Epidemiological survey on Cryptosporidium in an Equine Perinatology Unit.

The present study aims to evaluate the prevalence, pattern of spread and risk factors for the transmission of cryptosporidiosis in foals and mares hospitalized in a University Equine Perinatology Unit, where a new subtype family of Cryptosporidium horse genotype was described by Caffara et al. (2013). Mares (36) and foals (37) hospitalized during the 2012 foaling season were included. Multiple sampling from each animal was performed (a total of 305 stool samples were collected). One hundred and eleven environmental samples (gauze swabs) were also collected before and after the breeding season. Fourteen foals were found positive for Cryptosporidium spp. by PCR in at least one sample; a total of 35 foal stool specimens were confirmed for the presence of the protozoa. Instead none of the stool specimens from mares were found positive. PCR-RFLP analysis shows Cryptosporidium parvum in 5 stool samples and Cryptosporidium horse genotype in 21. In 9 specimens, from 4 different foals, the profile was suggestive for a mixed infection. The subtyping at gp60 locus showed 2 strains as members of the subtype family IId and six of the subfamily IIa of C. parvum. Twenty isolates were identified as Cryptosporidium horse genotype subtype VIaA15G4. Five gauze swabs collected from the walls of the boxes where the animals were hosted out of 111 environmental samples examined were PCR positive for Cryptosporidium spp. Cryptosporidium parvum was detected in one sample collected before the foaling season, while Cryptosporidium horse genotype profile was observed in 4 wall samples collected at the end of the 2012 foaling season. The prevalence observed in foals (37.8%) was higher than that reported in other studies. These features and the diffusion of the same genotype point out as the EPU, where critically ill foals are hospitalized, can support the spread of cryptosporidiosis. Therefore, the manual tasks and the activities carried out in these facilities are of great importance, as they might favor the diffusion of the infection.

Cortisol levels in cats' hair in presence or absence of Microsporum canis infection.

The purpose of this work was to perform a preliminary screening in the domestic cat to assess the concentration of cortisol in hairs by radioimmunoassay technique (RIA) in presence or absence of Microsporum canis infections. A total of 245 cats (7 with cutaneous lesions referable to dermatophytosis and 238 apparently healthy) coming from 14 shelters were examined. M. canis was isolated in 126 (51.4%) cats. The cortisol levels were significantly higher in cats with lesions or without lesions but with a high number of colonies in the plates (≥ 10 CFU) than in cats negative or with a lower number of colonies. The results obtained seem to highlight that chronic high levels of cortisol in cats could possibly promote the dermatophytes infections. Furthermore, in High-CFU asymptomatic cats, it could be present a state of infectious, and they, therefore, represents not a simple mechanical carrier.

Comparison of diagnostic methods to detect piroplasms in asymptomatic cattle.

This study was carried out to compare different diagnostic techniques to reveal the presence of piroplasms in asymptomatic cattle kept at pasture. Nineteen blood samples were collected from animals of two different areas of Emilia Romagna Region of Italy and processed for microscopic observation, PCR, serological test (IFAT) for Babesia bovis and Babesia bigemina antibodies and in vitro cultivation. The cultures were performed on both bovine and ovine erythrocytes. Seventeen blood smears (89%) were positive for piroplasms, while PCR was positive on 18 samples (95%). DNA sequencing of 18S rRNA identified the piroplasms as Theileria spp. In vitro cultures were successful for 6 samples (32%) cultured on bovine blood and subsequent identified these as Babesia major by PCR. On IFAT analyses of 16 samples, 36.8% resulted positive for B. bovis and 31.6% positive for B. bigemina. These results show, in the same animals, the co-infection with Babesia spp. and Theileria spp.; the detection of B. major was possible only using the in vitro cultures.

New insights into the epidemiology of bovine piroplasmoses in Italy.

Few studies have been published on bovine piroplasmoses in Italy, and therefore a clear picture of the epidemiology of these infections is difficult to obtain. Vertebrate and invertebrate hosts in Central and Northern Regions of Italy were investigated in 2005 and 2006, when microscopy, molecular tools and serological tests were applied to 468 blood samples drawn from cattle in order to evaluate the presence of these protozoa and identify possible risk factors. Ticks were also collected, identified and analyzed by molecular techniques. Microscopy identified 6.5% of the animals as positive, whereas PCR detected piroplasm DNA in 21.6%. BLAST analysis showed 67 amplicons (17.0%) referable to the Theileria sergenti/buffeli/orientalis group, 17 (4.3%) to Theileria annae, and 1 to Babesia divergens. Serology evidenced a prevalence of 45.4% for Babesia bovis, 17.4% for Babesia bigemina, and 34.9% for B. divergens. The 127 collected ticks were identified as belonging to 5 species, mostly represented by Rhipicephalus bursa, Hyalomma marginatum and Ixodes ricinus. Molecular analyses evidenced the presence of B. bovis and B. bigemina, in 3 and 5 ticks, respectively. Our findings suggest that different species of piroplasms are circulating in bovine populations in Central and Northern Italy, and provide new insights into the complex epidemiology of bovine piroplasmoses in Italy.

Evidence for bovine parafilariosis in Italy: first isolation of Parafilaria bovicola (Tubangui, 1934) from autochthonous cattle.

In May and July 2010 and in March 2011, the presence of multiple bleeding nodules at the level of the neck, shoulders, withers, back and rump in five cattle from three herds in north-central Italy were observed. A diagnosis of parafilariosis was made. Fragments of adult females of Parafilaria bovicola could be identified from the derma of three to five bioptic extirpates. Larvated eggs and free larvae were observed in serohemorrhagic exudates. This paper is the first report of the occurrence of parafilariosis in Italy and subsequent to the various outbreaks reported, it can now be said that bovine parafilariosis is also currently present in Italy.

Detection and molecular characterization of Theileria sp. in fallow deer (Dama dama) and ticks from an Italian natural preserve.

The prevalence of piroplasms in a closed population of fallow deer (Dama dama L.) living in the Italian preserve of "Bosco della Mesola"--Ferrara (Mesola wood) was investigated. Blood samples and ticks were collected from 62 fallow deer. On microscopic observation, 28 (45.0%) blood samples were positive for piroplasms while PCR provided evidence for piroplasms infection in 47 (75.8%) fallow deer. The 67 ticks, collected from positive and negative animals, were identified as Ixodesricinus L., 1758 (89.6%) and Haemaphysalisconcinna Koch, 1844 (10.4%). At the PCR, four samples of I. ricinus were positive for piroplasms. The sequences of the 18S rRNA gene from both blood and ticks were identical and showed high identity (99.6%) with Theileria sp. 3185/02 (DQ866842) and Theileria capreoli (AY726011) from roe deer. Interestingly, the phylogenetical analyses evidenced differences between the Theileria strain from Mesola wood and the ones isolated in fallow deer from other Italian areas.

Prevalence of Anaplasma phagocytophilum in fallow deer (Dama dama) and feeding ticks from an Italy preserve.

Up to date, information concerning the Anaplasma phagocytophilum infection in fallow deer is scant, therefore, to verify its prevalence in these ungulates serological and PCR screenings were performed on blood of 72 fallow deer hunted in a Central-Northern Italian preserve. Molecular analyses were also performed on 90 ticks removed from the animals. A. phagocytophilum infection in fallow deer was confirmed in 20 out 72 by IFA assay and in 11 out 72 by PCR. The sequence obtained revealed a complete genetic homology among the blood samples and strong degrees of homology with other European isolates. Considering the 90 ticks collected we found that 7.3% of Ixodes ricinus harboured A. phagocytophilum specific DNA. The data obtained confirmed that fallow deer can be a competent host for A. phagocytophilum and, therefore, that may represent a biological reservoir playing an important role in the epidemiological scenarios of the infection, in the geographical areas where is widespread.

Canine piroplasmosis in Italy: epidemiological aspects in vertebrate and invertebrate hosts.

In Italy, canine piroplasmosis is believed to be widespread, but few data are available on its presence in most areas. In 2005 and 2006, vertebrate and invertebrate hosts were investigated in Central and Northern Regions of the Country. Microscopy on blood smears, molecular tools and serological tests were applied to 420 blood samples collected from dogs, in order to evaluate the presence of these protozoa and to identify possible risk factors. Moreover, ticks were analyzed by molecular techniques. Microscopy identified as positive 2.8% of the animals, all from Central Italy, and PCR detected 'piroplasm' DNA in 6.0%. Serology evidenced a mean prevalence of 34.0% with a decreasing trend from Central to Northern areas. The 507 collected ticks were identified as belonging to 8 species, mostly represented by Rhipicephalus sanguineus (n=376) and Ixodes ricinus (n=58). Molecular analyses evidenced the presence of babesial parasites (Babesia canis canis, B. canis vogeli, B. microti-like) in 25 (4.9%) of them; in Rh. sanguineus there was also demonstration of the vertical transmission of B. canis canis. Statistical analysis identified 'kennel' as risk factor for Babesia infection. Our findings evidenced that different species of piroplasms potentially infectious for dogs are circulating in Italy, and that epidemiological aspects of these infections are more complex than expected. Vector importance of both Rh. sanguineus and I. ricinus is hypothesized, but further investigation is needed.

Epidemiology and variability of Malassezia spp.

A short review on Malassezia spp., completed with our experience, is made. The main epidemiological characteristics with particular regard to the diffusion in several animal species, the characteristics of skin colonization (in particular of the dog) and the distribution of the different Malassezia spp. in some hosts are discussed. Lastly the main phenotypic and genotypic characteristics, referred to M. pachydermatis especially, were described, showing their high variability and differentiation.

Animal babesiosis: an emerging zoonosis also in Italy?

In Italy, babesiosis is widespread in several Central and Southern Regions, but few data are available on its presence in most Italian areas. In 2004 a project was financed by the MIUR to investigate on the babesiosis epidemiology in vertebrate and invertebrate hosts, and on the transmission risk for humans in Central and Northern Regions of the country. Microscopy and/or molecular tools were applied to blood samples of wild animals, livestock and pets, and to 1,677 ticks collected on animals or in the environment, with the aim of detect babesial parasites. Moreover, serological tests were used to evaluate the circulation of these protozoa among animals and people at risk. Microscopy identified as positive 5.0% of the animals, mostly living in Central Regions, but also in Northern areas considered Babesia-free. Serology evidenced the same general trend. PCR detected "piroplasm" DNA in 13.8% of the animals, and sequencing identified babesial parasites in 101/233 samples. The ticks were identified as belonging to 12 species, mostly represented by Ixodes ricinus, Rhipicephalus sanguineus and Dermacentor marginatus. Molecular analyses evidenced babesial parasites in 3.8% of them; in Rh. sanguineus was also demonstrated the vertical transmission of Babesia canis canis. To date 30 human sera have been analysed: 3 showed antibodies to B. microti. Animal babesiosis is largely present among pets, wild and farm animals, whereas goats seem refractory to the infection. In wild ungulates have been found the B. divergens-like, and the Babesia EU1 strains (reported in Italy in humans). Our findings evidenced the low reliability of microscopy in epidemiological studies, and the need of new/improved immunological tests to face diagnostic problems. The monitoring of infected areas and infection rates, joined to appropriate control programs, seems necessary to avoid the transmission of babesiosis to humans.

Mycetes and urban areas.

Mycetes are ubiquitous organisms that can cause mycoses in human and animals. The role of animals in the epidemiology of human mycoses in urban areas is multiform, but here will be discuss only two features: A) animals as vectors of mycoses and B) animal substrates as growth factor of pathogenic fungi. A) Animals as vectors of mycoses: this role is important as zoofilic dermatophytes are very important agents of zoonosis; the urban dermatophytozoonoses are prevalent caused by Microsporum canis which is prevalent in cats and dogs. Cats are often asymptomatic carriers. The pattern of human dermatomycoses has changed in Italy during the past century: at the beginning of the century anthropophilic fungi were prevalent while at present the zoophilic fungi are the most important causes. B) Animal substrata as growth factor of pathogenic fungi: soil "animalization" (i.e., the addition of such debris as hair, skin scales, dropping and other organic matters) creates an optimal substratum for the growth and the multiplication of geophilic or saprophyitic fungi, such as Microsporum gypseum and Cryptococcus neoformans. The present human lifestyle, which favours a an overpopulation of birds, wild animals, domestic mammals and sinanthropic together with man in crowded areas seems to favour the formation of environments adapted to the abundant growth of some pathogenic fungi with consequent infection for man and animals. Finally, an environment heavily populated by fungi can cause allergic pulmonary reactions as well as reactions in other organs and tissues. The control of human and animal fungi, and the efficient use of a monitoring system require ample knowledge of mycological problems both in human and veterinary medicine and of efficient laboratories capable of resolving the needs of both disciplines. Close collaboration between veterinarians, doctors and mycologists is necessary in order to resolve health problems linked to mycosis.

Survey on helminthofauna in pheasants from Eastern Europe.

In order to assess the parasitological status in imported pheasants, 51 birds (Phasianus colchicus) coming from Poland and Rumania and used in Italy in repopulation interventions for hunting purposes were examined. From each animal the trachea, oesophagus, crop and intestine were collected and examined for the presence of nematodes. The examination of the oesophagi and crops of 5 birds revealed the presence of parasites pertaining to the family Capillariidae: Eucoleus contortus was found in all of the 5 animals, E. annulatus was present along with the previous parasite in one animal coming from Poland. The examination of the tracheae revealed the presence of Syngamus trachea in 5 animals (9.80%). Adult or larval stages of Heterakis gallinarum (37.25% of birds) and Capillariidae (35.29%) were found in the intestinal tracts. Aonchotheca caudinflata was detected only in one bird coming from Poland; the capillarids found in all of the remaining pheasants exhibited morphological characteristics referable to Capillaria phasianina, a species never reported in Italy. The release of game from foreign countries, therefore, may always constitute a risk for the autochthonous one due to the spread of new parasitic infections.

[Update on the diagnosis of dermatomycosis]. / Attualità sulla diagnosi delle dermatomicosi.

Dermatomycosis are mycotic diseases of skin caused by a few mycetes: dermatophytes, and some opportunistic fungi as Malassezia, Candida (not C. albicans), Trichosporon, Rhodutorula, Cryptococcus or Aspergillus, Geotrichum, Alternaria, etc. Dermatophytes are a group of closely related filamentous fungi that invade keratinized tissue (skin, hair, nails) of humans and other animals and produce infection called dermatophytosis or ringworm or "tinea". The etiological agents of dermatophytosis are classified in three genera: Microsporum, Trichophyton and Epidermophyton (Deuteromycetes). On the basis of their primary habitat dermatophytes are divided in Anthropophilic dermatophytes (parasitic organisms that infect humans), Zoophilic dermatophytes (parasitic organisms that infect animals, but also humans: agents of zoonosis) and Geophilic dermatophytes (saprobic fungi associated with keratinous materials in soil). In the soil there are also structure associated with contagion, ("spore", "arthroconidium", or "clamydospore") of anthropophilic and zoophilic dermatophytes that may persist for years, in the environment, in hair or skin scales. Since on the skin of animals there are many saprobic organisms (Malassezia) and many fungi may infect the fur, it is important to make an accurate diagnosis. Dermatophytosis are communicable diseases acquired from infected animals or from fomites. Infections caused by dermatophytes is a ringworm. These infections may range from mild and superficial, almost subclinical, to a few areas of scaling to a highly inflammatory reaction with extensive areas of scarring and alopecia. Granuloma formations (mycetoma-like) may occur especially in cats. Dermatophytes, as filamentous fungi, undergo radial fungi: collection of skin material is best made by collecting the scales near the edges of the rings. Hairs are best sampled by plucking; a scalpel may be used to scrape scales; brushes have also been used. Sample materials are best transported in dry packet. The Wood's light may be used to identify infected fluorescent hairs. Direct microscopy, although false negative up to 50% of cases, is a highly efficient screening technique. Scraping and hairs should mixed to 10-15% KOH. Culture is a valuable adjunct to direct microscopy and is essential to identify more dermatophytes. A medium selective against most nondermatophytic moulds and bacteria is used as a primary isolation medium. Many typical isolates of common dermatophytes can be identified directly from primary isolation media. Identification characters include: colony pigmentation, texture, morphological structure (macroconidia, microconidia, spirals, pectinate branches, etc). Nutritional requiment, growth in special media, "in vitro" perforation, mating studies are procedures used to identify atypical isolates. Serological approaches have revealed difficulties. Many kinds of molecular biologic techniques have been made available for clinical diagnosis recently; almost all of these techniques involve the polymerase chain reaction (PCR).

Dermatitis associated with "hypopodes" in a horse: the first case reported in Italy.

The authors report, for the first time in Italy, a case of dermatitis of "hypopodes" origin in a horse. The hypopodes are a particular nymphal stage of mites of the suborder Astigmata. The "hypopus" is non-feeding, lacks a mouth and has a ventral suctorial plate with suckers and conoids for attaching itself to insects as a mode of dispersal. Some of these larval stages can enter into the hair follicles and into the subcutaneous layers causing lesions similar to mange. There are few reports of dermatitis in horses specifically associated with the presence of hypopodes and these have been attributed to an allergic response to the body fluid of crushed hypopial stage. It may be that infestations of these nymphal stages are widespread, but the nature of the condition is not yet clear; therefore, they may be underdiagnosed.

Mosquitoes as vectors of Setaria labiatopapillosa.

An infected bovine-baited trap was utilised in summer 1994 to catch possible intermediate hosts of S. labiatopapillosa in northeastern Italy. Collections were made for 21 nights from 8.00 p.m. to 6.00 a.m. every 2 h and, after 12 September, every 30 min. Among the 16,159 mosquitoes sampled, 11,052 were freshly blood-fed. Most of the unfed females and a representative sample of those which had fed were identified as follows: Culex pipiens, Aedes caspius, A. vexans, Culiseta annulata, Anopheles maculipennis s.l., A. claviger and Coquillettidia richiardii. In spite of having the highest relative density, C. pipiens is the species which fed the least frequently and showed lower susceptibility and efficiency. The 2 Aedes species appear to act as vectors, particularly A. caspius, which proved to be the most efficient vector (K.I. = 0.8). A. claviger and A. maculipennis contribute to S. labiatopapillosa transmission, but their scarce presence reduces their epidemiological relevance. The other species identified showed a complete refractoriness to the infection. The risks for veterinary and medical health are discussed.

Development of Dirofilaria and Setaria nematodes in Aedes albopictus.

The development of Dirofilaria repens, D immitis and Setaria labiatopapillosa up to L3 stage was documented in a laboratory colony of Aedes albopictus originating from specimens collected in Civitavecchia (Central Italy). The susceptibility of Ae albopictus and its plastic trophic habits, strongly suggest that this mosquito may contribute to the spreading of these nematodes in the country. It is particularly emphasized the danger for human health of an increased probability of transmission of Dirofilaria in urban areas.