Spread of Toxoplasma gondii among animals and humans in Northern Italy: A retrospective analysis in a One-Health framework.

Toxoplasmosis occurs worldwide and is considered one of the most important food-borne parasitic zoonoses. The consumption of undercooked meat containing viable tissue cysts and ingestion of environmental oocyst are the most important sources of infection. The aim of this retrospective study was to evaluate the spread of Toxoplasma gondii in the province of Bologna (Emilia-Romagna region) in northern Italy, with a One Health approach, comparing seropositivity rates in different animal species and in humans over the last 19 and 4 years respectively. Analyses were performed on serological data collected over different periods at three separate locations: Istituto Zooprofilattico Sperimentale della Lombardia e della Emilia-Romagna (IZSLER); Veterinary University Hospital Clinical Pathology Service, Department of Veterinary Medical Sciences, University of Bologna; and Unit of Microbiology, St. Orsola Hospital, Bologna. Most relevant seropositivity rates observed in animals were 15.5% (wild boar), 25% (roe deer), 18.7% (goat), 29.9% (sheep), 9.7% (pigs), 42.9% and 21.8% in cat and dog, respectively. A comprehensive screening was conducted on a population of 36,814 individuals, revealing a prevalence of 20.4%. Among pregnant women, a frequence of 0.39% for active toxoplasmosis was observed. Despite certain limitations, this study provided valuable insights into the extensive distribution of this parasitic infection among diverse animal species and human populations in the province of Bologna. These findings underscore the importance of implementing consistent and proactive toxoplasmosis screening protocols during pregnancy, while emphasizing the critical need for adopting a One Health approach for effective control of this parasitic disease.

Could honey bees signal the spread of antimicrobial resistance in the environment?

The honey bee has long been known to be a bioindicator of environmental pollution and the use of antimicrobials in the beekeeping industry is strictly regulated. For these reasons, this paper was aimed to evaluate for the first time the role of Apis mellifera as a possible indicator of environmental antimicrobial resistance (AMR). The study isolated and analysed the resistance patterns of Enterobacteriaceae from a pool of honey bee guts located in five different environmental sites (ES), where different antimicrobial selective pressures were hypothesized. In all, 48 isolates were considered for identification and underwent analyses of AMR to ampicillin, amoxicillin/clavulanic acid, cefazolin, ceftazidime, tetracycline, imipenem, enrofloxacin, amikacin and trimethoprim/sulfamethoxazole. In all, 12 isolates out of 48 (25%) showed resistance to at least one antimicrobial drug. There were no significant differences between the resistance rates observed in the ESs, even if the highest percentage of resistance was found in ES4. Resistances to amoxicillin/clavulanic acid resulted significantly higher than those detected towards the other antimicrobials. Amoxicillin/clavulanic acid is not commonly used in beekeeping but it is extensively used in animals and in humans, suggesting an environmental origin of this resistance and supporting the hypothesis that honey bees could be used as indicators of AMR spread in the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, a possible role of honey bees as indicator of environmental antimicrobial resistance is hypothesized. Enterobacteriaceae were isolated from bees living in different environmental sites (ES) where different antimicrobial selective pressures were hypothesized. Even if no differences between the resistances in the five ES were observed, the resistance rates for amoxicillin/clavulanic acid, compared to other antimicrobials, were significantly higher. Since amoxicillin/clavulanic acid is not used in beekeeping but it is extensively used in animals and in humans, an environmental origin of this resistance is suggested that supports our hypothesis.

Cryptosporidium parvum: From foal to veterinary students.

This paper describes the transmission of a zoonotic subtype of Cryptosporidium parvum between two foals hospitalized in an Equine Perinatology Unit (EPU) linked to an outbreak of cryptosporidiosis in veterinary students. Fecal specimens of 36 mares (105 samples) and 28 foals (122 samples) were subjected to Ziehl-Neelsen staining, nested PCR of 18S rDNA. Two foals tested positive for Cryptosporidium; PCR restriction fragment length polymorphism (PCR-RFLP) analysis and subtyping by nested PCR of the 60kDa glycoprotein (gp60) gene revealed C. parvum subtype IIdA23G1. The introduction of Cryptosporidium into the EPU is suspected to be in a foal showing no initial clinical signs that tested positive for C. parvum during an asymptomatic phase. A second foal, hospitalized afterwards for perinatal asphyxia syndrome complicated with failure of passive transfer and sepsis, showed severe watery diarrhea after 4 days of hospitalization and was positive for the same subtype. During this period, six students attending the EPU complained of abdominal pain and diarrhea and were positive for the same subtype of C. parvum. To the authors' knowledge, this is the first description of this subtype in foals and the first report of evidence of zoonotic transmission of cryptosporidiosis from foals to human.

Molecular and phylogenetic analysis of the filarial nematode Micipsella numidica from the hare Lepus europaeus in Italy.

The genus Micipsella comprises three species of filariae to date identified in lagomorphs only, whereas the other genera belonging to the subfamily Splendidofilariinae are described as parasites of birds, reptiles and mammals. In the present study seven specimens of Micipsella numidica (Seurat, 1917), collected from the hare Lepus europaeus in Italy, were characterized genetically by molecular amplification of the mitochondrial genes (12S rDNA; cox1) and the 5S rDNA gene spacer region. Phylogenetic trees inferred using available sequences from filariae and those identified in this study evidenced a close relationship between M. numidica and Splendidofilariinae of other mammals and reptiles (Rumenfilaria andersoni and Madathamugadia hiepei). The present findings, apart from adding new data about the hosts in Italy, support the taxonomic position of M. numidica and highlight the substantial biological and molecular differences existing between Splendidofilariinae and other Onchocercidae. The study also contributes to our knowledge of the molecular/genetic diagnosis of filarial parasites of veterinary and medical concern in any vertebrate or invertebrate host.

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.

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.

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.

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.