Roberto Amerigo Papini

New insights into the morphology, molecular characterization and identification of Baylisascaris transfuga (Ascaridida, Ascarididae)

Species ranked within the genus Baylisascaris (Ascaridida, Ascarididae) have been implicated in clinical and subclinical intestinal diseases in their natural hosts (e.g., raccoons and bears) as well as in life-threatening larva migrans syndromes in a number of incidental hosts, including humans. Following the diagnosis of Baylisascaris transfuga infestation in two captive polar bears, living in the zoo park of Pistoia (Tuscany, Italy), nematodes (n=300; both sexes) have been characterized by morphological and molecular methods by sequencing and analysing ribosomal (large ribosomal DNA (28S) and internal transcribed spacer region 1 and 2 (ITSs)) and mitochondrial (cytochrome c oxidase subunit 1 (cox1) and cytochrome c oxidase subunit 2 (cox2)) target regions. In addition, seven faecal samples were collected from the animal enclosure and submitted to copromicroscopic and molecular examination. All nematodes were morphologically identified as B. transfuga and their main distinctive features are here presented. No variation in size and nucleotide polymorphisms was detected within each target sequence among all samples analysed. These data contribute to facilitate an accurate diagnosis of this little known nematode infestation in order to apply appropriate anthelmintic strategies.

Seroprevalence, Detection of DNA in Blood and Milk, and Genotyping of Toxoplasma gondii in a Goat Population in Italy

Toxoplasma gondii is the causative agent of a major zoonosis with cosmopolitan distribution and is known to be transmitted mainly by the ingestion of undercooked or raw animal products. Drinking unpasteurized goats milk is a risk factor associated with human toxoplasmosis. However, very little is known about the excretion of DNA in goat milk. Aim of the present study was to determine the seroprevalence of T. gondii infection using a modified agglutination test (MAT), to detect T. gondii DNA by nested-PCR (n-PCR) in samples of blood and milk from seropositive goats, and to genotype DNA isolates using 11 molecular markers in 127 adult lactating goats from 6 farms in Italy. Positive MAT results were found in 60.6% of goats while 13% of blood and milk samples from seropositive goats were positive to n-PCR. A kappa coefficient of 1 indicated a perfect agreement between blood and milk n-PCR. Genetic characterization of isolates revealed the occurrence of genotype III (n = 7), genotype I (n = 1), and atypical genotypes with hints for genotype I (n = 2). Our results suggest that the risk of excretion of Toxoplasma tachyzoites might frequently occur in milk of seropositive goats testing positive to n-PCR on blood.

High infection rate of Trichophyton verrucosum in calves from Central Italy.

Recent epidemiological features of the zoonotic dermatophyte Trichophyton verrucosum were investigated in 294 calves from 20 farms located in Central Italy. By the combination of microscopic examinations and fungal cultures, the total prevalence rate of T. verrucosum infection was found to be high, with 87.7% of samples yielding positive results and 100% of farms being infected. Farm to farm prevalence of infection varied from 25% to 100% of the examined calves. Prevalence rates were higher in females than males (91.6% versus 84%), in >3–≤6 than >6–≤9 month old calves (89.8% versus 85.7%), in purebred than in crossbred (88.5% versus 81.8%), and in symptomatic than asymptomatic ones (95.7% versus 80.4%). T. verrucosum was as prevalent in semi‐intensive as in intensive breeding system (88.2% versus 87.6%) but more prevalent in calves raised for meat rather than milk production (91.1% versus 84.9%), and reared under conventional than organic production management system (89.3% versus 82.6%). Prevalence rates were also higher when the cattle present in the farm were of mixed origin, rather than when the whole stock was purchased or was born in the farm (92% versus 88.2% and 85.7%), when other cattle in the farm were showing skin signs (90% versus 84.2%), and at least one case of human ringworm was recorded in the farm (90.9% versus 86.1%). No statistically significant association was found. Therefore, T. verrucosum infection may be more widespread in calves from Central Italy than it was previously thought to be. Calves of any sex, breed, age, and of any of the farm condition considered, are highly exposed to the risk of infection. As calves may play a considerable role as reservoirs of the infection, our results highlight the need to determine the incidence and the importance of T. verrucosum ringworm in personnel involved with cattle care in Italy and elsewhere.

Isolation of keratinophilic fungi from the floors of private veterinary clinics in Italy

To evaluate the presence of keratinophilic fungi in the environment, 400 samples were collected from the floors of 50 private veterinary clinics using 55-mm-diameter ‘contact plates’, containing mycobiotic agar. After incubation for 15 days at 25°C, the following species were isolated: Microsporum canis, Trichophyton terrestre, Chrysosporium keratinophilum, Chrysosporium sp., Microsporum gypseum, Trichophyton ajelloi, Chrysosporium tropicum, Trichophyton mentagrophytes, Chrysosporium state of Arthroderma tuberculatum and Chrysosporium pannorum. It is concluded that the keratinic material shed by infected pets may contribute to the development and propagation of dermatophytes and related fungi in veterinary clinics. Therefore, such veterinary clinics may represent sites where pets and humans are exposed to risk of infection with keratinophilic fungi from the environment.

Detection of Giardia assemblage A in cats in Florence, Italy.

In the present study, the assemblage of Giardia isolates from stray and owned cats was investigated. Faeces were randomly collected from 27 cats, screened by microscopy and examined by molecular methods that included DNA extraction, small subunit rRNA gene amplification and DNA sequence analysis. Ten cats were found to be infected with Giardia and harbouring cysts belonging to the zoonotic assemblage A. The cat-specific assemblage F was not found. Such a high proportion of Giardia assemblage A in cats may pose for a different geographical distribution of assemblages in the world. These findings provide insights that may be useful to map the epidemiological risk of the infection worldwide, as this may be instrumental in safeguarding human health.

Survey of keratinophilic fungi isolated from city park soils of Pisa, Italy

A survey of geophilic dermatophytes and related keratinophilic fungi isolated from city park soils of Pisa is reported. Twenty-three (48%) soil samples out of 48 were positive by hair baiting. The following species were isolated: Microsporum gypseum (39%), Trichophyton ajelloi (31%), Chrysosporium keratinophilum (14%), T. terrestre (8%), M. fulvum, Ch. luteum, Ch. indicum (5% each) and M. cookei (2%). The presence of the different species is discussed in relation to the risk of fungal skin infections.

Detection and genotyping of Toxoplasma gondii DNA in the blood and milk of naturally infected donkeys (Equus asinus).

BackgroundToxoplasma gondii is a worldwide zoonotic protozoan. Consumption of raw milk from infected animals is considered a risk factor for acquiring toxoplasmosis in humans. Recently, donkey milk has been indicated for therapeutic and nutritional purposes and T. gondii infection is common in donkeys. The purpose of the present paper was to detect the presence of parasite DNA in milk of T. gondii positive donkeys.FindingsAntibodies to T. gondii were found in 11 out of 44 healthy lactating donkeys by IFAT. T. gondii DNA was detected by PCR in blood of 6 and milk of 3 seropositive jennies. Results of limited RFLP-PCR genotyping indicated the presence of T. gondii genotype II or III, commonly found in Europe.ConclusionsThe occurrence of T. gondii DNA in milk suggests that the consumption of raw milk from seropositive donkeys could be a potential source of human infection.

SEROLOGIC, MOLECULAR, AND PATHOLOGIC SURVEY OF TOXOPLASMA GONDII INFECTION IN FREE-RANGING RED FOXES (VULPES VULPES) IN CENTRAL ITALY

We tested 191 sera of red foxes (Vulpes vulpes; 78 females and 113 males) for antibodies to Toxoplasma gondii using an indirect immunofluorescent test. Tissue samples of myocardium, lymph nodes, and brains from antibody-positive animals were tested for T. gondii DNA using specific PCR and processed for histopathology and immunohistochemistry (IHC) to detect T. gondii antigen. Of 192 (53.4%) antibody-positive animals, eight were positive by PCR on myocardium and one on brain. All DNA extracts were genotyped. Histopathology showed lesions characteristic of protozoan encephalitis; IHC did not show T. gondii antigen in examined tissues. The high antibody prevalence found in our study, which is the first in Italy, and the occurrence of polymorphic strains (combination of different type I and III alleles) divergent from typical T. gondii strains, suggests red foxes may be a sentinel of T. gondii in the environment.

Occurrence and cyst burden of Giardia duodenalis in dog faecal deposits from urban green areas: Implications for environmental contamination and related risks.

To investigate the prevalence and cyst burden of Giardia duodenalis as well as the genetic identity and possible zoonotic potential of isolates in canine faeces left on soil, a total of 143 freshly passed faecal specimens were collected in four different green urban areas and examined by Real-time PCR. Overall, 30.8% of faeces from different dogs that defecated in urban areas were found to harbour Giardia cysts, with single area positivity proportions ranging from 4.2% to 47.8%; significant (P<0.05) and highly significant (P<0.01) differences were found in the prevalence among different areas investigated. The number of cysts ranged from 2 to 1428 per gram of faeces. Results emphasize that a high risk of dog-to-dog Giardia transmission may occur in public areas where high quantities of faeces from infected dogs are daily left to cumulate on the ground. Results suggest there is a low risk for zoonotic transmission of Giardia: only 0.6% of canine faeces contaminating urban areas were carrying subgenotype A1, which is considered as the most potentially transmissible to humans.

Endoparasite Infections in Pet and Zoo Birds in Italy

Faecal samples were individually collected from pet (n = 63) and zoo (n = 83) birds representing 14 orders and 63 species. All the samples were examined by faecal flotation technique. In a subgroup of samples (n = 75), molecular assays were also used to detect Cryptosporidium oocysts and Giardia duodenalis cysts. Overall, 35.6% of the birds harboured parasites (42.2% of zoo birds and 27% of pet birds), including Strongyles-Capillarids (8.9%), Ascaridia (6.8%), Strongyles (5.5%), G. duodenalis Assemblage A (5.3%), Coccidia (4.1%), Cryptosporidium (4%), Porrocaecum (2.7%), Porrocaecum-Capillarids (2%), and Syngamus-Capillarids (0.7%). The zoonotic G. duodenalis Assemblage A and Cryptosporidium were exclusively found in Psittaciformes, with prevalences of 10.3% and 7.7% within this bird group. Zoo birds were more likely to harbor mixed infections (OR = 14.81) and symptomatic birds to be parasitized (OR = 4.72). Clinicians should be aware of the public health implications posed by zoonotic G. duodenalis Assemblages and Cryptosporidium species in captive birds.