Catalunya) identified the need to provide its meat inspectors with a support structure to improve diagnostic capacity: the Slaughterhouse Support Network (SESC). The main goal of the SESC was to offer continuing education to meat inspectors to improve the diagnostic capacity for lesions observed in slaughterhouses. With this aim, a web-based application was designed that allowed meat inspectors to submit their inquiries, images of the lesions, and samples for laboratory analysis. This commentary reviews the cases from the first 6 years of SESC operation (2008-2013). The program not only provides continuing education to inspectors but also contributes to the collection of useful information on animal health and welfare. Therefore, SESC complements animal disease surveillance programs, such as those for tuberculosis, bovine cysticercosis, and porcine trichinellosis, and is a powerful tool for early detection of emerging animal diseases and zoonoses.

Extra- and intra-articular synovial chondromatosis and malignant transformation to chondrosarcoma.

Intra- and extra-articular primary synovial chondromatosis (SC) was observed in a five-year-old, entire male German Shepherd. Thousands of small cartilaginous nodules were removed from the stifle joint as well as from several adjacent muscles. Diagnosis of SC was established based on clinical, radiographic and biopsy results. The owner declined to have a new surgery performed for complete nodule removal and partial synovectomy. Nine months after the initial presentation, a proximal pathological intra- articular tibial fracture was observed and malignant transformation to chondrosarcoma was diagnosed after limb amputation. No metastasis was observed after 1.5 years of follow-up.

Dermatomycosis in a pet inland bearded dragon (Pogona vitticeps) caused by a Chrysosporium species related to Nannizziopsis vriesii.

A Chrysosporium sp. related to Nannizziopsis vriesii was isolated in pure culture from squames and biopsies of facial lesions in a pet inland bearded dragon (Pogona vitticeps) in Spain. The presence in histological sections of morphologically consistent fungal elements strongly incriminates this fungus as the aetiological agent of infection. Lesions regressed following treatment with oral ketoconazole and topical chlorhexidine and terbinafine until the lizard was lost to follow up 1 month later. The ITS-5.8S rRNA gene of the isolate was sequenced and a search on the GenBank database revealed a high match with the sequences of two Chrysosporium sp. strains recently isolated from green iguanas (Iguana iguana) with dermatomycosis, also in Spain. Phylogenetic analysis of the sequences revealed that all these strains are related to N. vriesii. This is the first report of dermatomycoses caused by a Chrysosporium species related to N. vriesii in a bearded dragon outside North America.

Little evidence of seasonal variation of natural infection by Leishmania infantum in dogs in Spain.

Leishmania infantum, the etiological agent of canine leishmaniosis in the Mediterranean region, is vectored by Phlebotomus spp sandflies, which are active during the warmer months of the year. In order to determine whether seasonality in transmission induces seasonal changes in the prevalence of infection by L. infantum and of parasite-specific immune response, two groups of dogs, one in February (n=37) and another in October (n=42), were studied. Clinical signs compatible with leishmaniosis, as well as presence of microscopic skin lesions in the muzzle were recorded for all dogs. Assays were also performed for detection of L. infantum parasites in muzzle skin samples (PCR, immunohistochemistry and culture), specific serum antibodies (ELISA), and specific lymphocyte proliferation and interferon-gamma production. Although prevalence of non-specific clinical signs increased significantly after the sandfly season, this was not the case for Leishmania-specific markers: positivity by PCR (24% vs. 21%) or immunohistochemistry (3% vs. 2%) of muzzle skin samples, as well as lymphocyte proliferation (59% vs. 50%) or interferon-gamma production (21% vs. 27%) were similar in February and in October. Only prevalence of positive specific antibody titers increased noticeably in October (8% vs. 20%), although this was not statistically significant. Overall, the sandfly season did not have a marked impact on the prevalence L. infantum infection or parasite-specific immune responses analyzed in this study.

Identification of group 1 coronavirus antigen in multisystemic granulomatous lesions in ferrets (Mustela putorius furo).

Tissues from nine ferrets with granulomatous lesions similar to those seen in feline infectious peritonitis were examined histopathologically and immunohistochemically. Four main types of lesions were observed: diffuse granulomatous inflammation on serosal surfaces; granulomas with areas of necrosis; granulomas without necrosis; and granulomas with neutrophils. Other less commonly seen lesions were granulomatous necrotizing vasculitis and endogenous lipid pneumonia. FCV3-70 monoclonal antibody produced immunolabelling of group 1 coronavirus antigen in tissue samples from eight animals, the antigen being present in the cytoplasm of macrophages in the different types of granulomatous lesions.

Outbreak of canine distemper in domestic ferrets (Mustela putorius furo).

In 2006 an outbreak of canine distemper affected 14 young domestic ferrets in Barcelona, Spain. Their clinical signs included a reduced appetite, lethargy, dyspnoea, coughing, sneezing, mucopurulent ocular and nasal discharges, facial and perineal dermatitis, diarrhoea, splenomegaly and fever. Late in the course of the disease, general desquamation and pruritus, and hyperkeratotic/crusting dermatitis of the lips, eyes, nose, footpads, and perineal area were observed. None of the ferrets developed neurological signs. Non-regenerative anaemia and high serum concentrations of alpha- and beta-globulins were the most common laboratory findings. Most of the animals died or were euthanased because of respiratory complications. Postmortem there were no signs of lung collapse. Distemper was diagnosed by direct immunofluorescence of conjunctival swabs or pcr of several organs, and histology revealed the characteristic eosinophilic intracytoplasmic and intranuclear inclusion bodies of canine distemper virus in several organs. The minimum incubation periods calculated for six of the ferrets were 11 to 56 days, and in 13 of the ferrets the signs of disease lasted 14 to 34 days. Inclusion bodies compatible with infection by herpesvirus were found in the lungs of one of the ferrets.

Laryngeal Granuloma due to Leishmania spp. Infection in a Dog.

A French bulldog with a previous history of leishmaniosis was presented due to respiratory distress associated with a laryngeal mass. The mass was excised and cytological and histopathological examination revealed pyogranulomatous inflammation with Leishmania spp. amastigotes. After surgery, the respiratory condition resolved; however, 3 months later the dog developed clinicopathological signs of leishmaniosis, which improved with systemic treatment. This case shows an atypical presentation of leishmaniosis with a focal tumour-like mass in the vocal folds as the only clinical sign.

Transmission and immunopathology of the avian influenza virus A/Anhui/1/2013 (H7N9) human isolate in three commonly commercialized avian species.

H7N9 virus infection is a global concern, given that it can cause severe infection and mortality in humans. However, the understanding of H7N9 epidemiology, animal reservoir species and zoonotic risk remains limited. This work evaluates the pathogenicity, transmissibility and local innate immune response of three avian species harbouring different respiratory distribution of α2,6 and α2,3 SA receptors. Muscovy ducks, European quails and SPF chickens were intranasally inoculated with 105 embryo infectious dose (EID)50 of the human H7N9 (A/Anhui/1/2013) influenza isolate. None of the avian species showed clinical signs or macroscopic lesions, and only mild microscopic lesions were observed in the upper respiratory tract of quail and chickens. Quail presented more severe histopathologic lesions and avian influenza virus (AIV) positivity by immunohistochemistry (IHC), which correlated with higher IL-6 responses. In contrast, Muscovy ducks were resistant to disease and presented higher IFNα and TLR7 response. In all species, viral shedding was higher in the respiratory than in the digestive tract. Higher viral shedding was observed in quail, followed by chicken and ducks, which presented similar viral titres. Efficient transmission was observed in all contact quail and half of the Muscovy ducks, while no transmission was observed between chicken. All avian species showed viral shedding in drinking water throughout infection.

Clinical, Cytological, Histological and Immunohistochemical Features of Cutaneous Mast Cell Tumours in Ferrets (Mustela putorius furo).

Cutaneous mast cell tumours (cMCTs) are one of the most common cutaneous tumours in ferrets (Mustela putorius furo). However, limited information is available regarding cytological and histological features of these tumours and studies evaluating KIT expression are lacking in this species. The aims of this prospective study were to describe the most common clinical, cytological and histological features of cMCTs in ferrets and to compare the usefulness of different staining techniques in the diagnosis of these tumours in ferrets as well as evaluating KIT expression in neoplastic mast cells (MCs) by immunohistochemistry. Macroscopically, the tumours were small, round to plaque-like and frequently associated with surface crusting. The most common locations were the extremities and the trunk. MC granules were stained in all cases using toluidine blue (TB) and Wright-Giemsa stains in cytological specimens, but none stained with modified Wright's stain. Haematoxylin and eosin and TB on histological sections failed to stain MC granules in all the cases. Cytological and histological examination revealed low to moderate anisocytosis and anisokaryosis. An infiltrative rather than a delineated or encapsulated growth pattern was noted histologically in all cases. Eosinophilic infiltration was not uncommon and 'collagenolysis' was detected on cytological and histological examination. KIT expression was detected in all cases evaluated. In approximately one third of the cases the MCs exhibited KIT labelling pattern I and in the remaining ferrets, KIT pattern III. No correlation was found between KIT expression pattern and biological behaviour.

Multiple cartilaginous exostosis in a Golden Retriever cross-bred puppy. Clinical, radiographic and backscattered scanning microscopy findings.

Multiple cartilaginous exostosis was diagnosed in a six-month-old Golden Retriever cross-bred male with a history of forelimb lameness and isolated, but very painful, acute episodes. Physical examination revealed a right forelimb lameness with a firm, painful palpable mass on the cranial aspect of the forearm. The radiological examination showed the presence of bony masses at the humerus and radius as well as several masses in the ribs and spinous processes of the thoracic vertebrae. Based on the history and radiographic findings, multiple cartilaginous exostosis was diagnosed. Treatment with non-steroidal anti-inflammatory drugs was commenced for two weeks without any effect. Due to the lack of a response to the treatment as well as to the progressive physical deterioration of the animal, the owners requested euthanasia of the dog. Histology of the different exostoses demonstrated the presence of a hyaline cartilage cup surrounding a central area, formed mainly by bone and cartilage trabecullae. Signs of malignancy were not observed. Back-scattered scanning electron microscopy (BEI-SEM) study revealed well ordered and progressively calcified cartilage trabecullae present underneath the non-calcified cartilage cap. At a greater depth, those cartilage trabecullae became osteochondral trabecullae, and the innermost were formed exclusively by woven and lamellar bone. The histological and back-scattered electron scanning microscopy results conclude that it was a well-arranged normal endochondral ossification process that followed a centripetal pattern inside the bony mass, confirming the diagnoses of multiple cartilaginous exostoses.

Scanning electron microscopy of swine lymphoid organs.

The aim of this investigation was to study by scanning electron microscopy the structure of several swine lymphoid organs (lymph nodes, Peyer's patches, and tonsil). Two groups of animals were used: six-month-old pigs and six- to nine-day-old piglets. Samples were jet-washed to eliminate most free cells in order to observe the reticular framework of these organs more clearly. Peyer's patches in piglets showed two types of villi. In one of them the cellular types were absorptive cells and goblet cells. The second type of villi were shorter and wider, with M cells characterized by presenting long, thick microvilli over their surfaces. Peyer's patches of pigs did not show this second type of villi but were usually covered by absorptive villi. The soft palate tonsil was similar in both groups of animals with its surface epithelial cells full of microfolds, partially and frequently obscured by microorganisms. The appearance of the surface epithelium in the same crypt was different depending on the area. There was a large number of holes through which cells apparently passed towards the crypt lumen. The medulla in the lymph nodes was at the periphery and showed a dense reticular framework. Cortex-like lymphoid tissue was formed by lymphoid follides and diffuse lymphoid tissue with high endothelid venules and lymphatic sinuses. The serosal surface of lymphoid organs was formed either by a typical mesothelial cell layer (small intestine) or by loosely arranged connective fibers (lymph nodes).

Immunocytochemical study of the pathogenesis of Pacheco's parrot disease in budgerigars.

Thirty-five budgerigars were infected with the Psittacid herpesvirus 1 (RSL-1 strain, ATCC) to study the pathogenesis of Pacheco's disease. Intramuscular (i.m.) and oral (p.o.) infection routes were used in 21 and 14 animals respectively. Animals were euthanized on days 1, 2, 3, 4, 6 and 8 post-inoculation (p.i.) and complete postmortem examinations and histological studies were performed. The presence of viral antigen in tissues was detected by immunohistochemical techniques using a rabbit polyclonal antibody. In the i.m.-infected birds, lesions were first detected on the third day p.i. and included necrotizing hepatitis and splenitis, both associated to the presence of viral antigen. Necrotic and inflammatory lesions as well as viral antigen were detected in many organs after the fourth day p.i. (oesophagus, crop, pancreas, kidney, adrenal gland, thyroid and parathyroid glands, thymus, ovary and feathers) proving generalization of the disease. Chronology of the infection was similar in the p.o.-infected birds. However, two main differences were observed between the groups: In the p.o. group, viral antigen was first detected in cloacal mucosa (3rd day p.i.), liver and spleen; and viral entry into target cells and dissemination to the rest of the tissues was slower in this group. In addition, detection of viral antigen in feather follicular epithelial cells and in granulosa cells of organ follicles are findings that could be of relevance to the transmission of the virus.

Ultrastructural study of turkey rhinotracheitis virus infection in turbinates of experimentally infected chickens.

Ultrastructural changes associated with turkey rhinotracheitis virus infection were studied in turbinates of chickens experimentally infected with the isolate CVL 14/86/1. Chickens were sacrificed at 3, 5 and 7 days after inoculation and samples of the middle turbinate were taken, fixed, dehydrated and embedded in an hydrophilic resin. An immunofluorescence technique on semithin sections was carried out and viral antigen was observed in the cytoplasm and associated to cilia of the turbinate epithelial cells, on days 3 and 5 after inoculation. Ultrastructurally, gold stained intracytoplasmic nucleocapsid aggregates of turkey rhinotracheitis virus were observed in ciliated and non-ciliated epithelial cells, as well as budding virus particles, at days 3 and 5 postinoculation. Different ultrastructural abnormalities, including cytoplasmic blebs, clumping and loss of cilia were observed in the apical cell membrane of many infected cells, associated with the presence of intracytoplasmic inclusions. On day 5 after inoculation, substitution of ciliated and non-ciliated epithelial cells was noted and many desquamated epithelial cells were observed within the lumina. Regenerative changes in the ciliated epithelium were observed by day 7 postinoculation. These results indicate that turkey rhinotracheitis virus is able to replicate in ciliated and non-ciliated epithelial cells causing severe alterations to the cell surface and ciliary apparatus of the turbinate epithelium. Viral-induced damage to the turbinate epithelium could enhance the susceptibility of epithelial cells to secondary bacterial infection.

Turkey rhinotracheitis virus and Escherichia coli experimental infection in chickens: histopathological, immunocytochemical and microbiological study.

The aim of this study was to evaluate the response of chickens to a combined infection with turkey rhinotracheitis virus (TRTV) and Escherichia coli O78:K80. Groups of specific-pathogen-free chickens were inoculated by eyedrop and intranasal routes with TRTV and/or E. coli O78:K80. Presence of E. coli O78:K80, histopathological changes and tissue distribution of viral antigen in the respiratory tract of chickens were evaluated. Dual infection resulted in increased severity of clinical signs, and macroscopic and microscopic lesions compared with those groups given single infections. All 36 chickens inoculated with TRTV plus E. coli O78:K80 showed severe rhinitis. Moreover, periorbital edema and fibrinous airsacculitis and pericarditis were observed in one of the three chickens inoculated with both agents and sacrificed at day 5 p.i. In addition, purulent material in the air spaces of the cranial bones was seen in three of the six animals from the same group sacrificed at days 5 and 7 p.i. The distribution of viral antigen in tissues was similar in groups inoculated with TRTV and TRTV plus E. coli, but viral antigen was detected only in main bronchi of chickens from the latter group. The quantity of E. coli O78:K80 isolated from the nasal cavity was greater in the group given dual infection. The results obtained suggest that TRTV may act as primary agent, enhancing E. coli multiplication. The lesions observed in the group inoculated with both agents could correspond to an initial stage of swollen head syndrome (SHS) and contribute to the hypothesis that SHS could be due to a mixed infection with TRTV and E. coli.

Adenovirus hepatitis in a boa constrictor (Boa constrictor).

A boa constrictor was submitted for postmortem evaluation. At necropsy, there were no substantial lesions except in the liver. Light microscopy revealed severe multifocal to coalescing coagulative necrotic hepatitis, with basophilic and eosinophilic intranuclear inclusions in hepatocytes within the necrotic foci. The histopathological findings suggested a viral hepatitis. An adenoviral infection was diagnosed by means of transmission electronic microscopy and in situ hybridization techniques.

Neospora caninum infection in a Napolitan mastiff dog from Spain.

Fetal neosporosis-associated myeloencephalitis was diagnosed in a 4-month-old Napolitan mastiff dog from Spain. Neospora caninum tachyzoites and tissue cysts were observed in lesions in the central nervous system and the diagnosis was confirmed by immunohistochemical staining with anti-N. caninum monoclonal and polyclonal antibodies.