Background: Bovine besnoitiosis, caused by the apicomplexan parasite Besnoitia besnoiti, is a chronic and debilitating cattle disease that notably impairs fertility. Acutely infected bulls may develop respiratory signs and orchitis, and sterility has been reported in chronic infections. However, the pathogenesis of acute disease and its impact on reproductive function remain unknown. Methods: Herein, we studied the microscopic lesions as well as parasite presence and load in the testis (pampiniform plexus, testicular parenchyma and scrotal skin) of seven bulls with an acute B. besnoiti infection. Acute infection was confirmed by serological techniques (IgM seropositive results and IgG seronegative results) and subsequent parasite detection by PCR and histological techniques. Results: The most parasitized tissue was the scrotal skin. Moreover, the presence of tachyzoites, as shown by immunohistochemistry, was associated with vasculitis, and three bulls had already developed juvenile tissue cysts. In all animals, severe endothelial injury was evidenced by marked congestion, thrombosis, necrotizing vasculitis and angiogenesis, among others, in the pampiniform plexus, testicular parenchyma and scrotal skin. Vascular lesions coexisted with lesions characteristic of a chronic infection in the majority of bulls: hyperkeratosis, acanthosis and a marked diffuse fibroplasia in the dermis of the scrotum. An intense inflammatory infiltrate was also observed in the testicular parenchyma accompanied by different degrees of germline atrophy in the seminiferous tubules with the disappearance of various strata of germ cells in four bulls. Conclusions: This study confirmed that severe acute besnoitiosis leads to early sterility that might be permanent, which is supported by the severe lesions observed. Consequently, we hypothesized that testicular degeneration might be a consequence of (i) thermoregulation failure induced by vascular lesions in pampiniform plexus and scrotal skin lesions; (ii) severe vascular wall injury induced by the inflammatory response in the testis; and (iii) blood-testis barrier damage and alteration of spermatogenesis by immunoresponse.

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Bovine besnoitiosis is continuing to spread in Europe. Therefore, the development of ruminant animal models of infection is urgently needed to evaluate therapeutic and prophylactic tools. Herein, we studied the effect of parasite dose and host age on the infection dynamics with Besnoitia besnoiti tachyzoites in cattle in two independent experimental infections. In experiment A, twelve 3-month-old male calves were inoculated intravenously with either three different doses of tachyzoites (G1: 10 ; G2: 10 ; G3: 10 ) or with PBS (G4). In experiment B, six 14-month-old bulls were inoculated with 10 tachyzoites based on results obtained in experiment A. In both trials, clinical signs compatible with acute and chronic besnoitiosis were monitored daily; blood and skin samples were collected regularly for 70-115 days post-infection (pi). Finally, animals were killed, and tissues were collected for lesion and parasite detections. Infected animals developed mild-moderate signs compatible with acute besnoitiosis. Lymphadenopathy and fever were observed in both calves (from 12 hr until 7 days pi) and bulls (from 6 days until 9 days pi). Seroconversion was detected at 16-19 days pi, and antibody levels remained high. Infected animals did not developed characteristic clinical signs and macroscopic lesions of chronic besnoitiosis. However, successfully, parasite-DNA was detected in a reduced number of target tissues: conjunctiva, ocular sclera, epididymis, skin of the scrotum and carpus in calves (n = 10, 6 of which belonged to G3), and pampiniform plexus and testicular parenchyma in bulls. Remarkably, one tissue cyst and mild microscopic lesions were also detected. In summary, inoculated animals developed the acute besnoitiosis and chronic infection was evidenced by microscopic findings. However, our results suggest that tachyzoite dose and host age are not key variables for inducing clinical signs and macroscopic lesions characteristic of chronic besnoitiosis. Thus, a further refinement of this model should evaluate other parasite- and host-dependent variables.

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