Histological findings in experimentally infected male calves with chronic besnoitiosis.

The histological findings associated to Besnoitia besnoiti infection were exhaustively studied in target tissues from experimentally and chronically infected calves. Calves were inoculated with 106 bradyzoites via intravenous, subcutaneous and intradermal route. Visible pathognomonic sclera cysts were observed in all infected animals. Tissue cysts were more abundant and lesions were more frequent in calves inoculated via intradermal. The most parasitized tissues were skin, including scrotum (40.81% of positive samples), nostril and nasal turbinate. Tissue cysts were already fully developed as the average tissue cyst diameter was 181.20 µm. Microscopic lesions were mainly detected in skin samples, followed by reproductive and upper respiratory tracts. Mild lesions compatible with both acute (thrombus, oedema and inflammation) and chronic besnoitiosis (skin lesions, hyperkeratosis and dilated sweat glands) coexisted. Vascular damage and inflammation were more frequently observed in skin (including scrotum) followed by testicular parenchyma, epididymis and pampiniform plexus. Histological findings evidenced a subclinical chronic besnoitiosis.

The route of Besnoitia besnoiti tachyzoites inoculation does not influence the clinical outcome of the infection in calves.

In a previous attempt, an experimental model of bovine besnoitiosis was established in calves that were intravenously inoculated with different doses of Besnoitia besnoiti tachyzoites. Despite the fact that all infected calves developed the acute stage of disease, only microscopic findings characteristic of chronic besnoitiosis were reported. In the present study, calves were inoculated by subcutaneous and intradermal routes with B. besnoiti tachyzoites with the aim of developing clinical signs and macroscopic lesions characteristic of chronic besnoitiosis. Nine 3-month-old male calves were randomly distributed into three groups of three animals each. Next, 106 tachyzoites were inoculated by either the subcutaneous (G1) or intradermal route (G2). The negative control group (G3) was inoculated with PBS. Daily clinical monitoring and regular blood collection were performed. At 70 days post-infection (pi), animals were euthanized, and tissues were collected to investigate lesions and parasites. Infected animals developed mild-moderate acute besnoitiosis characterized by lymphadenopathy from four days to 47 days pi, and sporadic fever peaks were only observed in one calf from G2. However, other clinical signs and macroscopic lesions characteristic of chronic besnoitiosis were not detected. Only nine tissue samples were B. besnoiti-DNA-positive, eight of which belonged to reproductive and respiratory tracts tissues from G1. Finally, the kinetics of the immune responses were similar in both infected groups. However, delayed and lower cellular and humoral immune responses were observed in G1 followed by G2 and were compared with intravenously inoculated calves. The differences observed among the three inoculation routes could be due to different effector mechanisms of the host early innate immune response against B. besnoiti. Accordingly, the inoculation route of B. besnoiti tachyzoites does not significantly influence the clinical outcome of the infection in calves. Thus, a further refinement of this experimental model of bovine besnoitiosis is needed to reproduce macroscopic lesions characteristic of chronic stage disease.

Bovine chronic besnoitiosis in a calf: Characterization of a novel B. besnoiti isolate from an unusual case report.

Bovine besnoitiosis, caused by the apicomplexan Besnoitia besnoiti, is a chronic and debilitating disease characterized by cutaneous and systemic manifestations that primarily affects adult beef cattle. Previous studies have reported that clinical besnoitiosisis is rare in calves. However, we isolated B. besnoiti from a chronically infected calf for the first time. The identity of the Besnoitia species was determined after parasite isolation and molecular genotyping. According to the results obtained in vitro the new isolate, named as Bb-Spain3, was characterized in a reproducible in vitro model and was categorized as a low invader and low prolific isolate with a slower lytic cycle compared to Bb-Spain 1 isolate. Specific traits that differentiate isolates obtained from adult animals from those infecting calves were not found. Next, we described the first case report of chronic besnoitiosis in a female calf less than 6 months-old with a low body condition. The disease was confirmed by the presence of specific anti-B. besnoiti antibodies and parasite detection in the skin. At post-mortem examination, tissue samples were collected for histological, immunohistochemical and molecular analyses. DNA-parasite was detected in 31 different calf's tissues, being the most highly parasitized tissues the skin and the respiratory and reproductive tracts. In addition, the parasite was also present in heart, eyes, lymph nodes and brain. The high parasite load, a wide intra-organic parasite distribution and the presence of both viable and degenerated cysts, were indicative of a rapid progression of the disease. This case report underlines the need to include the inspection of young animals in besnoitiosis control.

A new lyophilized tachyzoite based ELISA to diagnose Besnoitia spp. infection in bovids and wild ruminants improves specificity.

Recent studies have reported that routinely used whole or soluble Besnoitia besnoiti tachyzoite (TZ) extract-based ELISAs potentially give rise to a high number of false-positive results, which may compromise control and the epidemiological studies of bovine besnoitiosis. Thus, western blot (WB) has been recommended as a confirmatory test. In the present study, a new ELISA test that employs lyophilized tachyzoites for the first time (BbSALUVET ELISA 2.0) was developed and validated with cattle sera (n=606) under a worst-case scenario. False positive and false negative, soluble TZ extract-based BbSALUVET ELISA 1.0 reactors were overrepresented, and WB was used as the reference test. One commercial test (PrioCHECK Besnoitia Ab 2.0, which employs whole TZ extract) and a recently developed membrane-enriched ELISA (APure-BbELISA) were also tested. The three ELISAs showed high AUC values (>0.9). However, the best diagnostic performance corresponded to the BbSALUVET ELISA 2.0 and the APure-BbELISA [(92% sensitivity (Se) and 98% specificity (Sp)] followed by PrioCHECK Besnoitia Ab 2.0 (88% Se, 98% Sp, and 4.5% doubtful results). In addition, the BbSALUVET ELISA 2.0 was validated with wild ruminant sera, and excellent performance (96% Se, 97% Sp, and 4% doubtful results) was obtained again. A different antigenic composition of the lyophilized tachyzoites, compared with whole or soluble tachyzoite extracts, may be responsible for the improved diagnostic performance. This study proposes the use of the BbSALUVET ELISA 2.0 in cattle prior to entry to herds free of the disease and in valuable samples prior to a selective culling without the need of a confirmatory Western Blot test in positive samples due to its excellent specificity.