Silent Infection of Highly Pathogenic Avian Influenza Virus (H5N1) Clade 2.3.4.4b in a Commercial Chicken Broiler Flock in Italy.

From October 2021 to January 2022, different incursions of clade 2.3.4.4b H5N1 HPAIV (Highly Pathogenic Avian Influenza Virus) occurred in several Italian regions with its main diffusion in Densely Poultry Populated Areas (DPPAs) of north-eastern Italy. Monitoring and control activities applied in the affected area clearly evidenced that turkeys and broilers were the most affected species, although several flocks of broilers at times resulted HPAIV H5N1 infected in absence of increased mortality and/or clinical signs. Thus, an approach based on sampling dead birds was adopted in the broiler sector to improve the early detection of infection; this protocol allowed us to confirm that 15 farms were HPAIV-infected with birds ready to be delivered to the slaughterhouse. The aim of this report is to describe the results of the diagnostic activities carried out in one HPAIV H5N1-infected broiler farm, three days after laboratory confirmation during the pre-movement testing without showing increased mortality or clinical signs. Thus, clinical signs, daily cumulative mortality rate (CMR), virus shedding, seroconversion, pathobiology of clade 2.3.4.4b H5N1 HPAIV as well as Avian Influenza Viruses (AIVs) environmental contamination were thoroughly examined in the infected holding. Such in-depth investigation demonstrated low infection prevalence in live birds, low environmental contamination, no seroconversion for AIVs, gross and microscopic findings compatible with systemic infection with peracute death in H5N1 HPAIV-infected birds.

Identification of Histopathological Criteria for the Diagnosis of Canine Cutaneous Progressive Angiomatosis.

The term angiomatosis is used to denote a group of well-known to poorly characterized proliferative vascular entities. In animals, cutaneous progressive angiomatosis (CPA) is a disorder with variable prognosis related to the extension and depth of infiltration of the surrounding tissues by vessels. CPA may share some microscopical features with other vascular proliferations such as low-grade well-differentiated capillaritic hemangiosarcoma (HS), making the diagnosis not always straightforward, especially in small biopsies. The aim of this study is to retrospectively assess the most common diagnostic microscopical features of CPA in dogs. In this work, 11 histopathological criteria were analyzed on 31 CPA and 11 primary cutaneous HS in dogs. Features significantly associated with CPA included: lobular growth, interposition of connective tissue and adnexa between the vascular proliferation, presence of nerve fibers, and a mixed vascular proliferative component. Absence of plump/prominent endothelial cells, lack of atypia, and lack of mitoses were also significant factors differentiating CPA from HS. Additional distinctive findings in CPA, although with no statistical association to CPA diagnosis, were vascular shunting, absence of necrosis, and endothelial cell piling up. In conclusion, the combined use of different microscopical clues allowed for the distinction of CPA from HS and was considered useful for the diagnosis of CPA.

Atypical Mycosis in Psittacine Birds: A Retrospective Study.

A retrospective study was conducted on parrots submitted from necropsy to the Department of Veterinary Pathology, School of Biosciences and Veterinary, University of Camerino, Italy, from 2007 to 2018. From a total of 2,153 parrots examined at post-mortem, four cases were diagnosed with atypical mycosis and were considered for determination of the fungus species by PCR. A Fischer's lovebird (Agapornis fischeri), Peach-faced lovebirds (Agapornis roseicollis), and two Blue and Gold Macaws (Ara ararauna) from four different aviaries died after some days of lethargy and ruffled feathers. Records of gross necropsy and histopathological exams (H&E, PAS, and Grocott stain) were described and biomolecular analyses were carried out. No specific gross lesions were appreciated at necropsy, while histopathology evidenced a systemic mycosis in several organs, particularly in the lungs. In affected organs, broad and non-septate hyphae, suggestive of mycoses, were observed. Molecularly, Mucor racemosus (Fischer's lovebird) and M. circinelloides (Peach-faced lovebirds) were identified from formalin-fixed and paraffin-embedded (FFPE) lung and liver tissue. In addition, Alternaria alternata and Fusicladium spp. (respectively in male and female Blue and Gold macaws) were identified in FFPE tissue from several organs; whereas the role of Mucor spp. as true pathogens is well-demonstrated, and the behavior of A. alternata and Fusicladium spp. in macaws as opportunistic pathogens have been discussed. To our knowledge, this report is the first one reporting mucormycosis caused by M. racemosus and M. circinelloides in lovebirds, and A. alternata and Fusicladium spp. in macaws.

Looking for Dog Blood Donors in an Endemic Area for Vector-Borne Infections of Central Italy.

Dogs are proved to be competent reservoir hosts for several vector-borne pathogens. Their prevalence varies according to the geographical area. Many vector-borne pathogens may be transmitted by blood transfusion. The purpose of this study was to determine the serological and molecular prevalence of some vector-borne pathogens in dog blood donors, living in central Italy. Blood samples of 126 donors (19 breeds) included were tested for a broad serological and DNA-base tests panel. The differences in pathogen prevalence according to age, sex, and breeds were tested (chi-square test, Fisher's exact test). Overall, 50 animals (39.7%) tested positive at PCR (polymerase chain reaction) and/or serology (IFAT, indirect fluorescent antibody test) for at least one pathogen. Three dogs were positive at both serology and PCR. A tendency of hemoplasmas to be more prevalent in older dogs (41.2%) compared to the younger ones (25.7%) was noted. We highlight the difficulties of selecting healthy blood donor dogs in an endemic area for vector-borne infections. It is important to choose the serological and biomolecular investigations panel that is most suited to the donor's environment. Close collaboration between clinician and parasitologists is important in the interpretation of IFAT and PCR results. Finally, we underline the important role of blood donors as an epidemiological tool for active surveillance against canine vector-borne diseases.

Herpetic Pneumonia in Indian Ringneck Parrots (Psittacula krameri): First Report of Novel Psittacid Alphaherpesvirus-5 Infection in Europe.

The first two European outbreaks of herpetic pneumonia caused by Psittacid alphaherpesvirus-5 were diagnosed based on gross pathology findings, histological examination, transmission electron microscopy visualization and genome sequencing. The outbreaks, characterized by high morbidity and high mortality rates, involved two parrot species, namely the Indian ringneck parrot (Psittacula krameri) and the Alexandrine parakeet (Psittacula eupatria). Clinical signs observed were ruffled feathers, dyspnea, tail bobbing, open wings while breathing, depression and anorexia. Necropsy was performed on Indian ringneck parrots only, and the most evident and serious gross lesion found in all the birds was a diffuse marked consolidation of the lungs associated with parenchyma congestion and oedema. Histological examination confirmed the existence of bronchopneumonia characterized by the presence of syncytial cells with intranuclear inclusion bodies. In one bird, fibrinous airsacculitis was observed as well. Lung tissue inspection through electron microscopy revealed the presence of virus particles resembling herpesviruses. Viral DNA was extracted, amplified using primers for Alloherpesviridae DNA polymerase gene detection, and then sequenced. BLAST analysis showed a 100% identity with the only previously reported sequence of PsHV-5 (MK955929.1).

A case report of a rapid development of fetal anasarca in a canine pregnancy at term.

A 5-year-old healthy pluriparous pregnant Flat-coated Retriever bitch was monitored by ultrasound on post-ovulation days 30 and 57: no deviation from normality picture were observed. On day 60, one of the three most caudal fetuses showed ultrasonographic signs of fetal anasarca: subcutaneous edema and anechoic fluid in thoracic and abdominal cavities. There was an increased volume of extra-fetal fluids. On day 64 a Cesarean section was performed and one of the seven pups that were delivered, a female, showed generalized subcutaneous edema and died soon after birth. She weighed 660 g, compared to a mean of 472 g for the other 6 normal fetuses. A total of 295, 40 and 27.5 mL of liquid were collected from subcutaneous tissue, abdominal and thoracic cavity, respectively. Liver showed sub-glissonian necrotic areas. Molecular analyses with PCR method for canine Herpesvirus, Parvovirus, Adenovirus, Leptospira interrogans, Chlamydia spp., Neospora caninum and Toxoplasma gondii from pools of organs (spleen, kidney and brain) and pleural effusion tested negative. This is the first reported case of fetal anasarca with a rapid onset diagnosed on day 60 post-ovulation just three days after observing a normal ultrasonographic pattern in Flat-coated Retriever. Ultrasonographic diagnosis of fetal anasarca is of primary importance when assisting parturition, due to its inherent risk of dystocia. Ultrasonographic monitoring in the immediate prepartum period may be useful in all breeds as it may help to detect ultrasonographic alterations occurring right before term such as anasarca.

Localization and genotyping of canine papillomavirus in canine inverted papillomas.

Numerous canine papillomaviruses (CPVs) have been identified (CPV1-23). CPV1, 2, and 6 have been associated with inverted papillomas (IPs). We retrieved 19 IPs from 3 histopathology archives, and evaluated and scored koilocytes, inclusion bodies, giant keratohyalin granules, cytoplasmic pallor, ballooning degeneration, and parakeratosis. IHC targeting major capsid proteins of PV was performed, and CPV genotyping was achieved by PCR testing. Tissue localization of CPV DNA and RNA was studied by chromogenic and RNAscope in situ hybridization (DNA-CISH, RNA-ISH, respectively). IPs were localized to the limbs (50%), trunk (30%), and head (20%), mainly as single nodules (16 of 19). In 15 of 19 cases, immunopositivity was detected within the nuclei in corneal and subcorneal epidermal layers. PCR revealed CPV1 in 11 IPs and CPV2 DNA in 3 IPs. Overall, 14 of 17 cases were positive by both DNA-CISH and RNA-ISH, in accord with PCR results. A histologic score >5 was always obtained in cases in which the viral etiology was demonstrated by IHC, DNA-CISH, and RNA-ISH. IHC and molecular approaches were useful to ascertain the viral etiology of IPs. Although IHC is the first choice for diagnostic purposes, ISH testing allows identification of PV type and the infection phase. RNA-ISH seems a promising tool to deepen our understanding of the pathogenesis of different PV types in animal species.

Angiostrongylosis in northeastern Italy: First report of two autochthonous fatal cases in dogs and first detection in a wild red fox.

Canine angiostrongylosis is an emergent cardio-pulmonary gastropod-borne helminthic infection caused by the metastrongyloid nematode Angiostrongylus vasorum. Clinically, it is characterized by a wide spectrum of non-specific signs and the red fox serves as the most important reservoir for dog infections. In Italy, this disease has been well documented both in northwestern and central-southern regions, whereas it is apparently poorly recognized in the northeastern area of the country. This report describes the diagnostic findings of two autochthonous cases of fatal canine angiostrongylosis and of one case in a wild red fox detected in northeastern Italy. Reporting cases is relevant to clinicians in order to increase their awareness for the prompt diagnosis of a potentially life-threatening disease that may go unnoticed or misdiagnosed.

The expression in plants of an engineered VP2 protein of Infectious Bursal Disease Virus induces formation of structurally heterogeneous particles that protect from a very virulent viral strain.

Infectious Bursal Disease Virus (IBDV), the etiological agent of Gumboro disease, causes mortality and immunosuppression in chickens and major losses to poultry industry worldwide. The IBDV major capsid protein VP2 is considered the best candidate for the production of novel subunit vaccines. This structural protein contains the major conformational epitopes responsible for the induction of IBDV neutralizing antibodies in chickens and has been demonstrated able to form supramolecular structures in yeast and insect cells. The aim of this study was to express an engineered version of the VP2 protein (His-pVP2) to verify its ability to self-assemble into virus-like particles in plants. The recombinant VP2 was transiently expressed by agroinfiltration in Nicotiana benthamiana and transmission electron microscopy of sucrose density gradient fractions revealed the presence of a mixed population of differently shaped particles ranging from spherical capsids, with a diameter between ~25 and ~70 nm, to tubular structures, with variable length (from 100 to 400 nm). The recombinant VP2-based particles when used for the intramuscular immunization of specific-pathogen-free chicks resulted able to induce the production of anti-IBDV specific antibodies at titers comparable to those induced by a commercial vaccine. Moreover, all the immunized birds survived to the challenge with a Moroccan very virulent IBDV strain with no major histomorphological alterations of the Bursa of Fabricius, similarly to what obtained with the commercial inactivated vaccine.

Rapid detection of asperphenamate in a hay batch associated with constipation and deaths in dairy cattle. The application of DART-HRMS to veterinary forensic toxicology.

Direct analysis in real time (DART) coupled to high-resolution mass spectrometry (HRMS) was applied for the first time to veterinary forensic toxicology to investigate the presence of toxic compounds in hay after an episode of acute intoxication in a dairy cattle farm. In addition to gross field necropsy and histological examination, microbial cultures, and heavy metals analysis, the molecular fingerprinting of the suspected hay batch was investigated by DART-HRMS. DART-HRMS revealed a distinct signal of m/z 507.2289 in the hay batch thought to be associated with the digestive complications. A search on chemical structure databases matched the ion with asperphenamate, a toxin produced by Penicillium spp. and Aspergillus spp. Liquid Chromatography-HMRS analysis and electrospray-HRMS-MS/MS of the hay extracts further characterized the structure and confirmed the identification of the compound as asperphenamate. Asperphenamate is fungal metabolite which can have cytotoxic and antitumor activity in humans, and it is classified as acute toxicant and harmful if swallowed.

Chromogenic in situ hybridization for the detection of lambda and kappa immunoglobulin light chains as a potential auxiliary diagnostic technique in canine plasmacytomas.

The heterogeneous morphologic features of canine plasmacytomas (PCTs) can make their differentiation from other round cell tumors challenging. Immunohistochemistry (IHC) for lambda (λ) and kappa (к) immunoglobulin (Ig) light chains is often equivocal because of high background staining. The chromogenic in situ hybridization (CISH) technique for light chains has shown higher sensitivity compared to IHC in human plasma cell tumors. Therefore, we aimed to validate automated CISH for light chains in canine tissues and to evaluate its diagnostic potential in canine PCTs, in conjunction with routinely used IHC markers. CISH for light chains demonstrated a clear signal in plasma cell populations of canine control tissues (lymph nodes, lymphoplasmacytic inflammation) showing a polyclonal pattern with a prevalence of λ-producing cells. CISH detected monotypic light chain expression in 33 of 53 (62%) PCTs, 31 expressing λ and 2 expressing к. CISH was more sensitive than IHC for λ light chain (58% vs. 47%, respectively) and more easily interpretable given the absence of confounding background staining. The absence of CISH staining for both λ and к in a considerable subset of tumors may be the result of lower light chain production by neoplastic cells. Multiple myeloma oncogene 1 (MUM1) was expressed by all but 2 PCTs (96%), which showed λ expression by CISH and IHC. The identification of poorly differentiated canine PCTs requires the assessment of a panel of IHC markers, with the potential support of CISH for Ig light chains.

CD30 Cross-Reactivity and Expression in Feline Normal Tissues and Lymphomas.

CD30 is a transmembrane glycoprotein of the tumor necrosis factor receptor superfamily included in the diagnostic algorithm of human cutaneous, anaplastic large cell and Hodgkin lymphomas and represents an optimal therapeutic target for CD30+ tumors. Similar diagnostic and therapeutic approaches are largely missing for feline lymphomas. Cross-reactivity of the antihuman CD30 receptor clone Ber-H2 was investigated in feline lymphomas. Comparative analysis of feline and human CD30 identified 61% identity of the amino acid sequence, with 100% identity of the main sequence of the epitope targeted by the antibody (RKQCEPDYYL). CD30 expression in normal feline tissues was restricted to rare lymphoid cells in perifollicular and interfollicular lymph node areas and in the thymic medulla. In feline lymphoma, CD30 was expressed in 4 of 33 (13%) T-cell lymphomas, 3 of 22 (14%) B-cell lymphomas, and 5 of 7 (71%) mixed-cell lymphomas, showing diffuse (1/5) or multifocal (4/5) positivity restricted to neoplastic multinucleated lymphoid cells and binucleated cells consistent with Reed-Sternberg-like cells. Based on the human classification system, cell morphology, expression of multiple markers (mixed cell components), and CD30 positivity, these cases were considered most consistent with classical Hodgkin-like lymphoma (HLL). The other 2 mixed-cell lymphomas were CD30 negative and thus most consistent with either T-cell-rich large B-cell lymphoma (TCRLBCL) or nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL). These findings provide multiple data supporting the cross-reactivity of the Ber-H2 anti-CD30 clone in feline tissues and give evidence of the usefulness of CD30 in the diagnostic evaluation of feline lymphoma.

Quantitative real time polymerase chain reaction (qRT-PCR) and RNAscope in situ hybridization (RNA-ISH) as effective tools to diagnose feline herpesvirus-1-associated dermatitis.

BACKGROUND: Felid herpesvirus type 1 (FHV-1)-associated dermatitis is characterized by facial and nasal involvement; clinical and histopathological manifestations may overlap with other dermatitides. OBJECTIVE: To evaluate the realibility of qRT-PCR-2- ΔΔC q and RNAscope in situ hybridization (RNA-ISH) methods to diagnose FHV-1-associated dermatitis, in formalin-fixed paraffin-embedded (FFPE) tissues. ANIMALS: Sixteen FFPE samples from cats with facial dermatitis and four controls were studied. METHODS AND MATERIALS: Based on histopathological features, cases were separated into: Group 1, samples with herpetic dermatitis (four); Group 2, samples with nonherpetic facial dermatitis (six); Group 3, samples with facial dermatitis of ambiguous nature (allergic or viral) (six); and Group 4, samples from healthy cats (four). A relative quantification using the 2- ΔΔC q method was used to estimate the "upregulation" of each FHV-1 target viral gene copies (glycoprotein-B and thymidine-kinase) relative to reference gene. Detection of FHV-1 mRNA was performed using the RNAscope 2.5 detection kit. RESULTS: By 2- ΔΔC q analysis, upregulation of both FHV-1 genes was observed in all samples from Group 1 and two of six from Group 3. No upregulation was identified in samples from groups 2 and 4. Positive mRNA hybridization signal was observed in all cases from Group 1 and two cases of Group 3. No positivity was observed in samples from groups 2 and 4. CONCLUSIONS AND CLINICAL IMPORTANCE: QRT-PCR 2-ΔΔCq analysis and RNA-ISH can identify the FHV-1 genome as causative agent of the associated dermatitis, even where inclusion bodies are not detectable. Both techniques are functional in retrospective studies, have greater specificity than conventional PCR, and may be proposed for research and diagnostic purposes.

Felis catus Papillomavirus Types 1, 2, 3, 4, and 5 in Feline Bowenoid in Situ Carcinoma: An In Situ Hybridization Study.

Several studies based on histopathology or molecular investigations suggest a causal relation between Felis catus papillomavirus (FcaPV-2) infection and bowenoid in situ carcinoma (BISC) in cats. Nevertheless, data on distribution of viral DNA for different F. catus papillomavirus types (FcaPV-1, 2, 3, 4, 5) in precancerous skin lesions are lacking. In this study, incisional and excisional skin biopsies from 18 cats with BISC were investigated for the presence of FcaPV DNA by quantitative polymerase chain reaction (qPCR) and chromogenic in situ hybridization (CISH) using specific probes to detect each of the FcaPVs that have been identified so far. By qPCR analysis, 15 of 18 samples were positive for FcaPV-2, 2 were positive for FcaPV-4, and 1 sample was negative for all FcaPVs studied. Two cases were positive for FcaPV-5 by qPCR only. FcaPV-1 and FcaPV-3 were not detected by either method. CISH positivity for FcaPV-2 and FcaPV-4 was 100% concordant with qPCR. FcaPV-2 CISH signal was observed as nuclear dots within grouped neoplastic keratinocytes in 12 BISCs and in the perilesional skin of 9 biopsies. In 3 of these 9 cases, the signal was not observed within the BISC. FcaPV-4 CISH positivity was detected only within BISCs in 2 cases. The overall rate of concordance for FcaPV detection between PCR and CISH was 97.8%. This study suggests that CISH is a reliable method to detect FcaPV-2 and FcaPV-4 infection in cats and provides useful information on the type, rate, and localization of infected cells.

Detection and Prognostic Relevance of Circulating and Disseminated Tumour Cell in Dogs with Metastatic Mammary Carcinoma: A Pilot Study.

In human breast cancer, both circulating tumour cells (CTCs) in peripheral blood and disseminated tumour cells (DTCs) in the bone marrow are predictive of short survival and may be used as liquid biopsy to guide therapy. Herein we investigate, for the first time, the feasibility to quantify CTCs and DTCs in canine metastatic mammary carcinoma (MMC) with the automated CellSearch platform, which identifies tumour cells by immune-magnetic enrichment and fluorescent labelling. Using this approach before start of treatment, we could detect at least 1 CTC per 7.5 mL of peripheral blood in 12 out of 27 evaluable samples (44.4%) and at least 1 DTC per 1 mL of bone marrow in 11 out of 14 evaluable samples (78.6%). Conversely, we did not find any CTCs in the healthy, negative control dogs (n = 5) that we analysed in parallel. Interestingly, the levels of CTCs/DTCs and the prevalence of positive dogs closely resemble results obtained by CellSearch assay in metastatic breast cancer patients at diagnosis. Moreover, in the canine cohort, the presence of CTCs was significantly associated with poor outcome. These observations identify the first actionable marker in veterinarian oncology to guide treatment of canine MMC. Furthermore, our findings have important implications for human research, since it reinforce the value of canine MMC as model useful to speed up pharmacological studies with primary endpoint of overall survival, given the reduced life-span of the canine species.

Isolation of Streptococcus agalactiae in a female llama (Lama glama) in South Tyrol (Italy).

BACKGROUND: Streptococcus agalactiae is pathogenic for both animals and humans. In dairy cattle it commonly causes mastitis, with great economic losses, and there is scientific evidence of mastitis, caseous lymphadenitis, contagious skin necrosis and purulent infections associated with S. agalactiae in camels (Camelus dromedarius) as well. In humans, it is a common component of the respiratory and gastrointestinal microflora, but it can also act as a pathogen, especially in elderly people and immunocompromised patients, as well as in pregrant women and newborns. CASE PRESENTATION: A 10-year old non-pregnant female llama (Lama glama) was conferred to the Institute for Animal Health Control, in Bolzano for necropsy after sudden death. The animal had not shown unusual behaviour and had a low to normal nutritional condition (body condition score 2/5). The breeder had reported a chronic suppurative subcutaneous infection in the intermandibular area, resistant to therapy (therapy unknown). After necropsy, several samples were processed for histological, bacteriological and parasitological examinations. CONCLUSIONS: This report describes, to the best of our knowledge, the first isolation of S. agalactiae in llamas (Lama glama). The animal came from a herd that counts approximately 200 South American camelids (llamas, alpacas) along with several horses, chicken, rabbits, cats and dogs; this farm offers services, such as trekking and pet therapy activities.

Immunohistochemistry by anti-cleaved-Lamin A: an improved approach to tackle the misuse of glucocorticoids in cattle.

The illegal use of glucocorticoids (GCs) as growth-promoters (GPs) is prohibited in farm animals in the European Union because the strong pharmacological activity of most synthetic GCs produces residues that are dangerous for human consumption. Among the alternative methods proposed to increase the efficacy of official controls, histology was the technique of choice in Italy on account of its high performance level. The aim of this study was to evaluate the reliability of immunohistochemistry (IHC) using anti-cleaved-Lamin A antibody to enhance the performance of the histological test applied to GC-related microscopic changes in the thymus. Veal calves (VC) and beef cattle (BC) were raised and both underwent different growth-promoting protocols or were left untreated. The morphology of the thymus parenchyma was evaluated for cortical atrophy with concurrent adipose tissue infiltration, and a score of 1 to 3 was attributed. A semi-quantitative IHC analysis was also performed by counting the number of positive thymocytes in 5 randomly selected high-power fields (HPFs). The distribution of the thymus atrophy scores was significantly different among the subgroups in both BC and VC. The IHC values were higher in untreated than in treated animals, for both BC and VC. The association between thymus atrophy score and IHC positivity showed higher median values in control than in treated animals (independently of the treatment protocol), for both BC and VC. Our data shows that IHC against anti-cleaved-Lamin A antibody is a reliable marker to detect illegal GC treatments, administered either alone or in association with other growth promoters, in both BC and VC. Combining IHC with the thymus atrophy score improves the accuracy of the histological method in correctly identifying treated animals and could represent a valuable, reproducible method to be applied to large-scale screening programmes.

A G1-lineage H9N2 virus with oviduct tropism causes chronic pathological changes in the infundibulum and a long-lasting drop in egg production.

Since 1997, G1-lineage H9N2 avian influenza viruses have been circulating in Asia and later on in the Middle East, and they have been associated to mild respiratory disease, drops in egg production and moderate mortality in chickens, in particular in the presence of concurrent infections. In this study, we investigated the importance of the G1-lineage H9N2 A/chicken/Israel/1163/2011 virus as a primary pathogen in layers, analyzing its tropism and binding affinity for the oviduct tissues, and investigating the long-term impact on egg production. Besides causing a mild respiratory infection, the virus replicated in the oviduct of 60% of the hens causing different degrees of salpingitis throughout the organ, in particular at the level of the infundibulum, where the detection of the virus was associated with severe heterophilic infiltrate, and necrosis of the epithelium. Binding affinity assays confirmed that the infundibulum was the most receptive region of the oviduct. The drop in egg production was at its peek at 2 weeks post-infection (pi) (60% decrease) and continued up to 80 days pi (35% decrease). On day 80 pi, non-laying birds showed egg yolk peritonitis, and histopathological analyses described profound alteration of the infundibulum architecture, duct ectasia and thinning of the epithelium, while the rest of the oviduct and ovary appeared normal. Our results show that this H9N2 virus is a primary pathogen in layer hens, and that its replication in the infundibulum is responsible for acute and chronic lesions that limits the effective functionality of the oviduct, compromising the commercial life of birds.