A Novel CARMIL2 Immunodeficiency Identified in a Subset of Cavalier King Charles Spaniels with Pneumocystis and Bordetella Pneumonia.

Pet dogs are a valuable natural animal model for studying relationships between primary immunodeficiencies and susceptibility to Pneumocystis and other opportunistic respiratory pathogens. Certain breeds, such as the Cavalier King Charles Spaniel, are over-represented for Pneumocystis pneumonia (PCP), suggesting the presence of a primary immunodeficiency in the breed. Here, we report the discovery of a CARMIL2 nonsense variant in three Cavalier King Charles Spaniel dogs with either PCP (n = 2) or refractory Bordetella pneumonia (n = 1). CARMIL2 encodes a protein that plays critical roles in T-cell activation and other aspects of immune function. Deleterious CARMIL2 variants have recently been reported in human patients with PCP and other recurrent pneumonias. In addition to opportunistic respiratory infection, the affected dogs also exhibited other clinical manifestations of CARMIL2 deficiencies that have been reported in humans, including early-onset gastrointestinal disease, allergic skin disease, mucocutaneous lesions, abscesses, autoimmune disorders, and gastrointestinal parasitism. This discovery highlights the potential utility of a natural canine model in identifying and studying primary immunodeficiencies in patients affected by PCP.

Molecular phylogenetic analysis of Echinococcus granulosus sensu lato infecting sheep in Italy.

Cystic Echinococcosis (CE), caused by the larval form of Echinococcus granulosus sensu lato, is a neglected zoonosis still threatening public health worldwide. In Italy different epidemiological scenarios were reported depending on the geographical area and associated socio-economic activities. Although in northern Italy the occurrence of E. granulosus is considered sporadic, in the southern regions and, particularly in Sardinia, CE prevalence reaches high levels. We analysed CE cysts collected from infected sheep from various areas of mainland Italy and the Sardinia island, with the main objective to investigate intergenotypic and intragenotypic variations at national level. CE cysts were collected from slaughtered sheep following post mortem inspection at local abattoirs. Total genomic DNA was extracted and amplification and sequencing of the partial mitochondrial genes nad5 and cox1 were performed. A Bayesian phylogenetic tree was estimated on a nad5 dataset (n = 260) composed of E. granulosus samples from this study (n = 126) and all the nad5 haplotypes available in GenBank (n = 134). In addition, haplotype network, diversity and neutrality analysis were performed on nad5 and cox1 sequences of Italian origin obtained in this study. E. granulosus sensu stricto (s.s.) was found to be the only Echinococcus species infecting sheep in Italy, mainly represented by G1 genotype (76 %) and, to a lower extent, by G3 genotype (24 %). Phylogenetic analyses revealed 40 nad5 and 33 cox 1 haplotypes, and the presence of two founder haplotypes, belonging to G1 and G3 genotype, showing 100 % similarity with DNA sequences from different geographic regions. The lack of geographical segregation, high haplotype and low nucleotide diversity coupled with significant negative values of Tajima's D and Fu's Fs observed in this study indicated high genetic variation and demographic expansion of E. granulosus s.s. in Italy.

Corrigendum: Comparing copromicroscopy to intestinal scraping to monitor red fox intestinal helminths with zoonotic and veterinary importance.

[This corrects the article DOI: 10.3389/fvets.2022.1085996.].

Oculosystemic pneumocystosis in 2 sibling Chihuahuas.

Sibling female and male Chihuahuas were evaluated for a 9-month history of tachypnea that failed to respond to fenbendazole, doxycycline, amoxicillin-clavulanate, and prednisone. Physical examination identified tachypnea, hyperpnea, and harsh bronchovesicular lung sounds. Fundic examination disclosed diffuse chorioretinitis, manifested as multifocal chorioretinal granulomas in the female dog and occasional chorioretinal scars in the male dog. Thoracic radiographs indicated moderate to severe interstitial to broncho-interstitial infiltrates in both dogs. Serum and urine antigen and antibody testing in the female dog failed to identify infectious agents, but cytologic assessment of hepatic lymph node, liver, and splenic aspirates identified Pneumocystis trophozoites. Infection was confirmed in both dogs by 28S rRNA PCR sequencing from multiple tissue samples. The female dog responded well to trimethoprim-sulfamethoxazole, but the male dog was euthanized because of liver failure, presumably related to antimicrobial treatment.

Giardia duodenalis Colonization Slightly Affects Gut Microbiota and Hematological Parameters in Clinically Healthy Dogs.

Giardia duodenalis (Giardia) is a worldwide cause of acute diarrheal disease both in humans and animals. The primary aim of this study was to investigate possible variations in gut microbiota in a population of asymptomatic dogs (n = 31), naturally infected or not by Giardia. Gut microbiota and the hematological, biochemical, and fecal parameters related to intestinal function were investigated. Giardia infection was associated with a significant shift of beta diversity, showing a relevant reduction of Gammaproteobacteria and an increase of Fusobacteria in male-positive dogs if compared with negatives. A significant imbalance of different bacterial taxa, with particular reference to the Erysipelotrichales, Lactobacillales, Clostridiales, and Burkholderiales orders, was observed, with the first two being higher in Giardia-positive dogs. Giardia-positive males displayed significantly higher values of cCRP than negative males as well as positive females, supporting the presence of a pro-inflammatory state. Taken together, these results indicate that the presence of Giardia does not substantially modify the microbial ecology of the intestine nor the hematological markers of disease. Thus treatments against Giardia should be considered with caution in asymptomatic subjects.

Environmental and pathological factors affecting the hatching success of the two northernmost loggerhead sea turtle (Caretta caretta) nests.

In recent years, the report of loggerhead sea turtle (Caretta caretta) Mediterranean nesting range has expanded together with new records of nests becoming northward on the Italian coastline of the Tyrrhenian and Adriatic seas. These areas are characterized by intensive human activities, such as tourism, fishery, and marine traffic, all possibly involved in the influence of the use of coastal habitat by marine species. These anthropic threats, in addition to the natural ones and the changing environmental characteristics of the beach, may influence the growth of microorganisms causing hatching failures. Among microorganisms, fungal infection by the genus Fusarium (Link, 1809) is considered one of the main causes of globally declining sea turtle populations. In summer 2021, the two northernmost worldwide loggerhead sea turtle nests were monitored along the Northern Adriatic coastline (Veneto, Italy). These first records may potentially candidate this area as suitable for a large part of the loggerhead turtle's life cycle and it could represent a minor sea turtle nesting area that, according to Prato and colleagues, remained unnoticed due to the lack of specific monitoring. Sea Turtle Egg Fusariosis (STEF) was deemed to have deeply compromised the hatching success of the northmost one. Climate change and anthropogenic impacts have been scored as one of the highest hazards to sea turtle health and could have played a role in the STEF development. Environmental changes, human activities, and emerging pathogens deserve the highest attention in terms of health research, and conservation management.

Eumycetoma Caused by Madurella pseudomycetomatis in a Captive Tiger (Panthera tigris).

A captive-kept adult male tiger presented with a large cutaneous and subcutaneous mass on the thigh with a fistula. During sedation, multiple nodules were detected and samples for a histopathological exam were collected. Histologically, granulomatous panniculitis and dermatitis were seen around dense aggregates of pigmented fungal hyphae, and a diagnosis of phaeohyphomycosis was made; considering the clinical features, it was classified as a eumycotic mycetoma. This is a rarely reported subcutaneous fungal infection in humans and animals, caused by dematiaceous fungi. Clinically, it is characterized by tumefaction, fistulous sinus tracts, and the formation of macroscopically visible grains. In the literature, only a few infections in wild felids have been reported. In this case, Fontana-Masson staining better showed pigmentation and panfungal PCR and sequencing identified Madurella pseudomyectomatis (OP623507) as the causative agent. Systemic therapy with oral administration of itraconazole was planned, but the patient died during the first period of treatment. The animal was not submitted for post-mortem examination. Visceral dissemination of the agent cannot be excluded. To the authors' knowledge, this is the first report of eumycotic mycetoma by Madurella pseudomycetomatis in a captive tiger.

Malassezia overgrowth in dogs in northern Italy: frequency, body distribution, clinical signs and effects of pharmacologic treatments.

The present study describes Malassezia populations in clinically healthy dogs (HD) and dogs with Malassezia overgrowth (MO), and evaluates the correlation with clinical signs and previous treatments. Thirteen clinically HD and 84 dogs with MO were enrolled. Clinical history and previous treatments were recorded. After a complete physical and dermatological examination, Canine Atopic Dermatitis Extent and Severity Index_03 scores were calculated. Samples for cytology and mycological cultures were obtained from four body regions and from skin lesions. Malassezia overgrowth was diagnosed by cytology. A global score (GS) for quantitative evaluation of the population of Malassezia was calculated. In dogs with MO, the highest frequency of yeast detection was found in skin lesions (82%, P < 0.001). Sum of GS (GSs) obtained from dogs with MO (68, 0­621) was significantly higher compared to those of HD (3, 0­48, P < 0.001). GSs in dogs previously treated with antibiotics (312.5, 30­975) was significantly higher compared to those of dogs that not have received antibiotics (80, 0­975, P = 0.015). No difference was found between dogs treated and those not treated with steroids.

Early start of seasonal transmission and co-circulation of West Nile virus lineage 2 and a newly introduced lineage 1 strain, northern Italy, June 2022.

In spring 2022, Europe faced an unprecedented heatwave, increasing the risk of West Nile virus (WNV) outbreaks. As early as 7 June 2022, WNV was detected in Culex mosquitoes in northern Italy, and - in the following days - in two blood donors, a patient with encephalitis, wild birds and additional mosquito pools. Genome sequencing demonstrated co-circulation of WNV lineage 2 and a newly introduced WNV lineage 1, which was discovered in the region in 2021.

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.

A highly endemic area of Echinococcus multilocularis identified through a comparative re-assessment of prevalence in the red fox (Vulpes vulpes), Alto Adige (Italy: 2019-2020).

Surveillance of Echinococcus multilocularis at the edge of its range is hindered by fragmented distributional patterns and low prevalence in definitive hosts. Thus, tests with adequate levels of sensitivity are especially important for discriminating between infected and non-infected areas. In this study we reassessed the prevalence of E. multilocularis at the southern border of its distribution in Province of Bolzano (Alto Adige, northeastern Alps, Italy), to improve surveillance in wildlife and provide more accurate estimates of exposure risk. We compared the diagnostic test currently implemented for surveillance based on coproscopy and multiplex PCR (CMPCR) to a real-time quantitative PCR (qPCR) in 235 fox faeces collected in 2019 and 2020. The performances of the two tests were estimated using a scraping technique (SFCT) applied to the small intestines of a subsample (n = 123) of the same foxes as the reference standard. True prevalence was calculated and the sample size required by each faecal test for the detection of the parasite was then estimated. True prevalence of E. multilocularis in foxes (14.3%) was markedly higher than reported in the last decade, which was never more than 5% from 2012 to 2018 in the same area. In addition, qPCR showed a much higher sensitivity (83%) compared to CMPCR (21%) and agreement with the reference standard was far higher for qPCR (0.816) than CMPCR (0.298) meaning that for the latter protocol, a smaller sample size would be required to detect the disease. Alto Adige should be considered a highly endemic area. Routine surveillance on definitive hosts at the edges of the E. multilocularis distribution should be applied to smaller geographic areas, and rapid, sensitive diagnostic tools using directly host faeces, such as qPCR, should be adopted.

Detection of a Novel Chlamydia Species in Invasive Turtles.

Trachemys scripta is a turtle species native to Central America. Since the 1950s, pond sliders have been imported worldwide as companion animals, but have often ended up in foreign ecosystems with great ecological consequences. Moreover, both autochthonous and invasive species of turtles can be carriers of pathogens, including Chlamydiaceae. In the present study, pulmonary tissues collected from four Trachemys scripta were tested with a 23S-targeting real-time PCR (rPCR) specific for the Chlamydiaceae family. The turtles were hosted in a rescue center for wild exotic animals located in northeastern Italy, and were found dead after the hibernation period. Two out of four individuals resulted positive in rPCR for the presence of Chlamydiaceae. Further characterization of this positivity was performed by phylogenetic analysis of the 16S rRNA and outer membrane protein A genes. The phylogenetic tree showed that these chlamydial strains are identical to a novel Chlamydia reported in 2017 in Polish freshwater turtles, and closely related to Chlamydia pneumoniae and to other chlamydial strains found in reptiles. This first finding evidences the presence of this Chlamydia strain in Italian turtles, but further studies will be necessary to confirm the presence and the strain pathogenicity and to evaluate its prevalence in the local turtles' population.

Pneumocystis Colonization in Dogs Is as in Humans.

Pneumocystis is an atypical fungus that resides in the pulmonary parenchyma of many mammals, including humans and dogs. Immunocompetent human hosts are usually asymptomatically colonised or show subtle clinical signs, but some immunocompromised people can develop florid life-threatening Pneumocystis pneumonia (PCP). Since much less is known concerning Pneumocystis in dogs, we posit the question: can Pneumocystis colonization be present in dogs with inflammatory airway or lung disease caused by other pathogens or disease processes? In this study, Pneumocystis DNA was detected in bronchoalveolar lavage fluid (BALF) of 22/255 dogs (9%) with respiratory distress and/or chronic cough. Although young dogs (<1 year-of-age) and pedigree breeds were more often Pneumocystis-qPCR positive than older dogs and crossbreds, adult dogs with other infectious conditions and/or a history of therapy-resistant pulmonary disease could also be qPCR-positive, including two patients with suppression of the immune system. Absence of pathognomonic clinical or radiographic signs render it impossible to convincingly discriminate between overt PCP versus other lung/airway disease processes colonised by P. canis. It is possible that colonisation with P. canis might play a certain role as a co-pathogen in some canine patients with lower respiratory disease.

Canine eumycetoma caused by Madurella pseudomycetomatis.

Canine eumycetoma is a rare granulomatous disease caused by dematiaceous fungi. A 2-year-old Great Dane dog had a subcutaneous mass in the right thigh that was surgically removed. Grossly, numerous black-grains were visible. Histologically subcutaneous pyogranulomas were centered on myriads of pigmented fungal elements. Madurella pseudomycetomatis was molecularly characterized.

Assessing Red Fox (Vulpes vulpes) Demographics to Monitor Wildlife Diseases: A Spotlight on Echinococcus multilocularis.

The assessment of red fox population density is considered relevant to the surveillance of zoonotic agents vectored by this species. However, density is difficult to estimate reliably, since the ecological plasticity and elusive behavior of this carnivore hinder classic methods of inference. In this study, red fox population density was estimated using a non-invasive molecular spatial capture-recapture (SCR) approach in two study areas: one in a known hotspot of the zoonotic cestode Echinococcus multilocularis, and another naïve to the parasite. Parasitological investigations on collected samples confirmed the presence of the parasite exclusively in the former area; the SCR results indicated a higher fox population density in the control area than in the hotspot, suggesting either that the relationship between fox density and parasite prevalence is not linear and/or the existence of other latent factors supporting the parasitic cycle in the known focus. In addition, fox spotlight count data for the two study areas were used to estimate the index of kilometric abundance (IKA). Although this method is cheaper and less time-consuming than SCR, IKA values were the highest in the areas with the lower molecular SCR density estimates, confirming that IKA should be regarded as a relative index only.

Comparing copromicroscopy to intestinal scraping to monitor red fox intestinal helminths with zoonotic and veterinary importance.

The red fox acts as reservoir for several helminthic infections which are of interest for both public and animal health. Huge efforts have been made for the assessment of the sensitivity of coprological tests for the detection of Echinococcus multilocularis, while less attention has been paid to other helminthic species. This study aimed at assessing the performance of two copromicroscopic techniques in the detection and prevalence estimation of gastrointestinal helminths in the red fox. Helminths were isolated from the small intestines of 150 red foxes from Bolzano province, Italy, with a scraping, filtration and counting technique (SFCT) and morphologically identified. Rectal contents were collected and submitted to simple flotation (FT) and, only for Taenids, a method based on the concentration of eggs and identification with multiplex PCR (CMPCR). Using SFCT as a reference standard, we estimated the sensitivity of the copromicroscopic tests. Three species of nematodes (namely, Toxocara canis, Uncinaria stenocephala and Pterygodermatites sp.) and five species of cestodes (E. multilocularis, Taenia crassiceps, T. polycantha, Hydatigera taeniaeformis, Mesocestoides sp.) were identified with SFCT, whereas eggs referable to the same taxa were detected with fecal diagnostics, except for Pterygodermatites sp. and Mesocestoides sp. The sensitivity of FT was low for all taxa, ranging from 9.8 to 36.3%, with lower values for Taeniidae. CMPCR was confirmed to perform better for the detection of Taeniidae eggs (23.5%) and the multiplex PCR on retrieved eggs was effective in the identification of the species. A meta-analysis of literature also suggested that our results are consistent with existing data, indicating that copromicroscopy tends to underestimate the prevalence of helminthic infections. The extent of such underestimation varies with taxon, being higher at high prevalence levels, in particular for cestodes. Irregular dynamics of egg shedding, and routine deep freezing of red fox feces may explain the frequency of false negatives with copromicroscopy. Low sensitivity of copromicroscopic tests should be accounted for when estimating prevalence and when defining the correct sample size for the detection of the parasites.

Detection of Toxocara cati Larvae from Ostrich and Wild Boar Meat Intended for Human Consumption.

Toxocara cati is a common roundworm of cats and wild felids and, together with T. canis, it is the main causative agent of human toxocariasis. Humans may become infected by ingestion of embryonated eggs via contaminated soil, food, or water, or by ingestion of raw or undercooked meat of paratenic hosts that are infected by Toxocara larvae. In this study, we report the detection of T. cati larvae from meat samples of ostriches and wild boars. These samples were inspected by enzymatic digestion, as part of the trichinellosis surveillance. As ostrich meat is intended for "carpaccio" preparation, a traditional Italian raw meat preparation, there is the need to make the consumption of this meat safe. For this purpose, it is recommended to freeze the meat before preparation. Our findings confirmed that T. cati larvae can contaminate muscle tissues of paratenic hosts, increasing the risk of infection due to the consumption of raw or undercooked meat.

A Three-Year Biocrime Sanitary Surveillance on Illegally Imported Companion Animals.

The illegal trade of companion animals in the European Union poses several legal, ethical and health issues to the entire community. In the framework of the Biocrime Interreg project between Italy and Austria, we surveyed puppies and kittens confiscated at the borders to identify the most frequent pathogens associated with (i) the risk of spread within the shelter, (ii) the development of fatal disease and (iii) the zoonotic potential. From January 2018 to December 2020, we examined a total of 613 puppies and 62 kittens coming from 44 requisitions. Feces, skin specimens and blood sera from confiscated animals were tested to verify the presence of major infections and to assess the rabies post-vaccination immunity. Out of the total of individuals under investigation, necropsies and laboratory investigations were also performed on 79 puppies and three kittens that had died during the observation period. Results indicated a high prevalence of Canine Parvovirus (CPV) and Giardia spp. infections, CPV as the most likely cause of fatal gastroenteritis in puppies and Salmonella and Microsporum canis as major zoonotic pathogens. Conversely, both extended spectrum beta lactamases Escherichia coli and methicillin resistant Staphylococcus pseudintermedius strains as rare findings. Results highlighted that illegal animal trade could expose the human population to potential zoonotic risk and naïve animal population to potentially disrupting epidemic waves, both of these issues being largely underestimated when buying companion animals.

A novel Filobacterium sp can cause chronic bronchitis in cats.

BACKGROUND: Cilia-associated respiratory bacillus (CARB; now known as Filobacterium rodentium gen. nov., sp. nov.) is a primary pathogen of rodents. A CARB-like organism was reported in post-mortem lung samples of cats using light and electron microscopy. Here we explore by molecular procedures if a Filobacterium sp. is a part of the normal feline lower respiratory microbiome and whether it could in some cats contribute to the development of chronic bronchial disease. METHODOLOGY: A Filobacterium sp. was identified in three Czech cats clinically diagnosed as having chronic neutrophilic bronchitis. Bronchoalveolar lavage fluid (BALF) specimens obtained from these cats were subjected to panbacterial 16S rDNA PCR followed by Sanger sequencing of the V5 to V8 region. After these cats were treated with specific antimicrobials, their clinical signs resolved promptly, without recurrence. Next, BALF specimens from 13 Australian and 11 Italian cats with lower respiratory disease and an additional 16 lung samples of Italian cats who died of various causes were examined using next generation sequencing (NGS). Subsequently, a Filobacterium-specific qPCR assay was developed and used to re-test BALF specimens from the 11 Italian cats and lung tissue homogenates from the additional 16 deceased cats. PRINCIPAL FINDINGS: An amplicon of 548 bp with 91.24% sequence agreement with Filobacterium rodentium was obtained from all three patients, suggesting the novel Filobacterium sp. was the cause of their lower respiratory disease. The novel Filobacterium sp., which we propose to call F. felis, was detected in 3/3 Czech cats with chronic neutrophilic bronchitis, 13/13 Australian cats and 6/11 Italian cats with chronic lower respiratory disease, and 14/16 necropsy lung specimens from Italian cats. NGS and qPCR results all showed identical sequences. The Filobacterium sp. was sometimes the preponderant bacterial species in BALF specimens from cats with lower airway disease. There was an association between the presence of large numbers (greater than 105 organisms/mL) of Filobacterium and the presence of neutrophilic and/or histiocytic inflammation, although only a subset of inflammatory BALF specimens had F. felis as the preponderant organism. CONCLUSION: The novel Filobacterium sp. comprises a finite part of the normal feline lower respiratory microbiome. Under certain circumstances it can increase in absolute and relative abundance and give rise to neutrophilic and/or histiocytic bronchitis, bronchiolitis and bronchopneumonia. These findings strongly suggest that F. felis could be an underdiagnosed cause of feline bronchial disease.

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.