Besnoitia besnoiti, an apicomplexan parasite causes economically important disease in cattle in many countries of Africa and Asia is re-emerging in Europe. Serological identification of infected cattle is important because introduction of these animals into naive herds seems to play a major role in the transmission of the parasite. We report new, simplified immunoblot-based serological tests for the detection of B. besnoiti-specific antibodies. Antigens were used under non-reducing conditions in the immunoblots, because reduction of the antigen with beta-mercaptoethanol diminished the antigenicity in both, tachyzoites and bradyzoites. Ten B. besnoiti tachyzoite and ten bradyzoite antigens of 15-45 kDa molecular weight were recognized by B. besnoiti infected cattle, but not or only weakly detected by cattle infected with related protozoan parasites, Neospora caninum, Toxoplasma gondii, Sarcocystis cruzi, Sarcocystis hominis, or Sarcocystis hirsuta. The sensitivity and specificity of B. besnoiti immunoblots were determined with sera from 62 German cattle with clinically confirmed besnoitiosis and 404 sera from unexposed German cattle including 214 sera from animals with a N. caninum-specific antibody response. Using a new scoring system, the highest specificity (100%) and sensitivity (90%) of the immunoblots were observed when reactivity to at least four of the ten selected tachyzoite or bradyzoite antigens was considered as positive. When a cut-off based on this scoring system was applied to both the tachyzoite- and the bradyzoite-based immunoblots, there was an almost perfect agreement with the indirect fluorescent antibody test with a titre of 200 as the positive cut-off. We identified and partially characterized 10 tachyzoite and 10 bradyzoite B. besnoiti antigens which may help to develop new specific and sensitive serological tests based on individual antigens and in the identification of possible vaccine candidates.

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BackgroundBovine besnoitiosis is an emerging protozoan disease in cattle. Neither vaccines nor chemotherapeutic drugs are currently available for prevention and treatment of Besnoitia besnoiti infections. Therefore the implementation of appropriate disease management strategies is of utmost importance. The aim of this longitudinal study was to complement current knowledge on the chronology of disease progression. This was realized by correlating clinical findings in early stages of naturally acquired bovine besnoitiosis with results of real-time PCR of skin biopsies and of two western immunoblots and an immunofluorescent antibody test (IFAT).Animals for this study were obtained by i) closely monitoring a cow-calf operation with a high prevalence of bovine besnoitiosis for cases of acute disease, and by ii) conducting a 12-week cohabitation experiment on pasture with five healthy heifers, a healthy bull and five B. besnoiti infected cows. A control group of six healthy heifers was kept at a minimal distance of 20 m. Further, the spectrum of potential insect vectors was determined.ResultsInfected cattle were followed up to a maximum of 221 days after first detection of B. besnoiti antibodies. Two severely affected cows developed visible and palpable alterations of skin, a decrease in body condition despite good feed intake, and chronic bovine besnoitiosis-associated laminitis leading to non-healing sole ulcers. The cows also had high reciprocal IFAT titers and high loads of parasite DNA in skin samples. Two heifers developed a mild clinical course characterized by few parasitic cysts visible in the scleral conjunctivae and vestibula vaginae. Both heifers became infected during the time of high insect activity of the species Musca domestica, Musca autumnalis, Haematobia irritans, and Stomoxys calcitrans. When a third heifer became subclinically infected, low insect activity was recorded. None of the six control heifers contracted a B. besnoiti infection.ConclusionsIn chronic besnoitiosis, the severe clinical course apparently corresponded with high reciprocal IFAT titers and high loads of parasite DNA in skin, whereas mild and subclinical cases displayed lower values. Bovine besnoitiosis-associated laminitis represents an important complication in severe chronic disease which severely impairs animal welfare.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-015-0344-6) contains supplementary material, which is available to authorized users.

BackgroundThe emerging disease bovine besnoitiosis is caused by the apicomplexan parasite Besnoitia besnoiti. Clinical signs of acute besnoitiosis are pyrexia, anorexia and subcutaneous edema. In subacute and chronic besnoitiosis parasitic cysts arise in a variety of tissues and affected cattle display skin lesions and weight loss. In all stages of bovine besnoitiosis, lesions can be found in many organ systems and therefore presumably alter a variety of laboratory parameters. In this study, the impact of naturally acquired acute, subacute and chronic bovine besnoitiosis on hematologic parameters, serum chemistry, and enzyme activities was investigated. Laboratory parameters of two Simmental heifers and two Limousin cows were monitored during acute, subacute and chronic besnoitiosis and in another Simmental heifer during subclinical besnoitiosis. To determine aberrations of laboratory parameters, values were compared with reference ranges obtained from B. besnoiti negative Simmentals (224 samples of nine animals) and Limousins (41 animals). Further, laboratory parameters of B. besnoiti seropositive Limousin cows (54 animals; 32 of these showing clinical signs) and healthy B. besnoiti seronegative Limousin cows (41 animals) were compared.ResultsDuring acute and subacute besnoitiosis, a reduction of leukocyte and erythrocyte concentrations, hematocrit, serum albumin, urea, magnesium, and calcium concentrations were observed. Serum total protein, globulin, total bilirubin and creatinine concentrations were increased and aspartate transaminase (AST) and creatine kinase (CK) activities were elevated. In chronic besnoitiosis, erythrocyte parameters were statistically significantly lower, and total protein and globulin concentrations were significantly higher in B. besnoiti seropositive compared with B. besnoiti seronegative Limousin cows.ConclusionsIn this study, altered laboratory parameters during the course of naturally acquired acute, subacute and chronic bovine besnoitiosis are described for the first time. Only a few animals were examined in acute and subacute besnoitiosis, however the alterations of laboratory parameters during these stages reflected i) the acute inflammatory state (e.g. high levels of serum globulin fractions), ii) clinical findings such as disturbed condition (e.g. bilirubin concentrations), and iii) lesions such as muscle necroses described in the literature (e.g. AST or CK activities). Chronic besnoitiosis led to typical alterations of chronic inflammatory diseases like hyper-(gamma)-globulinemia or reduced erythrocyte concentrations.