Reply to Navarro-Fernandez, I. Nailing down Clinical Nuances. Comment on "Crotti et al. A Terbinafine Sensitive Trichophyton indotineae Strain in Italy: The First Clinical Case of tinea corporis and onychomycosis. J. Fungi 2023, 9, 865".

We have read with interest the comments and observations made regarding the paper "A terbinafine sensitive Trichophyton indotineae strain in Italy: the first clinical case of tinea corporis and onychomycosis" [...].

A Terbinafine Sensitive Trichophyton indotineae Strain in Italy: The First Clinical Case of tinea corporis and onychomycosis.

Trichophyton indotineae is an emerging dermatophyte species that plays a relevant role in human healthcare. It has been associated with severe chronic skin infections and a high level of terbinafine resistance. T. indotineae is endemic to India, Iran, and Iraq but several cases have been reported in Europe, recently. In this manuscript, the authors report the first clinical description of a tinea corporis and onychomycosis due to T. indotineae. The patient was a 42-year-old female from India that has lived in Umbria (Central Italy) for the last two years. Firstly, a dermatological examination suggested dermatophytosis: mycology isolation from cultures and macro- and microscopical features identified the colonies as belonging to the T. mentagrophytes/T. interdigitale species complex. Subsequently, ITS1/ITS4 end-point PCR and Sanger sequencing identified the strain as T. indotineae. Lastly, a DermaGenius® Resistance Multiplex real-time PCR assay was carried out, targeting the mutations in the SQLE gene to establish terbinafine resistance or susceptibility of the strain. The melting curve observed was compatible with wild-type positive control, identifying the strain as T. indotineae terbinafine-sensitive. An oral terbinafine treatment was associated with a topical ciclopirox nail solution, resulting in remission in its clinical manifestation. On 3 July 2023, the local Prevention Service notified the case to the Ministry of Health that then reported the information at national and international levels.

The role of Mycoplasma ovipneumoniae and Mycoplasma arginini in the respiratory mycoplasmosis of sheep and goats in Italy: Correlation of molecular data with histopathological features.

Mycoplasma infections are commonly found in the respiratory system of small ruminants; the species most commonly detected are Mycoplasma ovipneumoniae and Mycoplasma arginini, associated with the so-called "atypical non-progressive pneumonia". The pathogenic role of M. ovipneumoniae in pneumonia has been demonstrated in sheep but still needs to be verified in goats; on the other hand, the role of M. arginini in sheep is not well understood, while in goats seems to be of low pathogenic value. The present study aims to investigate the aetiology of pneumonia in sheep and goats that died from respiratory disease using anatomopathological, histopathological, and molecular investigations and to clarify the role of respiratory mycoplasmas by the association of molecular data with histopathological features. First, to better understand which histological changes are actually suggestive of atypical pneumonia in sheep and goats, the study identified the histological lesions significantly associated with Mycoplasma spp. infection. Then, the histological score of lesions considered suggestive of atypical pneumonia was used to estimate the pathogenicity of each mycoplasma detected. The results showed that M. ovipneumoniae and M. arginini (alone or in mixed infections) are pathogenic both in sheep, as well as in goats with similar histology and severity of lesions. Moreover, young animals were statistically more susceptible to M.ovipneumoniae and M. arginini infection than adults. Animals appeared more at risk to the development of M. ovipneumoniae and M. arginini infection in summer.

Molecular Screening of Echinococcus spp. and Other Cestodes in Wild Carnivores from Central Italy.

Tapeworm infections are among the most relevant parasitic diseases in humans and animals. Tapeworms from the Genus Echinococcus are particularly important as they can cause cystic or alveolar echinococcosis. A molecular screening was performed on 279 fecal samples collected from carcasses of wild carnivores from Central Italy using PCR targeting diagnostic fragments of nad1, rrnS, and nad5 genes. Samples positive for either Taenia spp. or Echinococcus granulosus were sequenced to taxonomically identify the parasitic DNA. Of the 279 samples, 134 (48.0%) gave positive results in the multiplex PCR. Only one (0.4%) sample from an Apennine wolf tested positive for Echinococcus granulosus sensu stricto (genotype G3), whereas no sample tested positive for E. multilocularis. The most frequently detected tapeworms were: Mesocestoides corti (syn M. vogae) (12.9%), M. litteratus (10.8%), Taenia serialis (9.3%), and T. hydatigena (6.5%), other tapeworms were rarely detected. The results suggest that Echinococcus infections in Central Italy do not seem to be sustained by sylvatic cycles, confirming the absence of E. multilocularis in Central Italy. The survey corroborates, yet again, the importance of passive surveillance of wild animals that can serve as reservoirs for zoonotic pathogens, especially on wild canids that in other areas are strongly implicated in the transmission of E. granulosus and E. multilocularis.

Tapeworms detected in wolf populations in Central Italy (Umbria and Marche regions): A long-term study.

Tapeworms are trophically-transmitted and multi-host parasites with a complex indirect life cycle, strictly depending on predator-prey interactions. Their presence in a free-living population, mainly definitive hosts, is arduous to study due to the complexity of collecting fecal samples. However, epidemiological studies on their frequency are crucial from a public health perspective, providing information on food habits and prey selection of predators. The present study aims to update the frequency of tapeworms detected in stool samples by molecular analysis in Italian wolf populations of Umbria and Marche regions collected from 2014 to 2022. Tapeworm's total frequency was 43.2%. In detail, Taenia serialis was detected in 27 samples (21.6%), T. hydatigena in 22 (17.6%), and Mesocestoides corti (syn. M. vogae) in 2 (1.6%). Three samples were identified as M. litteratus and E. granulosus s.s. (G3) and T. pisiformis, with a proportion of 0.8%, respectively. The low frequency of E. granulosus in a hyperendemic area is discussed. The results show for the first time a high frequency of Taenia serialis not comparable to other Italian studies conducted on wild Carnivora; thus, a new ecological niche is conceivable. These findings suggest a plausible wolf-roe deer cycle for T. serialisin the investigated area.

Preliminary Investigation about Aspergillus spp. Spread in Umbrian Avian Farms.

Among the fungi responsible for deep mycosis, the genus Aspergillus plays a predominant role both in human and veterinary medicine. From a "One Health" perspective, infections by Aspergillus spp. often represent a public health problem linked to specific occupational categories that could have a greater risk of inhaling spores and developing any respiratory disease. This preliminary investigation allowed to acquire information about the spread of Aspergillus spp. in avian livestock of the Umbria region (Central Italy), their sensitivity to antifungals, and the presence of mycotoxins in the considered farms. Environmental, feed, animal, and human samples were collected for mycological investigations; chemical analyses were also performed in feed samples. Moreover, prevalence estimated of the fungal isolates were provided for each individual farm sampled. Direct fungal identification was possible in 298 out of the 559 total samples; 162 of the samples were positive for Aspergillus spp. Mycotoxins were detected in 5 out of the 21 feed samples collected. All the aspergilli tested for antifungal susceptibility were resistant to fluconazole. The results obtained show how much the genus Aspergillus is widespread in the investigated farms; therefore, the poultry livestock represents a favorable environment for the maintenance and spread of fungal spores and their potential transmission to animals and humans.

Identification and Characterization of Clostridium perfringens Atypical CPB2 Toxin in Cell Cultures and Field Samples Using Monoclonal Antibodies.

A direct sandwich enzyme-linked immunosorbent assay (sELISA) was developed for the detection of the atypical ß2-toxin (CPB2) of Clostridium perfringens. Polyclonal (PAbs) and monoclonal (MAbs) antibodies were previously obtained employing recombinant CPB2 produced in the baculovirus system as antigen. In the current study, PAbs were used as capture molecules, while purified MAbs conjugated to horseradish peroxidase (MAbs-HRP) were used for the detection of atypical CPB2 toxin. MAbs 5C11E6 and 2G3G6 showed high reactivity, sensitivity and specificity when tested on 232 C. perfringens cell culture isolates. In addition, a reactivity variation among different strains producing atypical CPB2 toxin was observed using the conformation-dependent MAb 23E6E6, suggesting the hypothesis of high instability and/or the existence of different three-dimensional structures of this toxin. Results obtained by sELISA and Western blotting performed on experimentally CPB2-contaminated feces revealed a time-dependent proteolytic degradation as previously observed with the consensus allelic form of CPB2. Finally, the sELISA and an end-point PCR, specific for the atypical cpb2 gene, were used to test field samples (feces, rectal swabs and intestinal contents) from different dead animal species with suspected or confirmed clostridiosis. The comparison of sELISA data with those obtained with end-point PCR suggests this method as a promising tool for the detection of atypical CPB2 toxin.

Health Status of the Eastern Grey Squirrel (Sciurus carolinensis) Population in Umbria: Results of the LIFE Project 'U-SAVEREDS'.

The introduction of the Eastern grey squirrel (Sciurus carolinensis) in Europe is one of the best-known cases of invasive alien species (IAS) colonisation, that poses a severe risk to the conservation of biodiversity. In 2003, it was released in a private wildlife park near the city of Perugia (Italy), where it is replacing the native Eurasian red squirrel (Sciurus vulgaris). The LIFE13 BIO/IT/000204 Project (U-SAVEREDS) was set up for the Sciurus vulgaris conservation in Umbria through an eradication campaign of grey squirrels. One hundred and fifty-four animals were analysed for bacteriological, mycological, virological, and serological investigations (C4 action). Sanitary screening showed that Sciurus carolinensis is a dermatophyte carrier, and therefore, it could cause public health issues for humans, considering its confident behaviour. Moreover, it has been marginally responsible for the spreading of Candida albicans, Coxiella burnetii, and Borrelia lusitaniae. Health status evaluation conducted on the Sciurus carolinensis population indicated that it is necessary to raise awareness of its impacts on biodiversity and human health. Moreover, the health status and behaviours of the IAS must be considered when control or eradication campaigns are planned.

Apple Pomace as Valuable Food Ingredient for Enhancing Nutritional and Antioxidant Properties of Italian Salami.

Nowadays, food fortification with bioactive compounds deriving from agri-food waste is of great interest all over the world. In this work, apple pomace (AP), the most abundant by-product of apple juice manufacturing, was characterised by chemical, chromatographic and spectrophotometric analyses. AP showed valuable antioxidant activity, due to the presence of phenolic compounds (8.56 mg gallic acid equivalents/g), including quercetin-3-O-galactoside, quercetin-3-O-arabinofuranoside, and phloridzin. Dried AP, at 7% and 14%, was added to pork meat to produce Italian salami, then subjected to 25 days of ripening. Physicochemical, colorimetric and microbiological analyses were carried out at days 0, 5, 11, 19 and 25, while nutritional and sensory evaluations were performed at the end of the ripening. The overall acceptability was slightly higher for 7% AP compared to 14% AP sample, and generally the replacement of a percentage of meat with apple pomace allowed the production of salami with sensory properties comparable to those obtained with classic recipes. The improved fibre and phenol content, together with the lower fat and calories, represent the most interesting characteristics of fortified salami. The results confirm that the addition of AP represents a valid approach to adding healthy compounds to salami.

A novel intermediate host for Taenia serialis (Gervais, 1847): The European roe deer (Capreolus capreolus L. 1758) from the Monti Sibillini National Park (MSNP), Italy.

Taeniids are multi-host parasites with an indirect life cycle that strictly depends on a predator-prey relationship. Parasites with a complex life cycle may exhibit different degrees of host-specificity at each life stage. Knowing the host breadth is a fundamental concept of the biology and epidemiology of these multi-host parasites. Morphological identification of tapeworms is challenging and occasionally may produce misdiagnosis. Thus, molecular investigations were carried out for the identification of parasitic cysts detected from muscle tissues in a male roe deer necropsied at the Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche "Togo Rosati" (Central Italy). Sanger sequencing showed 99% query cover, 2e-109 e-value, and 100% identity with Taenia serialis. The exact definitive host was not revealed in this report, but red foxes and Italian wolves may play a significant role as being widespread within the area. Wildlife surveillance is crucial to monitor for human and animal health since global distribution and flexibility in intermediate hosts of many and even more critical taeniids species may enlarge their host range.

Presumptive Zoonotic Kerion by Nannizzia gypsea: Case Report.

Nannizzia gypsea (formerly Microsporum gypseum) belongs to geophilic dermatophytes, fungi involved in keratin degradation in the soil; however, they are also found in dogs and cats. Transmission to humans can occur directly by contact with soil, but indirect transmission via domestic animals is reported too. The exact source of the infection is usually difficult to assess and in most cases only hypothesised and rarely investigated. This case report describes a kerion caused by N. gypsea in a 2-year-old boy, where the contagion was probably secondary to domestic healthy carrier dogs. A "One-Health" approach involving human dermatologists and veterinarians, combined with the use of conventional and molecular-based techniques, allowed tracing of the epidemiological chain and managing of not only the treatment but also the prevention of a recurrence. The child's lesion began to regress after about 8 weeks of treatment with both systemic and topical therapy, while the dogs were given chlorhexidine and miconazole baths. No recurrences nor new infections occurred, demonstrating the effectiveness of the strategies used.

Diversity of yeasts and moulds in dairy products from Umbria, central Italy.

In this research communication we report on the diversity of yeast and mould species in 69 samples of milk and different dairy products from three plants located in Umbria, central Italy. Isolates were characterised both macroscopically and microscopically and then identified by PCR and genome sequencing of the ITS region and the D1-D2 domain of the large-subunit rRNA gene for filamentous fungi and yeasts, respectively. Out of the 69 samples analysed, 51 (73.9%) tested positive for the presence of yeasts, whereas moulds were detected in 25 (36.2%) samples. A total of 9 yeast species belonging to 8 different genera and 13 mould species belonging to 6 different genera were isolated. The most common genera isolated were Debaryomyces and Kluyveromyces among the yeasts and Penicillium and Galactomyces among the moulds. Microbiota play a key role in the formation of flavour, aroma, texture and appearance of dairy products. This complex microbial ecosystem includes both cultured and external bacteria, yeasts and moulds. Some of them have an important role in the production of cheeses, whereas others are responsible for dairy product spoilage, resulting in significant food waste and economic losses. Some species can produce mycotoxins, representing a potential hazard for the consumer's safety. This study provides interesting information on the diversity of fungi species in dairy products from central Italy that can be of major importance to identify these products and to develop adequate strategies for fungal spoilage control and consumer safety.

Fatal systemic toxoplasmosis in a 3-month-old young tibetan goat (Capra hircus).

BACKGROUND: Toxoplasmosis is one of the most common parasitic infections in both humans and animals. It is a frequent cause of abortion and stillbirth in intermediate hosts, especially sheep and goats but rarely causes fatal clinical form in adult animals. CASE PRESENTATION: In contrast, the study reports an unusual fatal case of toxoplasmosis in a young goat naturally infected with type II strain of Toxoplasma gondii. A three-month-old female goat was presented with dyspnea and died few days later. Grossly, lungs were firm, edematous and mottled with disseminated whitish areas. Generalized lymphadenopathy was found. The histopathological examination showed necrotic interstitial bronchopneumonia and necrotizing lymphadenitis with intralesional free and clustered within macrophages tachyzoites of T. gondii. DNA extracted from lungs and lymph nodes was positive for T. gondii by a fast qPCR. PCR-RFLP analysis and sequencing of GRA6 gene showed that the isolated strains belonged to type II genotype. CONCLUSIONS: This is an unusual report of acute systemic toxoplasmosis caused by the type II strain of T. gondii with a fatal outcome in a young goat.

Molecular Characterization of Clostridium perfringens Strains Isolated in Italy.

Clostridium (C.) perfringens is the causative agent of several diseases and enteric infections in animals and humans. The pathogenicity of the bacterium is largely mediated by the production of a wide range of toxins. Individual C. perfringens strains produce only subsets of this toxin repertoire, which permits the classification in seven toxinotypes (A-G). In addition, a variety of minor toxins further characterizes the single strains. The aim of this work was to evaluate, using Polymerase Chain Reaction (PCR) assays, the diversity of 632 C. perfringens strains isolated in Italy over 15 years. The genotyped strains were analyzed to determine the presence of major and minor toxins (cpe, consensus, and atypical cpb2), their geographical origins, and the source of isolation (animal species or food). Our study shows that toxinotype A had the greatest representation (93%) and correlated mainly with consensus cpb2 in a variety of animal species, as well as with atypical cpb2 in the five food samples. Type D, associated with cpe and atypical cpb2 minor toxins, was identified in 3% of the cases, and type F was identified in 2.5%. Seven type C isolates (1.1%) were detected in cattle, whereas the only type B atypical cpb2 isolated in Italy was detected in a goat, and one type E cpe+atypical cpb2 was detected in a sheep. Type G was not detected.

Reproductive Traits of an Invasive Alien Population of Grey Squirrel (Sciurus carolinensis) in Central Italy.

The reproductive cycle of an invasive alien Italian grey squirrel population was studied to understand its adaptation and limit its spread, in order to conserve the autochthonous red squirrel. Female and male genital traits were evaluated throughout the reproductive cycle, including the ovary, uterus, testicle, epididymis, seminiferous tubule morphometry, and germinative epithelium histology. Moreover, individual female fecundity was determined by counting uterine scars. Ovary width and uterus weight, length, and width reached their highest values in the luteal and pregnancy phases. On conducting a histological evaluation of the testicular germinal epithelium, four morphotypes related to the different reproductive phases of the male squirrels were identified: immature, pubertal, spermatogenesis, and regressive. Testicle and epididymis weights and seminiferous tubule diameters reached their largest values during spermatogenesis. Uterine scar analysis showed that 69% of the females had given birth to one or two litters, while 31% had no uterine scars. Litters were larger in the first breeding period than in the second; annual fecundity was 4.52 ± 1.88 uterine scars/female. Umbrian grey squirrels have adapted to their non-native range, showing two annual mating periods at times similar to those in their native range, and high reproductive success.

Bovine malignant catarrhal fever: case reporting in Central Italy.

A case of malignant catarrhal fever (MCF) occurred in a 4­month­old calf housed in a semi­intensive herd in central Italy is described. The herd was in strict cohabitation with a group of domestic sheep. The calf displayed clinical signs that resembled the acute form of MCF and, after a few days of antibiotic and anti inflammatory therapy, died in September 2016. The diagnosis was confirmed in vivo in blood by detection of ovine herpesvirus type 2 DNA through real­time PCR. At necropsy, the gross post­mortem findings were typical of MCF and the histological and molecular assays confirmed the presence of the virus. The sheep flock was suspected to be the source of the infection. In Italy, as well as in Europe, there is little data regarding the epidemiology and the recurrence of the disease in herds of cattle, due to the lack of an active surveillance plan and to a major consideration of MCF between differential diagnosis.

A multi-screening Fast qPCR approach to the identification of abortive agents in ruminants.

Abortion in ruminants represents an important economic concern for farmers. Microbial agents, such as Brucella spp., Chlamydia spp., Coxiella burnetii, Leptospira spp., Neospora caninum, Salmonella spp. and Toxoplasma gondii, are among the main infectious causes of abortion and require rapid and reliable diagnosis. This study describes the development of a multi-screening assay using Fast Real-Time PCR (Fast qPCR) that allows, in a single test, the simultaneous identification of the above-mentioned abortive agents. This multi-screening approach is characterized by a mean diagnostic sensitivity and specificity of 100% and 97%, respectively; it has a limit of detection (LOD) ranging from 5 × 103 to 4 × 104 genomic copies/g of tissue and a very good concordance with traditional end-point PCR assays used in routine diagnostic activity. The proposed method represents a rapid approach to the simultaneous detection of the main abortive agents in ruminants that allows to make an accurate diagnosis and to set up appropriate control measures in a short period of time.

Toenail onychomycosis by Trichophyton rubrum and concurrent infestation with Tyrophagus putrescentiae.

A remarkable case of onychomycosis by Trichophyton (T.) rubrum combined with nail infestation by Tyrophagus (T.) putrescentiae in an elderly diabetic farmer is described and discussed. Large numbers of eggs and mites in all development stages were present in nail debris, reflecting active reproduction on site. Treatment with ivermectin 0.1% cream and environmental decontamination cleared the mite infestation, while onychomycosis responded well to oral terbinafine and ciclopirox 8% nail lacquer. Such a combination of onychomycosis and mite infestation of the same nail is an exceptional finding reported only twice in the literature.