EPIDEMIOLOGY OF SELECTED PROTOZOAN INFECTIONS IN DOMESTIC RUMINANTS

Among Apicomplexa, particularly three protozoa infecting domestic ruminants (i.e. Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti) were selected in the current studies. All of them are nowadays considered (re-) emerging and under-reported in Europe but they are proven to have important consequences on public health and food security, in addition to animal welfare and health. Moreover, an economical aspect concerning zootechnical activities is to be highlighted: T. gondii and N. caninum are considered major infective cause of reproductive failure in small ruminants, with huge economic impact, although few data are available mostly on neosporosis in small ruminants. Similarly, bovine besnoitiosis is responsible of economic losses in affected herds and epidemiological surveys are necessary to monitor the presence of B. besnoiti in Europe. Therefore, the aim of my doctoral project was to investigate on the selected protozoan infections in small ruminants and cattle, following different research lines. A serosurvey was carried out to update epidemiological data on T. gondii and N. caninum infections in small ruminants bred in Northern Italy. High seroprevalence values were registered for both protozoa at individual and farm level, consistent to those registered in previous survey carried out in European countries. Further, spatial analysis was performed in order to determine geographical and climatic features able to facilitate the maintenance of T. gondii and N. caninum infections within the study area. For T. gondii infection in goats an area of high risk of infection was identified and climatic variables, such as mild temperature and rainfall, resulted predictors of infection. Geospatial analysis and analysis on climatic data revealed useful tools for the identification of clusters of infection, although data obtained should be always interpreted considering individuals and herds factors. A deeper investigation on T. gondii infection in small ruminants\u2019 foodstuff was planned. Milk and meat were selected as products potentially posing a risk for consumers. Firstly, a commercial ELISA was validated to analyze goats\u2019 milk sample. In a \u201ccase study\u201d caprine farm naturally infected by T. gondii, variation in antibodies anti-T. gondii both in serum and milk during lactation was analyzed. Different curves of antibodies level in sera and milk were observed. Finally, two epidemiological surveys were carried out on ovine and caprine products: antibodies anti-T. gondii were detected in caprine bulk tank milk and in meat juice obtained by slaughtered goats and sheep, registering quite high prevalence values. Finally, an epidemiological survey on bovine besnoitiosis was performed on dairy and beef cattle bred in northern Italy (Lombardy and Piedmont), Liguria region and Sardinia Island. Only five independent foci of infection were registered in Lombardy region. However, when a subsequent in-depth analysis was performed in a seropositive farm applying different diagnostic tools (serology, histology and immunohistochemistry), high intra-herd prevalence and several clinical cases were recorded. Concluding, high values of Toxoplasma gondii and Neospora caninum seroprevalence were registered in small ruminants in Northern Italy; geospatial analysis revealed a useful tool for an inclusive comprehension of predictors involved in the infections. T. gondii infection deserves a major attention considering the sanitary risk posed by caprine and ovine foodstuff. Alternatively to classic serological methods, analysis on individual and tank bulk milk and on meat juice samples were confirmed a valid tool for a fast and affordable screening of T. gondii infection in small ruminants. Finally, an important contribution to the epidemiology of an emerging disease as bovine besnoitiosis was given, with the update of epidemiological data and the deeper investigation on an infected farm, confirming the necessity to plan monitoring controls

Toxoplasma gondii infection in meat-producing small ruminants: Meat juice serology and genotyping

The consumption of ovine and caprine meat is considered one of the major transmission routes for Toxoplasma gondii infection in humans. The present study aimed at obtaining epidemiological and molecular data on T. gondii infection in small ruminants slaughtered or commercialized in Italy. Meat juices from 227 sheep and 51 goats were analyzed with a commercial ELISA and antibodies were detected in 28.6% sheep and 27.5% goats. A significant difference was highlighted between adult sheep and the other considered categories (young sheep, young and adult goats) concerning the detection of antibodies (94.1%; p-value = .008). Muscles of positives samples were submitted to molecular analysis, and T. gondii DNA was detected in 15 sheep and three goats; sequencing of B1 gene showed that all belonged to Type II. The present study confirmed small ruminants' meat as a possible source of T. gondii infection for consumers eating raw or undercooked meat, particularly in those countries where the consumption of sheep and goats' meat products is a traditional gastronomic habit

Intestinal parasites of owned dogs and cats from metropolitan and micropolitan areas: prevalence, zoonotic risks, and pet owner awareness in northern Italy

Intestinal parasites of dogs and cats are cosmopolitan pathogens with zoonotic potential for humans. Our investigation considered their diffusion in dogs and cats from northern Italy areas, specifically the metropolitan area of Milan and two micropolitan areas of neighboring provinces. It included the study of the level of awareness in pet owners of the zoonotic potential from these parasites. A total of 409 fresh fecal samples were collected from household dogs and cats for copromicroscopic analysis and detection of Giardia duodenalis coproantigens. The assemblages of Giardia were also identified. A questionnaire about intestinal parasites biology and zoonotic potential was submitted to 185 pet owners. The overall prevalence of intestinal parasites resulted higher in cats (47.37%-60.42%) and dogs (57.41%-43.02%) from micropolitan areas than that from the metropolis of Milan (dogs: P = 28.16%; cats: P = 32.58 %). The zoonotic parasites infecting pets under investigation were T. canis and T. cati, T. vulpis, Ancylostomatidae, and G. duodenalis assemblage A. Only 49.19% of pet owners showed to be aware of the risks for human health from canine and feline intestinal parasites. Parasitological results in pets and awareness determination in their owners clearly highlight how the role of veterinarians is important in indicating correct and widespread behaviors to reduce risks of infection for pets and humans in urban areas

Gastrointestinal nematodes of goats: host–parasite relationship differences in breeds at summer mountain pasture in northern Italy

Introduction: The Orobic goat is a hardy breed native to the Orobic Alps (Lombardy, northern Italy). The aim of the study was the assessment of gastrointestinal nematode (GIN) egg excretion in Alpine and Saanen (cosmopolite breeds) and Orobic grazing goats, after a strategic treatment with eprinomectin in late June. Material and methods: Individual faecal samples from a mixed flock of cosmopolite and Orobic goats were collected and analysed by the FLOTAC double technique every three weeks from June to September. Results: Strongylida was the primary GIN infection observed in goats that grazed on Alpine pastures; a strategic treatment with eprinomectin led to a prolonged reduction of egg excretion during the whole study period. Egg excretion was also influenced by breed. Pluriparous Orobic does were able to control reinfection better than the pluriparous cosmopolite does. Regarding Nematodirus sp. eggs per gram of faeces (EPG), the autochthonous Orobic breed presented higher values than the cosmopolite breeds. However, cosmopolite goats presented higher EPG values of Strongyloides papillosus than their Orobic counterparts in August. Conclusions: Further studies on genetic features of local autochthonous goats, such as the Orobic breed, are needed, since they could reveal peculiar characteristics of susceptibility, resistance or resilience to GIN infection, providing genetic resources for selection

First molecular subtyping and phylogeny of Blastocystis sp. isolated from domestic and synanthropic animals (dogs, cats and brown rats) in southern Iran

Background: Blastocystis sp. is a common intestinal protist that infects humans and many animals globally. Thus far, 22 subtypes (STs) have been identified in mammalian and avian hosts. Since various STs are common to humans and animals, it was suggested that some human infections might arise from zoonotic transmission. Therefore, the aim of this study was to assess the presence of Blastocystis sp. in domestic (dogs and cats) and synanthropic animals (rats) of Fars Province, Iran, and to genetically characterize the samples. Methods: A total of 400 fresh faecal samples from 154 dogs, 119 cats, and 127 rats were inspected by direct microscopy, Wheatley's trichrome staining, in vitro culture, and 18S rRNA gene nested-PCR. Finally, sequencing and phylogenetic analyses were performed. Results: Out of 400 samples, 47 (11.8%) and 61 (15.3%) samples were detected as positive by direct wet mount and culture, respectively. Molecular analysis detected a larger number of positive samples (n = 70, 17.5%): nested-PCR showed that 29 (18.8%) dogs, 21 (17.7%) cats, and 20 (15.8%) rats were infected by Blastocystis sp. Sequence analysis of positive samples indicated the presence of zoonotic STs in all investigated host species. Specifically, ST2 (allele 9), ST3 (allele 34), ST4 (allele 94), ST7 (allele 99), ST8 (allele 21), and ST10 (allele 152) were detected in dogs; ST1 (allele 2), ST3 (allele 34), ST4 (allele 94), ST10 (allele 152), and ST14 (allele 159) were detected in cats; and ST1 (allele 2), ST3 (allele 34), and ST4 (allele 92) were detected in rats. Conclusions: Our data suggest that domestic dogs and cats can serve as possible reservoirs for in-contact humans, especially those who handle shelter-resident and client-owned animals. Moreover, rats as synanthropic animals can function as a potential source of human infections. Conversely, humans can act as a source of infections to animals. These results should be reinforced in future molecular epidemiological studies.[Figure not available: see fulltext.

Investigating on Besnoitia besnoiti (Apicomplexa, Sarcocystidae) in naturally infected dairy cattle by an integrated approach.

Bovine besnoitiosis, caused by Besnoitia besnoiti, is a (re)emerging disease in Europe (Cortes et al., 2014), including Italy (Gazzonis et al., 2014; 2017). However, its economic impact is scarcely considered and generally underestimated and there are still little studied aspects concerning both the parasite and the disease. Following a natural outbreak of besnoitiosis in a dairy herd, a study was planned to characterize B. besnoiti infection in cattle through a multidisciplinary approach.Suspicious abortions and clinical cases of besnoitiosis were reported in a dairy farm (September 2017, Northern Italy) housing 216 Holstein cattle. Blood samples were collected; haematological and serological analyses (ELISA and confirmatory WB) were performed (Fernandez-Garcia et al., 2009). Histology and molecular (endpoint ITS-1 PCR (Cortes et al., 2007) and sequencing) analyses of tissues from a slaughtered cow with chronic besnoitiosis were carried out. Out of 59 ELISA-positive animals, 50 (23%) were confirmed by WB. B. besnoiti prevalence was higher in cows (41%) than in calves (12%); any heifer did not result positive. Considering haematological parameters, a significant shift in the differential leucocyte formula from lymphocyte to granulocyte was recorded in infected cows (Mean±S.D.:L=46.1±18.4,G=53.9±18.4) if compared to negative animals (Student’s T-test,p=0.012). This finding could be helpful in diagnosis, treatment and control of besnoitiosis. Histology revealed a high load of B. besnoiti tissue cysts in skin, vulva, muzzle, sclera, eyelid, respiratory tract, emphasizing the possibility of parasite transmission through direct contact among animals. B. besnoiti was confirmed by PCR in other organs (heart, liver, aorta wall, tonsil) and especially in ovary, uterus and vulva, suggesting that the infection could affect cows’ fertility. Parasite DNA was also found in masseters posing an important question for food security, even if B. besnoiti is not considered zoonotic. The study suggests that to investigate the dynamics of bovine besnoitiosis is mandatory associate clinical and laboratory tests, including the genetic characterization of the parasite and its eventual correlation with the disease outcome

Oestrus ovis L. (Diptera: Oestridae) Induced Nasal Myiasis in a Dog from Northern Italy

A companion dog from Milan province (northern Italy), presenting with frequent and violent sneezing, underwent rhinoscopy, laryngoscopy, and tracheoscopy procedures. During rhinoscopy, a dipteran larva was isolated from the dog and identified as first instar larval stage of O. ovis by morphological features. Reports of O. ovis in domestic carnivores are sporadic and nevertheless this infestion should be considered as a possible differential diagnosis of rhinitis in domestic carnivores living in contaminated areas by the fly as consequence of the presence of sheep and goats. This report described a case of autochthonous infestion in a dog from an area where O. ovis was not historically present but it could be affected by a possible expansion of the fly as a consequence of climate change. This is the first record of Oestrus ovis infestion in a dog in Italy and, at the same time, the most northerly finding of larvae of sheep bot fly in the country

Detection of Leishmania spp. in Chronic Dermatitis: Retrospective Study in Exposed Horse Populations

Leishmania infantum is a protozoan causing human zoonotic visceral leishmaniasis (ZVL) and visceral–cutaneous canine leishmaniosis (CanL) in the Mediterranean Basin. L. infantum is able to infect a large number of wild and domestic species, including cats, dogs, and horses. Since the 1990s, clinical cases of equine leishmaniasis (EL), typically characterized by cutaneous forms, have been increasingly diagnosed worldwide. The aim of the present study was to evaluate the presence of clinical forms of EL in CanL-endemic areas in Italy, where exposure of equine populations was ascertained from recent serological surveys. For this purpose, formalin-fixed and paraffin-embedded skin biopsies of 47 horses presenting chronic dermatitis compatible with EL were retrospectively selected for the study and subjected to conventional and q-PCR. A singular positivity for L. infantum was found; BLAST analysis of sequence amplicons revealed a 99–100% homology with L. infantum sequences. The histological examination revealed a nodular lymphoplasmacytic and histiocytic infil-trate; immunohistochemistry showed rare macrophages containing numerous positive amastigotes. The present retrospective study reports, for the first time, a case of a cutaneous lesion by L. infantum occurring in an Italian horse. Pathological and healthy skin samples should be investigated on a larger scale to provide information on the potential clinical impact of EL in the practice, and to define the role of horses in epidemiological ZVL and CanL scenarios