Toxoplasma gondii in small ruminants in Northern Italy - prevalence and risk factors.

OBJECTIVE: The aim of the survey was to evaluate Toxoplasma gondii seroprevalence in small ruminants and possible risk factors associated with the infection. MATERIALS AND METHODS: Sera from 474 goats and 502 sheep reared on 42 farms in northern Italy were collected and tested for IgG antibodies to T. gondii by IFAT (indirect immunofluorescence antibody test). To identify risk factors, a binary logistic regression analysis of the variables was performed. An audit form about farm management was used. RESULTS: Antibodies to T. gondii were found in 96.6% of goat farms and in 87.5% of sheep farms; 41.7% goats and 59.3% sheep resulted positive. Seroprevalence was significantly higher in sheep than in goats. Seroprevalence values were similar in goats from eastern and western areas, whereas goats from the southern area were at lower risk of infection. Saanen goats presented the lowest seroprevalence (30.7 %), whereas cross-breed exhibited the highest rate (48.7%). Goats from farms housing both sheep and goats had an infection risk 1.39 times higher than goats from farms that did not house sheep. Animals bred on intensive farms showed lower prevalence (22.1%) in comparison with those from extensive (45.6%) or semi-intensive farms (60%). Sampling area was one of the strongest predictors of T. gondii infection in sheep flocks. Transhumant flocks showed a higher risk of infection by T. gondii compared with semi-intensive farms (66.8% vs. 38.4%). CONCLUSIONS: The highest T. gondii seroprevalence values were registered in transhumant flocks of sheep and in family businesses rearing goats. As these traditional activities represent an important resource for the conservation of the territory and its economy, management practices for a better control of the disease should be improved.

Molecular and serological detection of tick-borne pathogens in donkeys (Equus asinus) in Italy.

Donkeys, owing to the frequent outdoor activity, are exposed to a high risk of infection with tick-borne pathogens. This work aimed to detect exposure to Theileria equi, Babesia caballi, Anaplasma phagocytophilum and Borrelia burgdorferi s.l. of donkeys reared in Central Italy. For this purpose 122 adult donkeys were selected within 11 herds and submitted to blood collection. IgG antibodies to T. equi, B. caballi, A. phagocytophilum and B. burgdorferi s.l. were detected by IFAT. Conventional PCRs targeting the genes MSP2 and the flagellin were used for the detection of A. phagocytophilum and B. burgdorferi s.l. respectively and a Real Time PCR Sybr Green was used to detect Babesia/Theileria spp…. The species identity was determined by amplicons sequencing. Forty eight (39.3%) and 58 (47.5%) animals tested positive for T. equi and B. caballi antibodies, respectively; nine animals (7.4%) were found positive for antibodies against A. phagocytophilum whereas negative results were obtained for B. burgdorferi s.l. Twenty-six (21.3%) animals showed antibodies for both T. equi and B. caballi. Twenty-three (18.8%) donkeys were positive to Babesia/Theileria spp. PCR assay. Out of 21 sequenced amplicons, 20 were identified as T. equi, belonging to three main groups designated A, B and D and one as B. caballi group A. Neither A. phagocytophilum nor B. burgdorferi PCR results were positive. The study showed a high exposure of donkeys to tick-borne pathogens and provides information on the genetic identity of the T. equi strains circulating in Central Italy.

Evaluation of the performance of a rapid enzyme-linked immunosorbent assay in the detection of Anaplasma phagocytophilum antibodies in horses.

The aim of this study was to evaluate the performance of a commercially available rapid enzyme-linked immonosorbent assay, the Snap® 4Dx test, in the detection of Anaplasma phagocytophilum antibodies in horses. Two hundred apparently healthy horses (asymptomatic) and 244 animals showing clinical symptoms (symptomatic), were tested for A. phagocytophilum immunoglobulin G (IgG) antibodies using both the Snap® 4Dx kit and an indirect fluorescence antibody test (IFAT), with the latter serving as a comparative test. Horses belonging to the symptomatic group were also tested for evidence of active infection with A. phagocytophilum by analysis of IFAT IgM titers and PCR assay amplifying a specific fragment of the 16S rRNA gene. The overall agreement between the results obtained using the two tests, as well as the relative performance exhibited by the Snap® 4Dx test in the two groups, was assessed. Forty of the 45 animals (89%) testing positive for IgG antibodies using IFAT were correctly identified using Snap® 4Dx testing. The agreement between the results of the two tests was very high (k>0.9), with almost identical performances in both symptomatic and asymptomatic animals. Conversely, within the symptomatic group, only 44% (no. 11/25) of Snap® 4Dx positives appeared to be associated with a state of active infection, whereas the remaining 56% (no. 14/25) were related both to not infected animals (no. 1) and to horses whose status of infection needed further evaluations to be confirmed (no. 13/25). This study suggests that the Snap® 4Dx test could represent a valid screening method for use during epidemiological surveys of equine populations. Nevertheless, in-clinic application of the test does not appear to be merited.

Trichophyton verrucosum infection in cattle farms of Umbria (Central Italy) and transmission to humans.

Trichophyton verrucosum is the most common ringworm agent in cattle. Epidemiology of cattle dermatophytoses in Central Italy is not clear. Its diffusion among cattle and herdsmen was investigated in 20 Umbrian farms, Central Italy. Hairs and scales were taken from 395 animals and 31 workers. Typical ringworm was present in 71.7% of cattle under 6 months and in 11% of animals over 6 months. T. verrucosum was isolated from 98.9% of symptomatic heads and was the most prevalent dermatophyte in all herds investigated (isolated in 18 of the 20 farms). T. mentagrophytes var. mentagrophytes was found in 16 symptomatic and in eight asymptomatic young animals. Prevalence of asymptomatic carriers of both species was significantly higher in young heads (21.1% vs. 8.1%) and the age below 6 months was the only statistically significant risk factor associated with dermatophytosis. About the workers, all the 14 men with lesions were positive for T. verrucosum; copresence of T. verrucosum and Microsporum gypseum was noticed in one case. Results indicate a high diffusion of T. verrucosum among both animals and humans in Umbrian farms and confirm the dermatophyte infection as a public health problem. Periodic epidemiological surveys, treatment of sick livestock and workers, cleaning/sanitisation of herds and vaccination programmes may be useful in controlling the infection.

Seroprevalence of Toxoplasma gondii in wild boars (Sus scrofa) from Central Italy.

Wild and farmed game meat consumption has been highlighted as an emerging risk factor for Toxoplasma gondii infection in humans. In Central Italy wild boar is widely distributed and is also one of the most popular game species. The main goal of the present study was to estimate the seroprevalence of T. gondii antibodies through a serological survey conducted on 400 hunted wild boars (250 males and 150 females) during three subsequent hunting seasons (2009-2011), using an Immunofluorescence Antibody Assay. The animals were sorted by age, determined on the evaluation of the dental table; 101 were <1 year old, 175 from 1 to 3 years, and 124 > 3 years. Antibodies against T. gondii were detected in 56 (14%) serum samples with titers ranging from 40 to ≥160; a significant association (p < 0.05) was found between seropositivity and age, but not gender, hunting districts, or year of sampling.

Nasal eucoleosis in a symptomatic dog from Italy.

A dog with chronic muco-purulent nasal discharge, sneezing, reverse sneezing and impaired scenting ability was diagnosed as being affected by nasal eucoleosis based upon rhinoscopic evidence of Eucoleus boehmi in situ, identification of the adult parasites in nasal biopsies, and eggs in the faeces by light and scanning electron microscopy. The dog was successfully treated with a single administration of moxidectin. A second course of moxidectin was required for about 10 weeks after the first treatment, because clinical signs recurred due to a likely re-infection. This second administration, along with measures undertaken to prevent geo- and coprophagic pica, resolved the parasitism, as demonstrated by negative copromicroscopic and rhinoscopic examinations, and prevented reinfestation for the next 4 months. To the best of the authors' knowledge, this represents the first report describing a clinical case of nasal eucoleosis with a demonstration of the adult parasites in situ in a dog from Italy where, until recently, infestation of E. boehmi was only detected by a coprological examination. Veterinarians and parasitologists should be aware of the occurrence of canine infection with E. boehmi. They should include this parasite in the differential diagnoses for animals suffering from upper airway distress and look systematically for it during rhinoscopic and copromicroscopic examinations.

Development of culture-based serological assays to diagnose Babesia divergens infections.

Babesioses are hematic tick-borne diseases that induce malaria-like disorders in domestic, wild animals, and humans. Although indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) commercial kits are available to test the presence of antibodies against most Babesia species, no kit exists to serologically diagnose the infections due to Babesia divergens, one of the most important zoonotic species. To fill this gap and to develop assays to detect animal and human infections, in vitro cultures (microaerophilous stationary phase system) of B. divergens were organized. Infected erythrocytes were adsorbed as corpuscular antigen (CA) on IFAT slides and ELISA microwells. The supernatant medium of the cultures (metabolic antigen, MA) was collected and employed in ELISA and western blot (WB) assays. B. divergens was also used to produce positive sera in Meriones unguiculatus and to infect a calf. Serological tests were set up with sera from experimentally/naturally infected animals, and possible cross-reactions were evaluated using heterologous sera from cattle positive to other piroplasms. Sera from clinically healthy people at risk of infection were also tested. As expected, assays based on the purified MAs from in vitro cultures proved more sensitive and specific than CA-IFAT and CA-ELISA. In fact, MA-ELISA provided satisfactory performances (even if 8.4%-15.7% cross-reactions were evidenced), and the WB developed proved totally sensitive and specific. WB indicated as immunodominant antigens two major protein bands at 33 and 37 kDa, which were also evidenced in 2.2% of the human sera tested, proving the parasite transmission to humans also in Italy.

Tick reservoirs for piroplasms in central and northern Italy.

Ticks, collected in central and northern Italy from pets, livestock, wild animals and the environment (n=2107), were identified by microscopy and processed by molecular diagnostics to determine the species that act as a reservoir for piroplasms. A total of 11 ixodid tick species were identified, with five of them proving to be piroplasm positive. Molecular diagnostics identified Theileria equi and eight Babesia species in 52 adult specimens, mostly (n=50) removed from piroplasm-free vertebrate hosts. Ixodes ricinus hosted the highest number of species, although the highest infection rate was recorded in Hyalomma marginatum (9.1%), followed by I. ricinus (5.1%), Dermacentor marginatus (5%), Rhipicephalus turanicus (3.1%) and R. sanguineus (1.2%). Novel tick/pathogen associations were detected, suggesting that certain tick species (such as Hy. marginatum, R. sanguineus and I. ricinus) are vector of more piroplasm species than previously thought. Trans-stadial maintenance of the piroplasms was observed in each positive tick species; vertical transmission of B. canis canis was demonstrated in R. sanguineus. Finally, the detection of Babesia sp., B. microti-like species and B. rodhaini, phylogenetically related to zoonotic species, suggests that the human population could be at risk of infection in the studied area.

Prevalence and diagnosis of Babesia and Theileria infections in horses in Italy: a preliminary study.

Babesia caballi and Theileria equi are the causative agents of equine piroplasmosis. In this preliminary epidemiological study, 412 horses reared in central and northern Italy were sampled and three diagnostic methods compared, namely, the microscopy, the indirect fluorescent antibody test (IFAT) and a PCR. Possible risk factors (such as area, season, breed, activity, sex, age, and grazing) associated with serological positivity were evaluated. A seroprevalence of 68.4% was found: 12.4% of the animals had anti-T. equi antibodies, 17.9% anti-B. caballi antibodies and 38.1% had antibodies against both species. Of the seropositive samples, 3.1% and 9.4% were positive to microscopy and PCR, respectively; 31.5% of the horses were IFAT-negative but 1.4% and 2.4% of the corresponding blood samples were positive to microscopy and PCR, respectively. Molecular techniques revealed that the species present were closely related to T. equi, Theileria sergenti, Theileria buffeli and the Babesia microti-like piroplasm provisionally named Theileria annae. Grazing was found to be a pronounced risk factor for equine piroplasmosis.

Field effectiveness of pyrantel and failure of Parascaris equorum egg count reduction following ivermectin treatment in Italian horse farms.

A study was carried out to assess the field efficacy of ivermectin (IVM) and pyrantel pamoate (PYR) against Parascaris equorum. Seventy-three foals (3-18 months old) from 5 stud farms, not treated with anthelmintics in the previous 10 weeks and with individual faecal egg counts (FEC) >200, were included in the study. For each stud farm, 5-7 foals were included in the IVM-treatment group (IVM 0.2%, 200 mcg/kg body weight) or in the PYR-treatment group (PYR 38%, 13.2mg/kg body weight) and 3 were untreated as controls. For each foal, FECs were carried out before treatment (Day 0) and on Days 7 and 21. An individually based estimation of efficacy was assessed by a bootstrap simulation applied to different previously suggested formulae to evaluate the percent reduction of faecal egg counts (FECR). Two thousand bootstrap resamples were constructed from individual FECRs and the parasite population was considered susceptible for FECs >or=90 and 95% confidence interval (C.I.) >or=95%, suspected resistant for FECRs comprised between 80 and 90% and 95% C.I. <95% and resistant when FECR

Wild ungulates as Babesia hosts in northern and central Italy.

Babesia and Theileria species were investigated in wild ungulates of Northern and Central Italy. Of 355 blood samples examined, 108 (30.4%) were positive to molecular diagnostics (polymerase chain reaction [PCR] with specific primers and sequencing). The sequence analysis showed that the roe deer is a susceptible host for several piroplasms belonging both to Babesia (31%) and Theileria (14.2%) species, whereas fallow deer and wild boar harbor only Theileria species (49% and 2.6%, respectively). Strains related to B. divergens are highly present (28.3%) in the roe deer, which, however, also harbors Babesia MO1 type and Babesia microti-like organisms. Babesia EU1 type is described for the first time in a roe deer in Italy. The finding in roe deer of Babesia species involved in human babesiosis is of concern for public health, mainly because ecological changes in progress cause the increase of both the deer species and the vector tick populations.

First report of oral colonization by Debaryomyces nepalensis in a dog.

A stray, young male, wire-haired pointing griffon dog, found in a street of Perugia (Italy), was examined in order to check his health status. Two oropharyngeal swabs were collected in 24 h and streaked onto Sabouraud agar and after 6 days the yeasts colonies were transferred onto Malt agar. Ascospores were observed on Potato Dextrose Agar medium. The major ubiquinone of an isolated yeast was identified as ubiquinone-9 (Q-9), and genetical analyses were performed together with the type strains of Debaryomyces hansenii (var. hansenii and var. fabry), C. psychrophila and D. nepalensis type strain. The base sequences of ITS1 and ITS2, and D1/D2 domains of LSU rDNA completely coincided with those of D. nepalensis. From these results, the isolated yeast was identified as D. nepalensis. RAPD patterns between the two strains were found to be identical. The results indicate the first colonization of D. nepalensis in a dog.

Diffuse cutaneous candidiasis in a dog. Diagnosis by PCR-REA.

The authors describe a clinical case of cutaneous candidiasis in a dog with dermatological lesions, characterized by persistent alopecia, crusts, ulcers and scales. Predisposing factors such as the use of corticosteroids, the concomitan presence of an autoimmune disease (pemphigus foliaceus) and an infection of ehrlichiosis caused by Ehrlichia canis were observed. Histopathological findings included signs of orthokeratotic hyperkeratosis, moderate follicular keratosis and light epidermic acanthosis. The reactive process included an infiltrative superficial dermatitis and a mural folliculitis with prevalent participation of macrophages and lymphocytes. The application of PCR-Restriction Enzyme Analysis (REA) method on cutaneous specimens in veterinary medicine is an extremely interesting diagnostic tool. Its use, together with other techniques, such as mycologic, cytologic and histological examinations, allowed us to identify Candida albicans as aetiological agent in this particular case.

Molecular characterisation of Babesia canis canis and Babesia canis vogeli from naturally infected European dogs.

The morphologically small Babesia species isolated from naturally infected dogs in Europe, Japan, and US are described as Babesia gibsoni despite the fact that molecular techniques show that they should be assigned to two or three separate taxons. The morphologically large Babesia isolated from dogs in Europe, Africa, and US were generally classified as B. canis until it was proposed to distinguish three related, albeit genetically distinct subspecies of this genus, namely B. canis canis, B. canis rossi, and B. canis vogeli. The insight into the molecular taxonomy of canine piroplasms is, however, limited because only partial small subunit ribosomal RNA (ssrRNA) sequence data exist for two species from the B. canis group. In this work, we molecularly characterised natural Babesia infections in 11 dogs from Croatia, France, Italy, and Poland. These infections were diagnosed as caused by B. canis canis and B. canis vogeli based on the analysis of the complete sequence of the ssrRNA genes. Phylogenetic analysis confirmed that the large Babesia species of dogs belong the to the Babesia sensu stricto clade, which includes species characterised by transovarial transmission in the tick vectors and by exclusive development inside the mammalian host erythrocytes. The new data facilitate the reliable molecular diagnosis of the subspecies of B. canis.