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Besnoitia besnoiti is an obligate intracellular parasite that is transmitted by direct contact or via mechanical transmission by flies as vectors. Besnoitiosis causes economic losses in the cattle industry and is regarded as a re-emerging disease in Europe. This study evaluated the seroprevalence of B. besnoiti in Korean cattle using a commercial ELISA kit. Among 558 serum samples, 19 (3.4%) tested seropositive for B. besnoiti. The statistically significant risk factors included age (≥ 2 years), sex (castrated males), and region (lower latitudes) (P < 0.05). The overall seroprevalence suggested a wide distribution of B. besnoiti infection in cattle reared in Korea. Thus, the practice of intensive cattle husbandry and the regionally different seroprevalence of B. besnoiti infection in cattle in Korea warrant routine monitoring and vector control to reduce economical losses due to bovine besnoitiosis in the country.

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.

Besnoitia darlingi, B. neotomofelis and B. oryctofelisi are closely related coccidian parasites with cats as definitive hosts. While B. darlingi uses opossums as intermediate hosts, B. neotomofelis and B. oryctofelisi have been described in Southern Plains woodrats (Neotoma micropus) from the USA and in domestic rabbits from Argentina, respectively. A comparison of the Internal Transcribed Spacer-1 (ITS-1) region of the ribosomal DNA (rDNA) of these Besnoitia spp. showed only a few differences. The present study aimed at developing a real-time PCR to detect B. darlingi, B. neotomofelis and B. oryctofelisi in tissues of intermediate and in faeces of definitive hosts in order to support studies of these organisms’ epidemiology and pathogenesis. The established PCR was based on primer regions distinct from the ITS-1 sequences of ungulate Besnoitia spp. and made use of a Besnoitia universal probe. To monitor inhibition, a heterologous internal control was established based on the enhanced green fluorescent protein gene. The real-time PCR reacted with B. darlingi, B. neotomofelis and B. oryctofelisi, while the novel PCR did not recognize ungulate Besnoitia spp. (B. besnoiti, B. bennetti, B. tarandi). DNA of Apicomplexa ascribed to other Besnoitia-related genera, including other gut parasites of cats (Cryptosporidium parvum, Giardia duodenalis, Tritrichomonas foetus), was not recognized. The real-time PCR had an analytic sensitivity of less than 1 tachyzoite per reaction. In feline faeces spiked with B. darlingi oocysts, the limit of detection was a DNA amount equivalent to 1 oocyst per PCR reaction. In B. darlingi infected ɣ-interferon knock-out mice, the lung was identified as the predilection organ. In conclusion, this real-time PCR should advance further studies on these parasites and may inspire research on related species, not only in the Americas, but also in other parts of the world., Graphical abstract Image 1, Highlights • A quantitative real-time PCR was established to detect Besnoitia darlingi. • Analytical sensitivity of the real-time PCR for tachyzoites and oocysts was high. • Lung tissue was the predilection site for B. darlingi in ɣ-interferon knockout mice. • Related parasites like B. neotomofelis and B. oryctofelisi are detected by this PCR, too. • PCR holds promise to assess the epidemiology of B. darlingi and related species.

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.

Bulls chronically affected by bovine besnoitiosis can suffer from sterility. There is limited information about the distribution of Besnoitia cysts and their associated lesions within the male genital organs. This work describes the gross and histological abnormalities in the genital organs of 6 bulls chronically infected with Besnoitia besnoiti, including both clinically (n = 4) and subclinically (n = 2) affected cases. Parasitic cysts were observed in the genital organs of all the clinically affected bulls. The tissue cysts were most commonly found within the pampiniform plexus (4/4), where they were often seen within venous vascular walls and associated with vasculitis, followed by epididymis (3/4), tunica albuginea (2/4), and penis (1/4). In decreasing order of their frequency, observed abnormalities included seminiferous tubule degeneration, testicular fibrosis, testicular necrosis, lack of/or diminished numbers of spermatozoa, testicular atrophy, and Leydig cell hyperplasia. Only one of the subclinically infected bulls had few Besnoitia cysts within the pampinoform plexus, which was associated to small areas of necrosis and mineralization in the ipsilateral testicle. Results indicate that Besnoitia cysts and genital abnormalities are frequent in bulls chronically affected by bovine besnoitiosis, while they are mild and scarce in subclinically affected ones. Moreover, present data show that Besnotia-associated testicular lesions can occur without the presence of cysts within the testicular parenchyma. B. besnoiti cysts seem to have a tropism for the vascular structures of the spermatic chord, which may cause testicular abnormalities via vascular damage, reduced blood flow, and/or impaired thermoregulation and subsequently lead to the observed testicular lesions. info:eu-repo/semantics/acceptedVersion

Serological cross-reactions represent a serious problem in some currently available tests to diagnose Besnoitia infections in many species including cattle, caribou and donkeys. False-positive results are due to the low positive-predictive value of these serological tests for besnoitiosis. These tests therefore have clear limitations if large herds are screened in areas with low prevalence, since increased numbers of false-positive reactions require confirmatory testing by alternative serological methods, e.g. immunoblotting, which are time-consuming and create extra costs. To overcome this problem, we aimed to develop a highly sensitive and specific competitive ELISA (cELISA) using a panel of 12 monoclonal antibodies raised against the tachyzoite stage of Besnoitia besnoiti. A cELISA set up with one of these antibodies (Bb-cELISA1) was screened with a large panel of B. besnoiti-positive bovine sera to estimate the diagnostic sensitivity of the test. Sera from herds with Neospora caninum- or Sarcocystis spp.-infected cattle were used to estimate its diagnostic specificity. Relative to a reference standard, which combined the results obtained in a previously established highly sensitive and specific ELISA, in the immunofluorescence antibody test and in B. besnoiti tachyzoite and bradyzoite immunoblots, the new Bb-cELISA1 revealed a diagnostic sensitivity of 99.2% (95% confidence interval: 97.1-99.9%) and a diagnostic specificity of 99.9% (95% confidence interval: 97.7-100%). This novel assay was tested on a variety of proven Besnoitia-positive sera from other species, including B. besnoiti-infected cats, rabbits or Besnoitia bennetti-infected donkeys or Besnoitia tarandi-infected caribou. The results obtained with the new Besnoitia-cELISA for these animal species also corresponded almost perfectly with those of the reference tests, which included immunoblot and immunofluorescence antibody tests. In conclusion, the novel Besnoitia-cELISA represents a valuable tool for the diagnosis and control of bovine besnoitiosis and for studies on the epidemiology of Besnoitia infections in a variety of host species, including naturally exposed wildlife and experimental hosts.

A form of generalized demodectic mange in two dairy cows infected with Besnoitia besnoiti is described. The herd was endemically infected with bovine besnoitiosis; an overall seroprevalence of B. besnoiti antibodies of 23.5%, that increased up to 43.5% considering only cows, was reported. 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. This is the first report of the co-infection of D. bovis infestation and bovine besnoitiosis in cattle. Furthermore, it was demonstrated that D. bovis circulates in the Italian cattle population, but subclinical forms could be underdiagnosed.

Cattle besnoitiosis caused by Besnoitia besnoiti (Eucoccidiorida: Sarcocystidae) is a re‐emerging disease in Europe. Its mechanical transmission by biting flies has not been investigated since the 1960s. The aim of this study was to re‐examine the ability of Stomoxys calcitrans (Diptera: Muscidae) to transmit virulent B. besnoiti bradyzoites from chronically infected cows to susceptible rabbits. Three batches of 300 stable flies were allowed to take an interrupted bloodmeal on chronically infected cows, followed by an immediate bloodmeal on three rabbits (Group B). A control group of rabbits and a group exposed to the bites of non‐infected S. calcitrans were included in the study. Blood quantitative polymerase chain reaction (qPCR) analyses, and clinical, serological and haematological surveys were performed in the three groups over 152 days until the rabbits were killed. Quantitative PCR analyses and histological examinations were performed in 24 tissue samples per rabbit. Only one rabbit in Group B exhibited clinical signs of the acute phase of besnoitiosis (hyperthermia, weight loss, regenerative anaemia and transient positive qPCR in blood) and was seroconverted. Parasite DNA was detected in four tissue samples from this rabbit, but no cysts were observed on histological examination. These findings indicate that S. calcitrans may act as a mechanical vector of B. besnoiti more efficiently than was previously considered., This is the first reproduction of the mechanical transmission of Besnoitia besnoiti by stable flies in a heterologous model.A batch of 300 stable flies was sufficient to transmit virulent B. besnoiti bradyzoites from chronically infected cattle to rabbit.Transmission was effective: one infected rabbit exhibited clinical signs of acute besnoitiosis and seroconversion. MVE-12356-toc-0001-c

Equine neosporosis is regarded to be caused either by Neospora hughesi or Neospora caninum and equine besnoitiosis is caused by Besnoitia bennetti, both of which are apicomplexan parasites. N. caninum is the only known Neospora species in Europe, where equine N. caninum infections have been reported as being associated to abortion and reproductive failure. N. hughesi is prevalent in North America and was predominantly linked to neurological disorders. B. bennetti is considered an emergent disease in donkeys in North America and evidence for B. bennetti infection was recently reported in Europe. Though N. caninum and Besnoitia besnoiti are prevalent in cattle in Portugal, little is known about neosporosis in horses and, to the best of our knowledge, no information was hitherto available for Besnoitia spp. The aim of this study was thus to carry out a serological survey to determine the seroprevalence of these parasites in naturally exposed horses in Portugal. A total of 385 animals were screened by the Indirect Fluorescent Antibody Test at the cut-off value 1:50 and positive results were confirmed by Western blot. Exposure to Neospora spp. and Besnoitia spp. was confirmed in 9.1% (95% Confidence Interval [CI]: 6.6–12.4%) and 0.3% (95% CI: 0.0–1.5%) of horses, respectively. Considering the putative economic and animal health impact of neosporosis in horses and the consequences of a possible spread of equine besnoitiosis in Europe and elsewhere, more comprehensive studies are needed to characterize the species detected in serological surveys, evaluate the geographical distribution and assess possible risk factors that could favor transmission.

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.

International audience; Stomoxys calcitrans (Diptera: Muscidae), is an important vector of lumpy skin disease and bovine besnoitiosis in Europe. Control of this biting fly could represent a keystone in the containment of this emerging disease. Reports of insecticide resistance in S. calcitrans are scarce in Europe. Therefore, the purpose of this study was to evaluate the phenotypic susceptibility to deltamethrin, cypermethrin and phoxim of five wild S. calcitrans populations from southwestern France, where transmission of bovine besnoitiosis is very prevalent. Adult S. calcitrans were caught at each study site and exposed to insecticide-impregnated filter papers under laboratory conditions. Quantities of active ingredients on filter papers corresponded to the recommended doses proposed by the manufacturers (37.5 mg a.i./m2 of cattle's skin, 125 mg a.i./m2 and 750 mg a.i./m2 for deltamethrin, cypermethrin and phoxim respectively) were tested. Knock-down effects (KD) (1 h after the onset of exposure) and mortality rates (24 h and 48 h after exposure) were evaluated. Phoxim showed a rapid and full efficacy in all populations. However, the KD effects (37.5 to 97.5%) and the mortality rates at 48 h (10 to 91.25%) induced by the exposure to pyrethroids varied greatly according to the study site but none of the populations showed full susceptibility. Therefore, the current recommended doses of these pyrethroids are probably less efficient than expected in the field and should be considered with caution in the control of bovine besnoitiosis in France.

Summary The dynamics of bovine besnoitiosis were studied in an area where the disease is endemic. A four-year longitudinal study was conducted for the first time in three infected beef cattle herds located in the Urbasa-Andia Mountains (Navarra, Spain). Each herd was visited four to seven times, and clinical and serological prevalence rates and incidence rates were estimated. Clinical inspections to identify compatible clinical signs with the disease stages were conducted at the beginning and end of the study. Serological assessment was initially performed by ELISA. Seronegative animals with clinical signs and seropositive animals with relative index per cent (RIPC) values lower than 30 that did not increase during the study period were analysed by Western blot to optimize the sensitivity and specificity of the ELISA test. Clinical prevalence rates were slightly higher (62% on average) than the seroprevalence rates (50% on average), and tissue cysts located in the vestibulum vaginae and sclera were the most frequently detected clinical signs. The proportion of seropositive animals with clinical signs varied from 16.7% to 73.6% among the herds, and 17% of cattle with clinical signs proved to be seronegative by both serological tests. An average 22% serological incidence rate was also reported in addition to clinical incidence rates that varied from 12.5% to 16.7%. Additionally, parasitemia was investigated in the herd that showed the highest clinical and seroprevalence rates. Only one PCR positive blood sample was detected. Thus, the role that blood may play in parasite transmission needs to be further investigated. Infected herds maintained both high prevalence and incidence rates in the absence of control measures and a high number of parasite carriers. Finally, economic impact studies on reproductive and productive losses associated with besnoitiosis need to be performed to implement a cost–benefit control programme.

The parasitic diseases discussed elsewhere in this issue of the Scientific and Technical Review are not the only ones to make use of biological vectors (such as mosquitoes or ticks) or mechanical vectors (such as horse flies or Stomoxys flies). The authors discuss two major groups of vector-borne parasitic diseases: firstly, helminthiasis, along with animal filariasis and onchocerciasis, which are parasitic diseases that often take a heavy toll on artiodactyls throughout the world; secondly, parasitic diseases caused by vector-borne protists, foremost of which is bovine besnoitiosis (or anasarca of cattle), which has recently spread through Europe by a dual mode of transmission (direct and by vector). Other protists, such as Plasmodium and Hepatozoon, are also described briefly.

International audience; Cattle besnoitiosis due to Besnoitia besnoiti is spreading across Europe and is responsible for severe economic losses in newly infected herds. Experimentally speaking, rabbits have been found to be susceptible to this parasite. The adaptation of B. besnoiti to rabbits may offer a new, easier and cheaper model of investigation for this disease. This study compared the virulence between tachyzoites and bradyzoites of B. besnoiti in rabbits. Eighteen New Zealand rabbits were allocated into three groups of six animals each. The rabbits from the control (group C), "tachyzoite" (group T) and "bradyzoite" (group B) groups were subcutaneously injected in the right flank with 66 mu g of ovalbumin, 6.10(6) tachyzoites (125th passage on Vero cells) and 6.10(6) bradyzoites (collected from a natural infected cow) of B. besnoiti, respectively. Clinical follow-up and blood sampling for serological survey and qPCR were performed during 10 weeks until euthanasia. Molecular and immunohistochemistry examination was achieved on 25 samples of tissue per rabbit. Seroconversion occurred in group T without any clinical signs. Rabbits of group B exhibited a febrile condition (temperature above 40 A degrees C from day 8 to day 11 following injection) with positive qPCR in blood. Cysts of B. besnoiti were found on skin samples and organs of rabbits from group B in tissue explored with threshold cycle (Ct) values below 30. These results suggest a higher virulence of bradyzoites in rabbits than Vero cell-cultivated tachyzoites. The proposed model could be used to assess the in vivo effectiveness of vaccine or drugs against cattle besnoitiosis.

An-eighteen months (one and half years) old heifer was presented with a progressive loss of weight leading to cachexia and was attended to by a team of doctors from the ambulatory clinic of the School of Animal Science and Veterinary Medicine, University of Rwanda. Clinical examination revealed scleroderma of both sides of the neck and pinpoint cysts in the sclera and conjunctiva as well as corneal opacity of the left eye. Histological examination of skin biopsy showed a mononuclear dermatitis with several cysts of 100–300 μm filled up with bradyzoites in the subcutis and dermis. These lesions are consistent with bovine besnoitiosis, a disease caused by Besnoitia besnoiti in cattle. To the best of our knowledge, this is the first confirmed case of bovine besnoitiosis in Rwanda.

Background Besnoitiosis is caused by different species of intracellular protozoan parasites belonging to the family Sarcocystidae and affecting multiple host species worldwide. Including B. besnoiti, ten species are described infecting animals. Among ungulates, Besnoitia bennetti infects horses, donkeys and zebras and was described in Africa and in the USA where donkey besnoitiosis is considered as an emerging disease. Case presentation A two-year-old male donkey was purchased in May 2016 in poor body condition (cachexia, alopetic areas and pruritus mainly on neck and head) by the present owner in Le Roeulx (Belgium) from a milk producing donkey farm in Frasnes-lez-Buissenal (Belgium). Shortly after its purchase and shearing, the donkey presented with crusts, hyperkeratosis (both flanks and neck) anorexia and cachexia. A treatment with phoxim was given with no improvement. A cutaneous biopsy of hyperkeratotic skin was performed in July. It showed a perivascular eosinophilic infiltrate with a large thick walled cyst located in the dermis containing numerous bradyzoites. This was highly suggestive of besnoitiosis. Several skin biopsy samples were obtained for qPCR analysis and confirmed the presence of Besnoitia spp. DNA. Further laboratory diagnosis tests were performed (western blot and rDNA sequencing) confirming Besnoitia bennetti aetiology for the male. For the female, the punch-biopsy, haematology and qPCR were negatives but the western blot showed the presence of antibodies directed to Besnoitia spp. Further clinical examination performed in August highlighted scleral pinhead sized cysts (pearl) in the right eye and between nares. Another ten-year-old female donkey purchased in France and sharing the same accommodation showed a good clinical condition, but a thorough clinical examination showed the presence of numerous cysts on the inner face of upper labial mucosa. A daily treatment based on sulfamethaxzole and trimethoprim (Emdotrim 60% Mix®, 30 mg/kg) was given orally and some improvement was noticed. Conclusion This is the first evidence of Besnoitia bennetti infection (Protozoa: Apicomplexa) in donkeys (Equus asinus) in Belgium. Electronic supplementary material The online version of this article (10.1186/s13071-018-2993-3) contains supplementary material, which is available to authorized users.

12 páginas, 3 figuras, 3 tablas.-- Diezma-Díaz, C. et. al., Bovine besnoitiosis is continuing to spread in Europe. Therefore, the development ofruminant animal models of infection is urgently needed to evaluate therapeutic andprophylactic tools. Herein, we studied the effect of parasite dose and host age on theinfection dynamics withBesnoitia besnoititachyzoites in cattle in two independentexperimental infections. In experiment A, twelve 3‐month‐old male calves were inocu-lated intravenously with either three different doses of tachyzoites (G1: 108; G2: 107;G3: 106) or with PBS (G4). In experiment B, six 14‐month‐old bulls were inoculatedwith 106tachyzoites based on results obtained in experiment A. In both trials, clinicalsigns compatible with acute and chronic besnoitiosis were monitored daily; blood andskin samples were collected regularly for 70–115 days post‐infection (pi). Finally, ani-mals were killed, and tissues were collected for lesion and parasite detections. Infectedanimals developed mild–moderate signs compatible with acute besnoitiosis. Lym-phadenopathy and fever were observed in both calves (from 12 hr until 7 days pi) andbulls (from 6 days until 9 days pi). Seroconversion was detected at 16–19 days pi, andantibody levels remained high. Infected animals did not developed characteristic clini-cal signs and macroscopic lesions of chronic besnoitiosis. However, successfully, para-site‐DNA was detected in a reduced number of target tissues: conjunctiva, ocularsclera, epididymis, skin of the scrotum and carpus in calves (n= 10, 6 of whichbelonged to G3), and pampiniform plexus and testicular parenchyma in bulls. Remark-ably, one tissue cyst and mild microscopic lesions were also detected. In summary,inoculated animals developed the acute besnoitiosis and chronic infection was evi-denced by microscopic findings. However, our results suggest that tachyzoite doseand host age are not key variables for inducing clinical signs and macroscopic lesionscharacteristic of chronic besnoitiosis. Thus, a further refinement of this model shouldevaluate other parasite‐and host‐dependent variables., This study was financed by the Spanish Ministry of Economy and Competitiveness (AGL2013‐46442‐R), CYTED (Thematic Network113RT0469 Protozoovac) and by the Community of Madrid (PLATESA S20137ABI‐2906). Carlos Diezma‐Díaz was financially sup-ported through a grant from the Spanish Ministry of Economy and Competitiveness (BES‐2014‐069839), and Alejandro Jiménez‐Meléndez was supported through a grant from the Spanish Ministry of Education, Culture and Sports (grant no. FPU 13/05481). We wish to acknowledge all members from the SALUVET group as well as, residents and students from the Department of Medicine and Surgeryof ruminants of the Faculty of Veterinary Sciences (ComplutenseUniversity of Madrid) for their excellent collaboration.

The apicomplexan parasite Besnoitia besnoiti is the causative agent of bovine besnoitiosis that affects livestock, particularly cattle. The definitive host of B. besnoiti is unknown and its transmission only partially understood. Here, we report the first draft genome sequence, assembly, and annotation of this parasite.

The recent spread of bovine besnoitiosis warrants further epidemiological investigations to improve the knowledge on disease development. Thus, a 4-year longitudinal open cohort study was conducted in the first German cattle herd naturally infected with Besnoitia besnoiti. At seven herd-visits between 2008 and 2012, fourteen breeding bulls (1.5 years) and 131 females (1 year) were examined clinically and serologically. In females, clinical and serological prevalences, incidence and remission rates were determined. In addition, the association of age, antibody levels and number of visible parasitic cysts with clinical and serological outcome was investigated. The seroprevalence (89.4%-100%) and serological incidence rate (140.5 per 100 animal-years) were considerably higher than the clinical prevalence (23.5%-36.6%) and clinical incidence rate (16.7 per 100 animal-years). Of 33 new clinical and 12 new serological cases, only 6.7% (3/45) attracted attention with clinical signs of acute bovine besnoitiosis. The apparent serological remission rate (1.9 per 100 animal-years) was considerably lower than the clinical remission rate (37.3 per 100 animal-years). A median cyst score of1 and mean immunofluorescent antibody test (IFAT) titre of ≤1,600 over the entire observation period was significantly associated with a negative clinical outcome at the end. Overall cyst score was not significantly associated with serological outcome and age had no significant influence on clinical and serological outcome. Within 4 years, there was a significant reduction in cyst scores and IFAT titres in the same animals, leading to eight clinically and serologically negative animals in the end. Two initially negative animals achieved clinical and apparent serological remission in about 2.5 years. In bulls, the time between herd entry and seroconversion was 7-30 months and the serological incidence rate was nearly identical to the rate in females (142.0 per 100 animal-years). This shows that a high B. besnoiti prevalence leads to infection of bulls within a short time period.

Bovine besnoitiosis, caused by Besnoitia besnoiti, is an economically important disease of cattle in many countries but its transmission remains a mystery. Wild felids are suspected to be its definitive hosts. The domestic cat (Felis catus) is known experimental definitive host for Besnoitia species of rodents. Here, we report for Besnoitia darlingi the first identification of a natural definitive host, the bobcat (Lynx rufus). Oocysts resembling Toxoplasma gondii (unsporulated; 10.9 ± 0.8 × 12.1 ± 0.2 μm; n = 5) were detected microscopically in the feces of two of 25 free ranging wild bobcats from Mississippi, USA. After detailed investigation, we identified these oocysts as B. darlingi and not T. gondii. The IFN-γ gene knockout (KO) mice fed oocysts from bobcats died of acute besnoitiosis and tachyzoites were found in their tissues. Oocysts were also mildly pathogenic to outbred Swiss Webster mice (SW) (Mus musculus). The SW mice fed oocysts became ill but generally survived and developed characteristic thick-walled Besnoitia tissue cysts in their tongue and heart muscles and brains. Two laboratory-raised domestic cats (Felis catus) excreted B. darlingi oocysts after ingesting murine tissues infected with bobcat-derived oocysts. The parasite was successfully cultivated in African green monkey kidney fibroblast cells (CV-1 cell line) seeded with infected murine tissue homogenate. The multilocus PCR-DNA sequencing (18S rDNA, 28S rDNA, and ITS-1) from culture-derived tachyzoites confirmed the parasite as B. darlingi. Our results suggest that bobcats may be an important link in the sylvatic cycle of Besnoitia species and bioassay or molecular tests are needed to differentiate Toxoplasma gondii-like oocysts in feces of felids, both domestic and wild cats.

Early in vivo diagnosis of bovine besnoitiosis is crucial for the success of control programmes. However, diagnosis in acutely infected animals is hindered by the low sensitivity of the available serological tools. In this study, a novel ELISA to detect specific anti-Besnoitia besnoiti IgM antibodies was developed. The usefulness of this tool together with an avidity ELISA were studied with a well-coded sera panel from experimentally and naturally infected cattle. First, the kinetics of specific IgM levels were determined in experimentally infected calves during the acute and chronic infection. Next, IgM levels were determined in naturally infected cattle with either acute or chronic infection. Finally, the IgG avidity index was monitored in both experimentally and naturally infected cattle. Specific IgM antibodies were detected prior to specific IgG antibodies (7-19 days vs. 17-26 days post-infection). A prompt IgM response was associated with the end of the febrile stage in experimentally infected calves. Naturally and experimentally infected animals with acute clinical signs tested IgM-positive but IgG-negative, followed by IgG seroconversion 2-3 weeks later. Chronically infected cattle developed both IgM and IgG specific antibodies. Moreover, a progressive increase in the avidity index (AI) was observed in all experimentally infected calves during the course of the experimental trials. However, a low AI coincided with visible tissue cysts. Low avidity values were also detected when naturally infected cattle with acute clinical signs seroconverted, in contrast to a high AI detected in chronically infected cattle. In summary, IgM and avidity ELISAs improved the early in vivo diagnosis of bovine besnoitiosis. IgM-positive but IgG-negative results were indicative of an acute infection, whereas IgG positive results accompanied by low avidity values confirmed a recent infection.

The pathogenesis of bovine besnoitiosis, a disease of increasing concern within Europe, is still incompletely understood. In this study, disease progression after natural infection with the causative apicomplexan Besnoitia besnoiti was monitored in histological skin sections of 5 individual female cattle over time. High-frequency skin sampling of 2 cattle with mild and 2 with severe acute, subacute, and chronic besnoitiosis, as well as from 1 animal during subclinical disease, enabled documentation from the beginning of the disease. Skin sections were stained with hematoxylin and eosin and Giemsa, periodic acid–Schiff reaction, and anti- Besnoitia immunohistochemistry. In all 4 clinically affected animals, tachyzoite-like endozoites could be detected for the first time by immunohistochemistry, and tissue cyst evolution was monitored. Besnoitiosis-associated lesions were not detected in the animal showing the subclinical course. Because of the inconsistency of the nomenclature of Besnoitia tissue cyst layers in the literature, a new nomenclature for B. besnoiti cyst wall layers is proposed: tissue cysts consist of a hypertrophied host cell with enlarged nuclei, an intracytoplasmic parasitophorous vacuole with bradyzoites, a sometimes vacuolated inner cyst wall, and an outer cyst wall in more developed cysts. Inner and outer cyst walls can be readily distinguished by using special stains. In 1 animal, extracystic B. besnoiti zoites were immunohistochemically detected during the chronic stage. At necropsy, the 2 severely affected cows displayed large numbers of B. besnoiti cysts in a variety of tissues, including the corium of the claws, contributing mainly to the development of chronic laminitis in these 2 cases.

The bovine Besnoitiosis is a disease caused by a sporozoan protozoan, Besnoitia besnoiti. It mainly affects to cattle but there have been also several cases in other ruminants. Because of the productive declines that it produces and it morbidity we can assure that it is a first magnitude emergent illness in Spain. To decrease it dissemination, it is important to recognize the typical clinic sings of the disease, carry out an early diagnostic in suspicious animals, set the sanitary status of the whole flock and take the management measures adapted to the real possibilities of the farm, needed for the checking.

Bovine besnoitiosis is a parasitic disease caused by the protozoan Besnoitia besnoiti. Described many decades ago, recent epidemiological studies reveal its important spread within Europe in the last years. To date, many epidemiological aspects related to life cycle, routes of transmission, incidence rates and associated risk factors are lacking; hence, the establishment of appropriate disease control programmes poses an important challenge. Thus, the aim of the present study was to determine the epidemiological pattern of the disease in an endemic herd reared under extensive conditions (Spanish Pyrenees) by identifying main factors associated with infection and clinical disease dynamics. The study population consisted of 276 Brown Swiss and Pirenaica adult animals and 145 calves born and weaned at the farm during the study. Three sampling time frames were used: January 2010, September 2010 and February 2011, which allowed us to differentiate two periods designated as mountain and valley periods. The data related to animals (breed, sex and age) and herd management (animal grouping and time in housing) were recorded. The data collection methodology was mainly based on clinical examinations and defining the serological status against bovine besnoitiosis by the immunofluorescent antibody testing of blood samples. The total prevalence among adult animals was 38.34% (CI95%: 34.53–42.07), with 18.54% of seropositive animals showing clinical signs. In regard to the cumulative incidence, 34.57% of new infections were detected during the mountain period, in contrast to the 24.59% observed in the valley period. The incidence density was 0.058 and 0.061 new infections per animal-month for the mountain and valley periods, respectively. According to the seroepidemiological study, the seroconversion probability of B. besnoiti infection was directly associated with the number of seropositive cows with whom an animal had been stabled as well as the housing period duration, supporting horizontal transmission by close contact as one of the most important methods of disease spread. In addition, the risk of developing the clinical course increased with age, and the presence of clinical signs was related to higher antibody responses. Among calves (from 3.1 to 7.1 months old) sampled once at weaning, the total seroprevalence was 15.17% (CI95%: 9.36–21.04), and the chronic stage was observed in three animals, supporting the ability of B. besnoiti to infect and even cause disease in animals less than 6 months old. Finally, the risk of calf seroconversion was positively related to the serological status of the cows, suggesting postnatal transmission between dams and offspring by contact during the suckling period.

BOVINE besnoitiosis is a chronic, debilitating disease characterised by both cutaneous and systemic manifestations with the possibility of severely affected animals dying (Fig 1). Economic losses are associated with sterility in bulls, the reduced value of the hides of affected animals, poor body condition, decreased milk production and occasionally abortions and deaths (Alvarez-Garcia and others 2014b, Cortes and others 2014). It has been suggested that transmission mainly occurs through direct contact between cattle (Alvarez-Garcia and others 2013). Moreover, mechanical transmission of the causative agent, Besnoitia besnoiti , probably occurs through blood-sucking insects or from reusing syringes (Bigalke 1968). FIG 1: ‘Elephant skin’ and alopecia in a bull severely infected with Besnoitia besnoiti Photograph: J. Balado This cattle disease gained the attention of veterinary health officials in Europe in 2010, and was considered as a re-emergent disease due to an increased number of cases as well as the geographical expansion of the disease (EFSA 2010, Alvarez-Garcia and others 2013). From the initial foci in the Pyrenees and Alentejo in Portugal at the beginning of the 20th century (Besnoit and Robin 1912, Franco and Borges 1915) the disease has spread almost all over Spain, France and Portugal over the past two decades (Alzieu and others 2007, Fernandez-Garcia and others 2010, Gentile and others 2012, Waap and others 2014). Since then the disease has propagated from …

Bovine besnoitiosis is an emerging disease in Europe, presenting quick spread toward central and southern Spain. Characterization of an outbreak in a free-ranging Limousin and Avilena beef cattle herd from southwestern Spain territories is attempted. Serological survey in the herd revealed increase of number of infected animals, from 34.3 % on first diagnoses/exams on December 2013 to 42.5 % in the second on April 2014. Blood analysis and serum biochemistry showed important alterations like leukocytosis (+33.2 % of mean value), with lymphocytosis (+205.3 %) and increase of LDH (+25.1 %), associated with tissue damage. Clinical cases were only observed in Limousin animals. Along with typical lesions of acute and chronic besnoitiosis, inflammatory and degenerative processes and parasitic cysts were present in the corpus cavernosum and the corpus spongiosum of penis. By using polymerase chain reaction (PCR) sequencing of 18S rDNA, Besnoitia besnoiti was confirmed as causative agent; microsatellite sequence analyses showed the homology of isolates with previously studied strains.

Besnoitia spp. are coccidian parasites that infect a variety of wild and domestic mammals as well as some reptiles. Although infection with Besnoitia is common in Virginia opossums (Didelphis virginiana), clinical signs or death due to Besnoitia is rare. This manuscript describes four Virginia opossums that had severe clinical disease and inflammation associated with besnoitiosis. Clinical signs included trembling, incoordination, circling, blindness, poor body condition, and sudden death. Gross lesions included parasitic cysts in eyes, skin, and visceral organs. Histologically, cysts were often degenerate and associated with mild to marked inflammation, and amyloidosis was noted in one animal. Polymerase chain reaction and sequencing confirmed Besnoitia darlingi in three of the four opossums.

Recently, Besnoitia neotomofelis was described from a southern plains woodrat (Neotoma micropus) from southern Texas. During May 2010, 1 of 55 southern plains woodrats trapped in Uvalde County, Texas, was diagnosed with besnoitiosis. Grossly, the woodrat had bilateral swellings of the cheeks, and numerous Besnoitia sp.-like cysts were observed in the tongue, facial region, musculature of the limbs, and subcutis of the dorsum and flanks. Little to no inflammation was noted around cysts. The cysts were morphologically similar to B. neotomofelis based on light and transmission electron microcopy. The sequence of the internal transcribed spacer region-1 was identical to the type isolate of B. neotomofelis. Attempts to isolate Besnoitia sp. in laboratory mice failed; however, Toxoplasma gondii was isolated in a Swiss Webster mouse. This represents the first report of besnoitiosis caused by B. neotomofelis in a southern plains woodrat and the first concurrent Besnoitia sp. and T. gondii infection in any host species.

The biology of Besnoitia besnoiti, the cause of bovine besnoitiosis, is poorly understood. Its definitive host is unknown, and information on potential intermediate hosts is scarce. In order to investigate potential definitive and intermediate hosts for European isolates of B. besnoiti, domestic dogs, cats, rabbits, guinea pigs (Cavia porcellus), gerbils (Meriones unguiculatus), common voles (Microtus arvalis) and NMRI-mice were inoculated with B. besnoiti isolated from naturally infected German cattle. Dogs and cats were fed 5×10(6)B. besnoiti tachyzoites (isolate Bb-GER1), or tissue cysts containing at least 2×10(7)B. besnoiti bradyzoites obtained from the skin of a naturally infected Limousin cow from the same herd where strain Bb-GER1 was isolated. Rodents and rabbits were subcutaneously inoculated with either 5×10(5) Bb-GER1 tachyzoites or 5×10(5) bradyzoites. Groups of 2-4 non-inoculated animals of each species were monitored as negative controls. Feces from all dogs and cats were daily examined by a sedimentation-flotation technique for at least 11 weeks after inoculation but no B. besnoiti oocysts were identified. Cats fed tachyzoites and dogs did not seroconvert, but specific antibodies to B. besnoiti tachyzoites were detected by IFAT (titer≥100) in 2 out of 3 cats fed tissue cysts since 5-7 weeks post infection. By immunoblot, these two cats exhibited a reaction pattern against tachyzoite antigens similar to that observed in naturally infected cattle. Antibodies against B. besnoiti tachyzoites were detected in all inoculated rodent species and rabbits by both, IFAT and immunoblot since 3 weeks post-inoculation. Rabbits and rodents, subcutaneously inoculated with same doses of inactivated bradyzoites remained serologically negative (IFAT titer50). Clinical signs observed in the inoculated rabbits included fever, serous conjunctivitis and transient swelling of the testes. No clinical abnormalities were noticed in the other tested animal species. Voles developed pneumonia as observed by histological examination. B. besnoiti-DNA was detected by PCR in blood from rabbits, gerbils and voles at 9 days post-infection, and in skin, heart, lung, striated muscle and kidney tissues from voles at 19-21 weeks post-infection. Domestic dogs and cats could not be shown to be definitive hosts of B. besnoiti, but cats seroconverted after feeding on B. besnoiti tissue cysts indicating that B. besnoiti stages had invaded the cats' tissues. The molecular and serological results from this study indicate that European B. besnoiti isolates may infect cats, rabbits, guinea pigs, gerbils, mice and voles; however a persistence of the parasite could be demonstrated only in voles.

Bovine besnoitiosis is caused by Besnoitia besnoiti, an apicomplexan parasite closely related to Toxoplasma gondii and Neospora caninum. In the acute stage of besnoitiosis, cattle suffer from pyrexia, swollen lymph nodes, anorexia and subcutaneous edema. In the chronic stage, tissue cysts are formed in a variety of tissues including the skin. Knowledge about the distribution of tissue cysts of different parts of the skin of infected animals is scarce. Four chronically infected cattle were euthanized and skin samples were taken from a total of 77 standardized cutaneous locations per animal. Portions of the dermis were taken, from which DNA was extracted and examined by real-time PCR. Cycle of transition (Ct) values reflecting the amount of parasite DNA in the samples were determined. For statistical analysis, samples were attributed to 11 larger skin regions ('OuterHindlegDistal', 'Rump, ForelegMiddle', 'NoseFrontEars', 'CheekEye', 'SideLowerPart', 'ForelegDistal', 'SideUpperPart', 'LegsInner', 'VentralHeadNeck', 'DorsalNeckWithersBackTail'). While all samples revealed a positive result in three female cattle, only 63.6% (49/77) of the samples of a bull showed positive results. For statistical analysis, a Ct value of 45 was assumed for samples with a negative result. The dams showed median Ct values of 16.1, 17.5 and 19.4, while in skin samples of the bull a median Ct value of 37.6 was observed. To determine the differences in DNA concentrations between different locations of the skin of the animals, a relative Ct (relCt) was determined by subtracting for each animal indv the MedianCt. indv from each sample Ct. Analyses of the relCt values showed that the highest relative parasite DNA concentrations were observed in the categories 'OuterHindlegDistal', 'Rump', 'ForelegMiddle' and 'NoseFrontEars'. The relCt values in these categories differed statistically significantly from those determined for the categories 'VentralHeadNeck' and 'DorsalNeckWithersBackTail'. The analysis showed clear differences in the distribution and the detectability of parasite DNA in the skin of cattle infected with B. besnoiti. In all four animals, samples from the 'Rump' region (Regio fermoris) showed high parasite DNA concentrations. Because this region is also easily accessible for veterinarians, this skin location appears to be optimal for taking skin biopsies for detection or isolation of B. besnoiti.

Bovine besnoitiosis is an exudative dermatitis characterized by thickening, folding of the skin, hair loss, cracks, scabs, edema, and enlargement of superficial lymph nodes. It is caused by Besnoitia besnoiti , an obligate intracellular protozoan parasite (apicomplexan). It occurs worldwide, affecting cattle over 6 month old, horses and donkeys. The disease is spread by biting flies but infection may originate from cats. Affected animals exhibit fever, photophobia, edema, diarrhea, and enlargement of superficial lymph nodes and scleroderma. Diagnosis includes demonstration of intradermal cysts containing bradyzoites with inflammatory infiltrates in skin sections. Treatment is difficult and of limited success but wound dressing and control of secondary infections may help. Oxytetracycline, antimony, or sulfanilamide show good results. Prevention is by separating sick animals from healthy ones or culling. A vaccine is available in South Africa.

International audience; Bovine besnoitiosis is a reemerging disease in Europe. The clinically Besnoitia besnoiti infection in bulls is characterized by fever, nasal discharge, and orchitis in the acute phase and by scleroderma in the chronic phase. However, in many bulls, B besnoiti infection remains at a subclinical stage. Bull infertility is an economically relevant consequence of besnoitiosis infection. It is not clear, however, if semen quality returns to normal levels when infected animals have clinically recovered. The aim of this study was to examine the relationship between chronic besnoitiosis and bull sexual function in a region of eastern France, where the disease is reemerging, by comparing semen quality and genital lesions in 11 uninfected, 17 subclinically infected, and 12 clinically infected bulls. The presence of anti-B besnoiti antibodies was detected by Western blot test. Semen was collected by electroejaculation. Bulls clinically infected with B besnoiti showed significantly more genital tract alterations than uninfected or subclinically infected bulls. No relationship was evidenced between besnoitiosis infectious status and semen quality, whereas a significant relationship was noted between genital lesions and semen score. This means that in the absence of moderate to severe genital lesions, chronic bovine besnoitiosis is unlikely to alter semen quality. However, as the presence of infected animals could lead to spread of the disease, culling or separation of clinically infected bulls from the remaining healthy animals is strongly recommended.

International audience; The parasitic diseases discussed elsewhere in this issue of the Scientific and Technical Review are not the only ones to make use of biological vectors (such as mosquitoes or ticks) or mechanical vectors (such as horse flies or Stomoxys flies). The authors discuss two major groups of vector-borne parasitic diseases: firstly, helminthiasis, along with animal filariasis and onchocerciasis, which are parasitic diseases that often take a heavy toll on artiodactyls throughout the world; secondly, parasitic diseases caused by vector-borne protists, foremost of which is bovine besnoitiosis (or anasarca of cattle), which has recently spread through Europe by a dual mode of transmission (direct and by vector). Other protists, such as Plasmodium and Hepatozoon, are also described briefly.

Besnoitia besnoiti is an apicomplexan parasite and the causative agent of bovine besnoitiosis which is considered as a re-emergent disease in Europe. A cross-sectional serological study was conducted to determine the seroprevalence and to identify risk factors associated with B. besnoiti infection in 68 dairy herds (n = 806 cows) in Jordan during the period from January to June 2007 and the spring of 2014. Data regarding herd’s management was obtained by filling questionnaires through personal interviews with farmers. An indirect ELISA test was used to detect antibodies against B. besnoiti. Chi-square analysis and multivariable logistic regression model were used to identify risk factors associated with seropositivity to B. besnoiti. At the individual cow and herd level, the true prevalence of seropositive animals was 6 and 28.7 %, respectively. Cows between 2 and 6 years of age had significantly higher seroprevalence of B. besnoiti than other age groups. The highest seroprevalence of B. besnoiti was found in Zarqa and Irbid governorates. Multivariable logistic regression model identified that exchanging visits by farm workers to neighboring farms as a risk factor for seropositivity to B. besnoiti, while smaller herd size and twice a day farm cleaning using sweeping and water hosing were identified as protective factors. This is the first study that investigated the seroprevalence of B. besnoiti infection in dairy herds in Jordan. Further studies are warranted to explore the clinical manifestation of B. besnoiti infection as well as to identify the possible presence of other Besnoitia species and definitive hosts for the parasite.

Besnoitia besnoiti, an obligate intracellular protozoan parasite belonging to the phylum apicomplexa, is the causative agent of bovine besnoitiosis. Besnoitiosis is responsible for significant losses in the cattle industry of Africa and Mediterranean countries due to the high morbidity rate, abortion and infertility in males. The acute stage of disease is associated with the proliferative forms (tachyzoites) and is characterized by fever, whimpery, general weakness and swelling of the superficial lymph nodes. During the following chronic stage, a huge number of cysts are formed mainly in the subcutaneous tissues. This process is non-reversible, and chronic besnoitiosis is characterized by hyper-sclerodermia, hyperkeratosis, alopecia and, in bulls, atrophy, sclerosis and focal necrosis that cause irreversible lesions in the testis. In this paper we report on the identification of large cysts in the skin of a cow and a bull in Portugal, which presented loss of hair and enlargement and pachydermis all over the body. The observation of a two-layered cyst wall within the host cell, the encapsulation of the host cell by a large outer cyst wall, and the subcutaneous localization of the cysts within the host, were characteristic for B. besnoiti. The parasites were isolated from the infected animals and successfully propagated in Vero cells without prior passages in laboratory animals. Morphological characterization of B. besnoiti tachyzoites and the amplification of the 149 bp segment from the internal transcribed spacer 1 (ITS1), aided with specific primers, confirmed the identification of B. besnoiti. Keywords: Besnoitia besnoiti; Bovine besnoitiosis; Portugal; Cyst; In vitro cell culture

Besnoitia besnoiti is a protozoan parasite responsible for bovine besnoitiosis. Indirect immunofluorescence showed that isolated B. besnoiti possesses a set of subpellicular microtubules, radiating from the apical end and extending for more than 2/3 of the cell body. Upon interaction with the host cell, B. besnoiti undergoes dramatic modifications of shape and surface, as revealed by atomic force microscopy, accompanied by a distinct tubulin labeling on the posterior region. In the host cell, the microtubule cytoskeleton shows a re-arrangement around the invading parasite suggesting a filamentous interaction with the parasite cytoskeleton during invasion.

Fourteen miniature donkeys (Equus asinus) in a mid-Michigan herd of 38 animals presented with clinical signs of besnoitiosis, including the presence of typical tissue cysts in the ocular sclera, the buccal and nasal mucosa, together with characteristic dermatitis in specific areas of the body. The common histopathological change seen was the presence of many 100-200-microm diameter, thick walled, typical Besnoitia sp. tissue cysts together with a chronic cellular response associated with degenerating cysts. Microscopy of isolated scleral cysts and skin biopsies showed the presence of protozoal organisms consistent in morphology with that of Besnoitia bennetti bradyzoites. Molecular analysis of these parasites indicates that they differ from previously described coccidia, including Besnoitia sp., from rabbits and opossums. Isolated cases of infection with this agent have been reported infrequently in equids; however, this is the first report of an outbreak in a herd of donkeys in the United States.

The Sarcocystidae comprise a diverse, monophyletic apicomplexan parasite family, most of whose members form intracellular cysts in their intermediate hosts. The extent of pathology associated with such cyst formation can range widely. We currently lack experimental animal models for many of these infections. Here we explored dexamethasone treatment as a means to render outbred mice susceptible to Besnoitia darlingi infection and demonstrated that this approach allows viable parasites to be subsequently isolated from these mice and maintained in tissue culture. Besnoitia bradyzoites recovered from crushed cysts derived from naturally infected opossums (Didelphis virginiana) replicated and reproduced the development of besnoitiosis in mice treated with dexamethasone (0.5 mg/ml drinking water) daily for 12 days post infection (DPI). Isolates recovered from the peritoneal exudates of these mice were viable and were maintained in long-term tissue cultures. In contrast, control mice given saline without dexamethasone and challenged with similar bradyzoites remained clinically normal for up to 70 DPI. An additional group of mice challenged with the same inoculum of bradyzoites and given dexamethasone at the same concentration and treated with sulfadiazine (1 mg/ml drinking water) daily for 12 DPI also remained normal for up to 70 DPI. Severe disease developed more rapidly in dexamethasone-treated mice inoculated with culture-derived B. darlingi tachyzoites than in those inoculated with cyst-derived bradyzoites. B. darlingi tachyzoite-infected, untreated control mice developed signs of illness at 18 DPI. In contrast, mice treated with sulfadiazine showed no clinical signs up to 50 DPI. Although dexamethasone treatment was required to establish B. darlingi infection in outbred mice inoculated with opossum-derived B. darlingi bradyzoites, no such treatment was required for mice inoculated with culture-derived B. darlingi tachyzoites. Finally, sulfadiazine was highly effective in protecting mice from infection with the tachyzoite stage of B. darlingi.

Besnoitia besnoiti is a protozoan parasite known to cause important economic losses in the cattle industry in Africa, Asia and the Mediterranean area. In the last years, (re-) emergence of the parasite has been reported in France, Germany, Hungary and Italy with in some cases, establishment of an endemic infection. In this article, the first case of besnoitiosis in Belgium in a Blonde d’Aquitaine bull imported from the south of France is described. Additionally, a brief overview of the epidemiology of the disease is provided.