EPIDEMIOLOGY AND MOLECULAR CHARACTERIZATION OF SELECTED PROTOZOA IN DOMESTIC RUMINANTS

Apicomplexa parasites are single-celled, obligate intracellular cyst-forming protozoa, infecting humans and animals, that pose major threats to world health and global economy. Among the most relevant species for farm animals, Besnoitia besnoiti, Neospora caninum and Toxoplasma gondii are parasites of medical (T. gondii) and veterinary (T. gondii, N. caninum, B. besnoiti) importance in domestic ruminants. Bovine besnoitiosis, caused by Besnoitia besnoiti, is a chronic and debilitating parasitic disease of cattle, characterized by both cutaneous and systemic manifestations, compromising animal welfare and responsible for economic losses on affected farms. In Europe, including Italy, bovine besnoitiosis is an emerging or re-emerging disease, with an increasing geographical distribution and the number of cases of infection. Neospora caninum, a coccidian protozoan, represents an important cause of bovine abortion. It was suggested that the parasite may have adverse effects on fertility and milk production, but only few and contrasting data are available to date. Besides, while only a single genotype of N. caninum exists worldwide, available parasite strains show considerable variation in vitro and in vivo, including different virulence in cattle. Microsatellite markers allow to fingerprint N. caninum isolates or DNAs and undertake population studies. Toxoplasmosis represents an important public health issue, with the consumption of raw or undercooked meat being a major way of human infection. The role of beef in the transmission of the parasite to humans is questioned due to lower quantity of tissue cysts compared with other meat-producing species. However, the habit of consuming raw beef is regionally diffused, and the risk posed by Toxoplasma gondii infection in cattle should not be overlooked. The aim of my doctoral thesis project was to investigate on the epidemiology and molecular characterization of selected protozoa parasites of medical and veterinary importance in domestic ruminants, i.e., B. besnoiti, N. caninum and T. gondii in cattle. A multidisciplinary approach based on clinical features, laboratory tests including serological and molecular techniques, was applied throughout the research project, to achieve a multi-level comprehension of the epidemiology of these parasite infections. Three main research lines were developed: Research Line 1: Exploring bovine besnoitiosis: a multi method approach. A case of bovine besnoitiosis in a dairy farm housing 217 cattle in Italy was reported. A serological screening was performed on the whole herd using the recommended approach of ELISA and confirmatory Western Blot. Seropositive animals were clinically examined to reveal symptoms and lesions of besnoitiosis. Risk factors and the effects of the parasite infection on reproductive and productive performances were evaluated. Histopathology and molecular analyses on tissues from a slaughtered cow affected by the chronic phase of the disease were carried out. An overall seroprevalence of 23.5%, which increased up to 43.5% considering only cows, was recorded. Clinical examination of 33 of the seropositive cows evidenced the presence of tissue cysts in at least one of the typical localizations (sclera, vulva, or skin) in 25 animals. Statistical analysis did not evidence any significative impact of the parasite infection on herd efficiency; however, a decrease of productive parameters was recorded in cows showing cutaneous cysts. Concerning the chronically affected cow, histopathology revealed B. besnoiti tissue cysts in the skin of the neck, rump, hind legs, eyelid and vulva, in the muzzle, in mucosal membranes of the upper respiratory tract, and in the lungs. Parasite DNA was detected also in masseter muscles, tonsils, mediastinal lymph nodes, liver, cardiac muscle, aorta wall, ovaries, uterus, and vulva. Moreover, alterations of laboratory parameters, i.e., hematological and biochemical parameters, enzyme activities and serum cortisol levels in Besnoitia besnoiti naturally infected cows were deeply investigated. Laboratory parameters of 107 cows, 61 seronegative and 46 seropositive to B. besnoiti, including 27 with clinical signs of bovine besnoitiosis, were compared. Generalized Linear Models were used to evaluate the effect of Besnoitia infection on the considered laboratory parameters. Hematological analyses revealed that B. besnoiti infection determined a significant alteration of the leukocyte differential with a higher percentage of neutrophil granulocytes and a lower percentage of lymphocytes in seropositive animals; Erythrocyte and Platelet counts did not show any difference between the considered groups of cows. Biochemistry evidenced that the parasite infection influenced serum protein values in seropositive animals and GLDH in clinically affected cows. No or only slight differences were revealed for all the other biochemical and enzyme activity parameters in B. besnoiti infected animals. Seropositive and clinically affected cows evidenced mild higher concentrations of serum cortisol values if compared to seronegative animals, indicating that bovine besnoitiosis could be related to stress in infected animals and that the parasite could compromise animal welfare and also influence disease onset and progression. Furthermore, a form of generalized demodectic mange in two dairy cows infected with Besnoitia besnoiti was described. Two out of the cows seropositive to B. besnoiti, at clinical examination presented skin nodules, widespread all over the body, and in particular in anterior regions. Skin biopsies from the region of the neck were collected and the nodules were microscopically examined through compression method. B. besnoiti tissue cysts were not revealed but a semi-solid yellowish content was evidenced with the presence of several mites, morphologically identified as Demodex bovis. Histological examination of skin biopsies evidenced slight acanthosis and hyperkeratosis of the epidermis and superficial dermatitis with oedema and macrophagic and eosinophilic infiltration. Cystic formations located in the deep dermis were lined by metaplastic squamous epithelium and severe cellular infiltration. A treatment with eprinomectin was attempted and clinical improvement of both cows was observed, particularly at the fifteenth day after treatment, with nodules reduced in size and mites in there degenerated. Moreover, an additional sub research line from this one was implemented to investigate on these Apicomplexa protozoa, particularly Besnoitia spp. infection, in Italian equids: Research Line 1 bis: Exposure of Italian equids to selected protozoa infections and investigation on clinical besnoitiosis in donkeys. A serosurvey was planned to estimate the prevalence of Sarcocystidae species (Besnoitia spp., Toxoplasma gondii and Neospora spp.) 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 Western Blot 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. Low prevalence of T. gondii and Neospora spp. in horses raised in Italy was reported. A case of clinical besnoitiosis in two donkeys was reported. Two donkeys, one male and one female, reared in northern Italy, showed suspected skin lesions and poor body condition. The animals were clinically examined, and endoscopy of upper respiratory tract and of the vagina for the female donkey was performed. Blood samples and skin biopsies were collected. Serum samples were analyzed by Western Blot to detect anti-Besnoitia spp. antibodies; a PCR targeting ITS-1 region and sequencing were carried out on DNA extracted from skin biopsies. Moreover, blood samples were examined for hematology, biochemistry, and enzyme activity. Clinical examination revealed numerous scleral pearls in the eyes of both animals; besides, alopecia and hyperkeratosis with skin nodules in the region of the neck, hind leg and on the pinnae were detected. No cysts were evidenced in the nares, in the upper respiratory tract and in the vagina and vulva in the female animal. Both animals resulted seropositive according to Western Blot results. Skin biopsies collected from the donkeys resulted positive for the presence of parasitic DNA. Sequencing demonstrated a homology of 100% with Besnoitia spp. sequences deposited in GenBank. Hematology evidenced light anemia, leukocytosis with eosinophilia, and lymphocytosis, whereas biochemistry and enzyme activity revealed hypoalbuminemia with decreased albumin/globulin ratio and elevated alkaline phosphatase values. This first clinical case of besnoitiosis in donkeys in Italy confirmed the circulation of Besnoitia spp. in Italian and European equids. Research Line 2: Genetic characterization of Neospora caninum isolates in cattle and impact of neosporosis on herd performances. With the aim of evaluating the spread of neosporosis and its effects on herd efficiency, an epidemiological study was designed in two dairy farms recruited as case-study. Both selected farms, located in Lombardy region, performed genetic improvement of Holstein Friesian, and reported cases of abortions ascribable to N. caninum. Blood samples were collected from 540 animals, including cows and heifers above 24 months, and analyzed by indirect immunofluorescent antibody test. Epidemiological data (individual, reproductive and productive data) were noted. Overall, 94 animals (17.4%) resulted positive to N. caninum (15.5% and 18.5% in Farm 1 and Farm 2), with differences between the farms concerning the antibody titres (Chi-square, p-value = 0.04), particularly in cows (Chi-square, p-value = 0.018). Regarding the episodes of abortions, a different pattern was depicted in the two investigated farms. Data on fertility and production were considered. The number of insemination necessary to get an animal pregnant resulted higher in seropositive animals (2.4 and 2.9) than in seronegative animals (2.1 and 2.4 in Farm 1 and 2, respectively). Similarly, particularly in Farm 1, the number of days in lactation of not-pregnant cows resulted higher in seropositive (167.7) than seronegative animals (133.4). Moreover, although the association between N. caninum infection and milk production is still unclear, both the daily production and the mature equivalent milk yield were lower in seropositive (31.02 and 11838.94) than seronegative cows (33.59 and 12274.88) in Farms 1. To genotype N. caninum in aborted bovine foetuses from Lombardy, the proportion of N. caninum PCR-positive aborted foetuses in this area was determined and the available isolates were characterized by multi-locus microsatellite genotyping. Aborted bovine foetuses were collected between 2015 and early 2019 from Italian Holstein Friesian dairy herds suffering from reproductive problems. A total of 198 samples were collected from 165 intensive farms located in Lombardy, northern Italy. N. caninum positive samples were then subjected to multilocus-microsatellite genotyping (MLMG) using ten previously established microsatellite markers. In addition to own data, those from a recent study providing data on five markers were included and analyzed. 55 aborted foetuses were positive for N. caninum by RT-PCR, yielding a prevalence of 27.8%; 43 farms recorded at least one positive fetus (26.1%). Of the 55 samples finally subjected to MLMG, 35 were typed at all or 9 out of 10 loci. Linear regression revealed a statistically significant association between the spatial distance of the sampling sites with the genetic distance of N. caninum MLMGs (P< 0.001). Including data from a previous north Italian study (eBURST analysis) revealed that part of N. caninum MLMGs from northern Italy separate into four groups; most of the samples from Lombardy clustered in one of these groups. Principle component analysis revealed similar clusters and confirmed MLMG groups identified by eBURST. These findings confirm the concept of local N. caninum subpopulations. The geographic distance of sampling was associated with the genetic distance as determined by MLMGs. Research Line 3: Evaluation of Toxoplasma gondii infection in beef cattle raised in Italy. To update information on T. gondii in cattle reared in Italy, a multicentric seroepidemiological survey was designed and implemented in four northern regions (Liguria, Lombardy, Piedmont, and Trentino Alto Adige) and Sardinia. A convenience sampling was performed, collecting 1444 serum samples from 57 beef cattle herds. Thirteen beef breeds were sampled, besides crossbreed; bovines age varied from 3 months to over 12 years. Sera were tested with a commercial ELISA for the detection of anti-T. gondii antibodies. Individual and herd data were analyzed by binary logistic regression analysis. A T. gondii seroprevalence of 10.2% was recorded, with differences among regions and values ranging from 5.3% in Liguria to 18.6% in the Piedmont region (p value = 0.0001). Both young and adult animals and males and females tested positive, without any significant difference (age and gender: p value >0.05). Lower seroprevalence values were recorded in cattle born in Italy (8.7%) if compared with animals imported from abroad (13.4%) (p value = 0.046). To obtain epidemiological and molecular data on T. gondii infection in cattle slaughtered in Italy, 80 animals were sampled from one of the biggest Italian slaughterhouses. Dairy (Holstein Friesian) or dual purpose (Alpine Brown and Pezzata Rossa Italiana) breeds or crossbreeds from 15 farms located in northern Italy were sampled; age of animals varied from six months to three years. Approximately 50 g of diaphragm was collected to obtain meat juice and muscle homogenate samples. Individual data were noted. Meat juice samples were analyzed with a commercial ELISA to detect anti-T. gondii antibodies. DNA extracted from muscle homogenate samples were subjected to B1 real-time PCR. Anti-T. gondii antibodies were found in 10 (12.5%) out of 80 examined animals, whereas parasitic DNA was detected in 26 diaphragm muscle samples (32.5%). Only seven samples scored positive in both test: a fair agreement between ELISA and B1 real-time PCR results was achieved (κ value = 0.254). Nevertheless, higher ELISA S/P% values were recorded in diaphragm samples scoring positive to PCR. Higher number of positive samples were found in younger than older animals considering both ELISA and B1 real-time PCR results. Similarly, considering the provenience, animals that have been acquired from other holdings scored more frequently positive to both ELISA and B1 real-time PCR compared to animals that have never left the holding of origin until slaughter. Statistical analysis showed an effect of ELISA S/P% values on B1 real-time PCR results, increasing the risk of parasitic DNA detection when increasing the S/P% values. The other considered variables (age and provenience) did not show any effect on neither ELISA nor B1 real-time PCR. In conclusion, obtained results from the studies of my PhD project allowed to update information on protozoa of medical and veterinary importance both in domestic ruminants and in equids. It was demonstrated that bovine besnoitiosis continues to spread in Italy: both clinical and laboratory tests are needed for an accurate diagnosis, and thus to implement plans for the control of the disease. Moreover, Besnoitia spp. infection circulates in Italian equids and besnoitiosis may be considered an emerging disease of donkeys in Italy and also in Europe. Serological screening of cows and molecular analysis of aborted foetuses confirmed the role of N. caninum in abortion and reproductive failure in dairy herds in northern Italy; besides, multilocus microsatellite genotyping confirmed the concept of local N. caninum subpopulations. The zoonotic importance of T. gondii should not be underestimated in animal species destined to human consumption, including cattle and horses. Serological data are useful to give an indication of the population exposure to the parasite, whereas molecular methods allow to detect tissue cysts in the edible parts reflecting the risk for human infection.