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.

Protothecosis in Dogs and Cats-New Research Directions.

Protothecosis refers to disease of humans and animals caused by infection with fungus-like, colourless microalgae of the genus Prototheca. Although protothecosis remains an uncommon infection, increasing numbers of human and animal cases are being diagnosed worldwide. This review summarises major new findings in basic science (sequencing analyses of sterol 14α-demethylase (CYP51/ERG11) genes and organelle genomes of Prototheca wickerhamii) to elucidate taxonomic features of this pathogen. Furthermore, this review updates and summarises the clinical features, diagnosis and treatment of protothecosis in dogs and cats. This content of this review is based on information presented at the medical phycology symposium held in the 20th Congress of the International Society for Human and Animal Mycology ( https://www.isham.org/ ).

Yeasts isolated from cloacal swabs, feces, and eggs of laying hens.

Domestic and wild birds may act as carriers of human pathogenic fungi, although the role of laying hens in spreading yeasts has never been investigated. We evaluated the presence of yeasts in the cloaca (Group I, n = 364), feces (Group II, n = 96), and eggs (Group III, n = 270) of laying hens. The occurrence and the population size of yeasts on the eggshell, as well as in the yolks and albumens, were assessed at the oviposition time and during storage of eggs at 22 ± 1°C and 4 ± 1°C. A statistically higher prevalence and population size of yeasts were recorded in Group I (49.7% and 1.3 × 104 cfu/ml) and II (63.8% and 2.8 × 105 cfu/ml) than in Group III (20.7% and 19.9 cfu/ml). Candida catenulata and Candida albicans were the most frequent species isolated. Candida famata and Trichosporon asteroides were isolated only from the eggshells, whereas Candida catenulata was also isolated from yolks and albumens. During storage, the yeast population size on the shell decreased (from 37.5 to 8.5 cfu/ml) in eggs at 22 ± 1°C and increased (from 4.6 to 35.3 cfu/ml) at 4 ± 1°C. The laying hens harbor potentially pathogenic yeasts in their gastrointestinal tract and are prone to disseminating them in the environment through the feces and eggs. Eggshell contamination might occur during the passage through the cloaca or following deposition whereas yolk and albumen contamination might depend on yeast density on eggshell.

Molecular detection of Pneumocystis in the lungs of cats.

The genus Pneumocystis comprises potential pathogens that reside normally in the lungs of a wide range of mammals. Although they generally behave as transient or permanent commensals, they can occasionally cause life-threatening pneumonia (Pneumocystis pneumonia; PCP) in immunosuppressed individuals. Several decades ago, the presence of Pneumocystis morphotypes (trophic forms and cysts) was described in the lungs of normal cats and cats with experimentally induced symptomatic PCP (after immunosuppression by corticosteroids); yet to date spontaneous or drug-induced PCP has not been described in the clinical feline literature, despite immunosuppression of cats by long-standing retrovirus infections or after kidney transplantation. In this study, we describe the presence of Pneumocystis DNA in the lungs of normal cats (that died of various unrelated causes; n = 84) using polymerase chain reactions (PCRs) targeting the mitochondrial small and large subunit ribosomal RNA gene (mtSSU rRNA and mtLSU rRNA). The presence of Pneumocystis DNA was confirmed by sequencing in 24/84 (29%) cats, with evidence of two different sequence types (or lineages). Phylogenetically, lineage1 (L1; 19 cats) and lineage 2 (L2; 5 cats) formed separate clades, clustering with Pneumocystis from domestic pigs (L1) and carnivores (L2), respectively. Results of the present study support the notion that cats can be colonized or subclinically infected by Pneumocystis, without histological evidence of damage to the pulmonary parenchyma referable to pneumocystosis. Pneumocystis seems most likely an innocuous pathogen of cats' lungs, but its possible role in the exacerbation of chronic pulmonary disorders or viral/bacterial coinfections should be considered further in a clinical setting.

Comparative pathogenicity of opportunistic black yeasts in Aureobasidium.

Aureobasidium pullulans and A. melanogenum are black-yeast-like surface colonisers and are commonly encountered as contaminants in the hospital. The species are able to produce melanin which play a role in protection against environmental stress and irradiation. Aureobasidium melanogenum shows higher frequency in opportunistic infections compared to A. pullulans. Comparative pathogenicity of opportunistic black yeasts between Aureobasidium pullulans and A. melanogenum to explain the observed differences in frequency in infection. Degrees of melanisation and thermotolerance were measured, and virulence of strains from different sources was examined in Galleria mellonela and murine infection models. Aureobasidium melanogenum responds with increased melanisation to temperature stress and generally survives at 37°C, A. pullulans on average scored less on these parameters. In the murine model, differences between species were not significant, but the melanised A. melanogenum group showed the highest virulence. This result was not reproducible in Galleria mellonella larvae at 25°C. The A. melanogenum black group showed higher pathogenicity in murine model, indicating that the combination of melanisation and thermotolerance rather than species affiliation is instrumental. Galleria larvae did not survive very well at 37°C, and hence, this model is judged insufficient to detect the small virulence differences observed in Aureobasidium.

Molecular diagnosis of Pneumocystis pneumonia in dogs.

Pneumocystis pneumonia (PCP) is a life-threatening fungal disease that can occur in dogs. The aim of this study was to provide a preliminary genetic characterisation of Pneumocystis carinii f.sp.'canis' (P. canis) in dogs and thereby develop a reliable molecular protocol to definitively diagnose canine PCP. We investigated P. canis in a variety of lung specimens from dogs with confirmed or strongly suspected PCP (Group 1, n = 16), dogs with non-PCP lower respiratory tract problems (Group 2, n = 65) and dogs not suspected of having PCP or other lower respiratory diseases (Group 3, n = 11). Presence of Pneumocystis DNA was determined by nested PCR of the large and small mitochondrial subunit rRNA loci and by a real-time quantitative polymerase chain reaction (qPCR) assay developed using a new set of primers. Molecular results were correlated with the presence of Pneumocystis morphotypes detected in cytological/histological preparations. Pneumocystis DNA was amplified from 13/16 PCP-suspected dogs (Group 1) and from 4/76 dogs of control Groups 2 and 3 (combined). The latter four dogs were thought to have been colonized by P. canis. Comparison of CT values in 'infected' versus 'colonized' dogs was consistent with this notion, with a distinct difference in molecular burden between groups (CT ≤ 26 versus CT range (26

Cryptococcus neoformans in the respiratory tract of squirrels, Callosciurus finlaysonii (Rodentia, Sciuridae).

Cryptococcosis is a fungal disease acquired from the environment, for which animals may serve as sentinels for human exposure. The occurrence of Cryptococcus spp. in the respiratory tract of 125 squirrels, Callosciurus finlaysonii, trapped in Southern Italy, was assessed. Upon examination of nasal swabs and lung tissue from each individual, a total of 13 (10.4%) animals scored positive for yeasts, 7 for Cryptococcus neoformans (C.n.) (5.6%) and 6 for other yeasts (4.8%). C.n. was isolated from the nostrils and lungs, with a high population size in nostrils. Two C.n. molecular types, VNI and VNIV, were identified, with C.n. var. grubii VNI the most prevalent. Phylogenetic analyses of ITS+ and URA5 sequences revealed that C.n. isolates were genetically similar to isolates from a range of geographical areas and hosts. Results suggest that C.n. can colonize or infect the respiratory tract of C. finlaysonii. The high occurrence and level of colonization of nasal cavities might be an indicator of environmental exposure to high levels of airborne microorganism. The close phylogenetic relationship of C.n. strains from squirrels with those from human and other animal hosts suggests a potential role for these animals as "sentinels" for human exposure.

Multilocus sequence typing (MLST) and M13 PCR fingerprinting revealed heterogeneity amongst Cryptococcus species obtained from Italian veterinary isolates.

Cryptococcosis represents a fungal disease acquired from the environment with animals serving as host sentinels for human exposure. The aim of this study was to investigate the genetic characteristics of Cryptococcus isolates from veterinary sources (cats, dogs and birds) to understand their epidemiology and the genetic variability of the casual isolates. Mating-type PCR in connection with MLST analysis using the ISHAM consensus MLST scheme for the C. neoformans/C. gattii species complex was used to genotype 17 C. neoformans isolates. In the absence of an MLST typing scheme Cryptococcus adeliensis, C. albidus, C. aureus, C. carnescens, C. laurentii, C. magnus and C. uniguttulatus strains were typed using M13 PCR fingerprinting. All C. neoformans isolates were MATα mating type, but hybrids possessed αADa and aADα mating and serotypes. Two C. neoformans molecular types VNI, VNIV and VNIII and VNII/VNIV hybrids were identified. Amongst the 66 non-C. neoformans strains investigated 55 M13 PCR fingerprinting types were identified. The wide variety of MLST types of C. neoformans and the occurrence of αADa and aADα hybrids in our study supports the notion of genetic recombination in the area studied. The heterogeneity of the non-C. neoformans isolates remains open to further investigations and should be taken into consideration when identifying emergent pathogens.

Molecular identity and prevalence of Cryptococcus spp. nasal carriage in asymptomatic feral cats in Italy.

Cryptococcosis is a life-threatening fungal disease that infects humans and animals worldwide. Inhalation of fungal particles from an environmental source can cause primary infection of the respiratory system. As animals can be considered a sentinel for human diseases, the aim of this study was to determine the prevalence and molecular identity of Cryptococcus spp. in the nasal cavity of feral cats. Cats from 162 urban and rural feral cat colonies were sampled over 3 years. Of 766 cats from which nasal swabs were obtained, Cryptococcus spp. were recovered from 95 (12.6%), including 37 C. magnus (4.8%), 16 C. albidus (2.0%), 15 C. carnescens (1.9%), 12 C. neoformans (1.6%), as well as C. oeirensis (n = 3), C. victoriae (n = 3), C. albidosimilis (n = 2), Filobasidium globisporum (n = 2), C. adeliensis (n = 1), C. flavescens (n = 1), C. dimnae (n = 1), C. saitoi (n = 1), and C. wieringae (n = 1) with prevalence <1%. Thirteen Cryptococcus species were identified by polymerase chain reaction and sequencing of internal transcribed spacer amplicons. Statistical analysis did not identify any predisposing factors that contributed to nasal colonization (eg, sex, age, season, or habitat). Results suggest that asymptomatic feral cats may carry C. neoformans and other Cryptococcus species in their sinonasal cavity. Genotyping of the specific cryptococcal isolates provides a better understanding of the epidemiology of these yeasts.

MALDI-TOF MS for the identification of veterinary non-C. neoformans-C. gattii Cryptococcus spp. isolates from Italy.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) offers an effective alternative to phenotypic and molecular methods for the rapid identification of microorganisms. Our aim in this study was to create an in-house library for a set of strains of nine uncommonly reported human and animal cryptococcal species, including Cryptococcus adeliensis, C. albidosimilis, C. albidus, C. aureus, C. carnescens, C. laurentii, C. magnus, C. victoriae and C. uniguttulatus, and to use this library to make timely and correct identifications using MALDI-TOF MS for use in routine laboratory diagnostics. Protein extracts obtained via the formic acid extraction method of 62 veterinary non-C. neoformans-C. gattii cryptococcal isolates were studied. The obtained mass spectra correctly grouped all 62 studied isolates according to species identification previously obtained by internal transcribe spacer sequence analysis. The in-house database was than exported and successfully uploaded to the Microflex LT (Maldi Biotyper; Bruker Daltonics) instrument at a different diagnostic laboratory in Italy. Scores >2.7 obtained from isolates reanalyzed in the latter laboratory supported the high reproducibility of the method. The possibility of creating and transferring an in-house library adds to the usefulness MALDI-TOF MS an important tool for the rapid and inexpensive identification of pathogenic and saprophytic fungi as required for differential diagnosis of human and animal mycoses.

Development of Highly Efficient Universal Pneumocystis Primers and Their Application in Investigating the Prevalence and Genetic Diversity of Pneumocystis in Wild Hares and Rabbits.

Despite its ubiquitous infectivity to mammals with strong host specificity, our current knowledge about Pneumocystis has originated from studies of merely 4% of extant mammalian species. Further studies of Pneumocystis epidemiology across a broader range of animal species require the use of assays with high sensitivity and specificity. To this end, we have developed multiple universal Pneumocystis primers targeting different genetic loci with high amplification efficiency. Application of these primers to PCR investigation of Pneumocystis in free-living hares (Lepus townsendii, n = 130) and rabbits (Oryctolagus cuniculus, n = 8) in Canada revealed a prevalence of 81% (105/130) and 25% (2/8), respectively. Genotyping analysis identified five and two variants of Pneumocystis from hares and rabbits, respectively, with significant sequence divergence between the variants from hares. Based on phylogenetic analysis using nearly full-length sequences of the mitochondrial genome, nuclear rRNA operon and dihydropteroate synthase gene for the two most common variants, Pneumocystis in hares and rabbits are more closely related to each other than either are to Pneumocystis in other mammals. Furthermore, Pneumocystis in both hares and rabbits are more closely related to Pneumocystis in primates and dogs than to Pneumocystis in rodents. The high prevalence of Pneumocystis in hares (P. sp. 'townsendii') suggests its widespread transmissibility in the natural environment, similar to P. oryctolagi in rabbits. The presence of multiple distinct Pneumocystis populations in hares contrasts with the lack of apparent intra-species heterogeneity in P. oryctolagi, implying a unique evolution history of P. sp. 'townsendii' in hares.

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.

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.

An improved molecular diagnostic assay for canine and feline dermatophytosis.

The few studies attempting to specifically characterize dermatophytes from hair samples of dogs and cats using PCR-based methodology relied on sequence-based analysis of selected genetic markers. The aim of the present investigation was to establish and evaluate a PCR-based approach employing genetic markers of nuclear DNA for the specific detection of dermatophytes on such specimens. Using 183 hair samples, we directly compared the test results of our one-step and nested-PCR assays with those based on conventional microscopy and in vitro culture techniques (using the latter as the reference method). The one step-PCR was highly accurate (AUC > 90) for the testing of samples from dogs, but only moderately accurate (AUC = 78.6) for cats. A nested-PCR was accurate (AUC = 93.6) for samples from cats, and achieved higher specificity (94.1 and 94.4%) and sensitivity (100 and 94.9%) for samples from dogs and cats, respectively. In addition, the nested-PCR allowed the differentiation of Microsporum canis from Trichophyton interdigitale (zoophilic) and geophilic dermatophytes (i.e., Microsporum gypseum or Trichophyton terrestre), which was not possible using the one step-assay. The PCRs evaluated here provide practical tools for diagnostic applications to support clinicians in initiating prompt and targeted chemotherapy of dermatophytoses.

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.

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.

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.].

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.

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.

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.