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

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.

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

Gastrointestinal nematodes of dairy goats, anthelmintic resistance and practices of parasite control in Northern Italy

Background: Gastrointestinal nematodes (GINs) is one of the main constraints to ruminant production worldwide. Anthelmintic resistance (AR) has been reported in goats throughout Europe, yet little is known about the AR status in Italy. The aims of the study were: i) determine the frequency of AR in GINs in goat flocks in Northern Italy, Italy, ii) survey goat farmers on the current practices of parasite control, iii) update the species composition of the gastrointestinal helminthofauna. Thirty three flocks were enrolled and 1288 individual fecal samples were collected. Based on the egg per gram (EPG), 15 flocks were selected to evaluate the presence of AR in GINs with the Fecal Egg Count Reduction Test (FECRT). A questionnaire surveyed 110 dairy goat farmers to acquire information about farm management and drenching practices against GINs. Further, the gastrointestinal tracts of 42 goats were analyzed. Results: The FECRs indicated that five of the 15 flocks had problems of AR, which was identified in all two of the anthelmintic classes tested. Resistance and suspected resistance was found in 40% of the flocks selected for AR testing that were treated with benzimidazoles while 20% of the flocks treated with eprinomectin had resistant GINs. Teladorsagia/Trichostrongylus L3 were isolated from the post-treatment coprocultures of all flocks with resistance but not from the flock with suspected oxfendazole resistance. Treatments against helminths were performed once annually in 73.63% of the flocks, but 20.00% of farmers declared not regularly treating their goats every year. Annual treatments usually occurred in autumn or winter at dose rate for sheep. Te. circumcincta, H. contortus, Tr. colubriformis, Skrjabinema caprae and Oesophagostomum venulosum were the most abundant and prevalent species of the gastrointestinal tract. Conclusions: Strategies to prevent the development of AR should be widely adopted in Northern Italy. Further, farmers and practitioners should be educated about the importance of using the correct dose rates in goats. In addition, the presence of highly pathogenic GINs coupled with high worm burden in all sector of gastrointestinal tract and the prevalence values further suggest that improved diagnosis and active surveillance of GIN infection is needed

Coinfection with Tritrichomonas foetus and Giardia duodenalis in Two Cats with Chronic Diarrhea

A Tritrichomonas foetus and Giardia duodenalis mixed infection was diagnosed in two Maine Coon cats aged six months. One of them presented a history of chronic liquid diarrhea and of several unsuccessful treatments. In both cats, G. duodenalis and trichomonads were detected in fecal smears from freshly voided feces; the presence of T. foetus was confirmed by a real-time PCR assay. The cats completely recovered after treatment with ronidazole. In a refrigerated fecal sample collected from the cat with chronic diarrhea, drop-shaped trichomonad pseudocysts smaller than G. duodenalis cysts were detected. They appeared brownish or light-bluish when stained with Lugol\u2019s solution or with Giemsa stain, respectively, and their morphological features were similar to those expressed by bovine T. foetus pseudocysts in vitro. Existence of pseudocysts even in feline trichomonads is noteworthy as they could represent a form of protozoan resistance due to unfavorable conditions whose detection in refrigerated feces can be a useful clue for clinicians

Ixodid ticks on wild donkeys in a Mediterranean nature reserve (Asinara National Park): diversity and risk factors

The Sardinian coloured donkey Equus asinus (Perissodactyla: Equidae) and its albino colour morph represent the wildlife species most typical of the island of Asinara. This Mediterranean island represents a favourable context for ticks and tick-borne diseases; however, knowledge of the tick fauna on Asinara is scarce. A total of 106 Sardinian donkeys were inspected for tick infestation from June to November 2015. All ticks found were collected, classified by stage and sex, and identified to species level. The level of infestation of each donkey was determined; both the overall tick infestation and infestations of each detected species were classified on a scale of 1\u20133 to give an infestation score (IS). Overall, 256 hard ticks were collected from 60 of 106 donkeys (56.6%). Rhipicephalus bursa, Haemaphysalis punctata and Hyalomma marginatum (all: Ixodida: Ixodidae) infested 26.4%, 28.3% and 6.6% of donkeys, respectively. Different variables affected the IS. With reference to overall tick infestation, a higher IS was observed in donkeys grazing on grassland and Mediterranean shrubland and in albino donkeys compared with coloured donkeys. The collected ticks included species involved in the transmission of pathogens to humans, which highlights the risks for public health in a tourist destination such as Asinara National Park

First detection of anti- Besnoitia spp. specific antibodies in horses and donkeys in Italy

Among Apicomplexa protozoa infecting equids, Besnoitia spp., Toxoplasma gondii and Neospora spp. represent important issues from a sanitary and zootechnical viewpoint. However, only scarce epidemiological data are available on the spread of the infections in horses and donkeys in Europe. Therefore, a serosurvey was planned to estimate the prevalence of these Sarcocystidae species in Italian equids. Serum samples from 268 horses and 18 donkeys raised in Italy were collected and serologically analyzed to detect anti-Besnoitia spp., anti-T. gondii and anti-Neospora spp. antibodies: an approach based on an initial screening by in-house ELISA followed by a confirmatory WB was used. Two horses (0.7%) and four donkeys (22.2%), showed antibodies anti-Besnoitia spp. Ten horses (3.7%) resulted positive to T. gondii and one of these (0.4%) was seropositive also to Neospora spp. This is the first detection of anti-Besnoitia spp. specific antibodies in Italian horses and donkeys. The study confirmed the circulation of Besnoitia spp. among equids in Europe. Low prevalence of T. gondii and Neospora spp. in horses raised in Italy was reported. Nevertheless, it is noteworthy to consider that consumption of horse meat could represent a source for human toxoplasmosis

Reliability of symmetric dimethylarginine in dogs with myxomatous mitral valve disease as kidney biomarker

The most common cause of heart failure in the canine population is myxomatous mitral valve disease, sometimes complicated by chronic kidney disease. Many studies have been done on the use of symmetric dimethylarginine as biomarker of renal impairment in dogs affected by chronic kidney disease, few studies have examined his reliability as biomarker in dogs affected by heart diseases. Aim of this study was to evaluate symmetric dimethylarginine in dogs affected by mitral valve disease in order to assess his reliability in heart diseases. This was a retrospective case-control study on a clinical population of dogs affected by mitral valve disease (cases) vs healthy dogs (controls). Both groups underwent a complete physical evaluation, echocardiographic examination, complete blood count, biochemical panel, including serum creatinine and urea and urine analysis with protein-to-creatinine ratio. Serum was frozen and sent to IDEXX laboratories for symmetric dimethylarginine determination. General linear model was applied to data. A total number of 24 cases and 7 controls were included. Symmetric dimethylarginine value was in the reference value in the 75% (n=18) of cases, and in the 43% (n=3) of controls. Once set symmetric dimethylarginine as dependent variable, no statistical significant differences were found for each variable considered (breed, age, sex, weight, class of cardiomyopathy, presence/absence of valvular disease, presence/absence of congestive heart failure, pharmacological therapy, creatinine and urea concentration). Blood concentration of SDMA resulted not influenced by the variables mentioned above, so it could be considered a reliable marker of early renal impairment in dogs affected by mitral valve disease