Bovine Besnoitiosis Research Articles

The first molecular investigation of Besnoitia besnoiti infections among cattle in Mosul, Iraq.

Bovine besnoitiosis (elephant skin disease) caused by Besnoitia besnoiti is a costly endemic disease in the Middle East, Asia, and tropical and subtropical Africa and is also emerging as a significant problem in Europe. This study is aimed at determining the prevalence of B. besnoiti in blood and skin biopsies of cattle as well as evaluating the risk factors associated with the infection among cattle in Mosul, Iraq. To achieve this aim, four hundred and sixty apparently healthy cattle of different breeds, ages, and sexes were sampled from seven different locations in Mosul, Iraq. Blood and skin biopsies were carefully collected from each cattle, and these samples were subjected to molecular analysis. The detection of B. besnoiti was molecularly confirmed by the presence of 231bp of ITS-1 in the rDNA gene of the protozoan. Besnoitia besnoiti DNA was present in 74 (16.09%; 95% CI = 13.01-19.72) and 49 (10.65%; 95% CI = 8.15-13.80) of the blood and skin biopsies, respectively, that were analyzed. Age, breed, and sex were significantly (p < 0.05) associated with the occurrence of B. besnoiti among cattle in the study area. Findings from this study will serve as baseline data in the epidemiology, prevention, and control of the protozoan among cattle in Iraq.

Besnoitia besnoiti tachyzoite replication in bovine primary endothelial cells relies on host Niemann-Pick type C protein 1 for cholesterol acquisition.

Besnoitia besnoiti is a cyst-forming apicomplexan parasite and the causal agent of bovine besnoitiosis. During early phase of infection, tachyzoites replicate within host endothelial cells in a host cell cholesterol-dependent process. By applying U18666A treatments, we here evaluated the role of Niemann-Pick type C protein 1 (NPC1) in both, intracellular B. besnoiti replication and host cellular cholesterol distribution. Additionally, B. besnoiti-driven changes in NPC1 gene transcription were studied by qPCR. Overall, U18666A treatments significantly reduced B. besnoiti proliferation and induced cholesterol accumulation in host cytoplasmic dense vesicles. However, NPC1 gene transcription was not affected by B. besnoiti infection.

Besnoitia besnoiti-induced neutrophil clustering and neutrophil extracellular trap formation depend on P2X1 purinergic receptor signaling.

Bovine besnoitiosis is a re-emerging cattle disease caused by the cyst-forming apicomplexan parasite Besnoitia besnoiti. Neutrophil extracellular trap (NET) formation represents an efficient innate immune mechanism of polymorphonuclear neutrophils (PMN) against apicomplexan parasites, including B. besnoiti. PMN purinergic signaling was proposed as a critical factor for NET formation. One important purinergic ligand is ATP, which is recognized as a danger signal and released into the extracellular space acting as an autocrine/paracrine signaling molecule. ATP-driven effects on PMN via the nucleotide P2 receptor family include chemotaxis, reactive oxygen species (ROS) production, and NET formation. So far, data on both PMN ATP concentrations and the role of ATP as a key modulator of purinergic signaling in B. besnoiti tachyzoite-triggered bovine NETosis is scarce. Current data showed that B. besnoiti tachyzoite exposure to bovine PMN neither changed total PMN ATP nor extracellular ATP quantities even though it significantly triggered NET formation. Moreover, B. besnoiti tachyzoite-exposed PMN revealed enhanced oxygen consumption rates (OCR) as quantified by the Seahorse metabolic analyzer. Exogenous supplementation of ATP or non-hydrolizable ATP (ATPγS) led to increased extracellular acidification rates (ECAR) but failed to alter tachyzoite-induced oxidative responses (OCR) in exposed PMN. In addition, exogenous supplementation of ATPγS, but not of ATP, boosted B. besnoiti tachyzoite-induced anchored NET formation. Referring to purinergic signaling, B. besnoiti tachyzoite-triggered anchored NET formation revealed P2X1 purinergic as receptor-dependent since it was blocked by the P2X1 inhibitor NF449 at an IC50 of 1.27 µM. In contrast, antagonists of P2Y2, P2Y6, P2X4, and P2X7 purinergic receptors all failed to affect parasite-driven NETosis. As an interesting finding, we additionally observed that B. besnoiti tachyzoite exposure induced PMN clustering in a P2X1-dependent manner. Thus, we identified P2X1 purinergic receptor as a pivotal molecule for both B. besnoiti tachyzoite-induced PMN clustering and anchored NET formation.

Epidemiological characteristics of bovine besnoitiosis (Besnoitia besnoiti) in a beef cattle farm: a cross-sectional serological assessment.

Bovine besnoitiosis is an emerging disease caused by the protozoa Besnoitia besnoiti that can have a serious economic impact on affected farms. The fact that there is no effective vaccine nor treatment, along with the lack of consistent epidemiologic data, renders the implementation of preventive medicine and control strategies much harder. A cross-sectional serological assessment was performed to better understand the distribution and prevalence of this parasite in a large beef cattle farm in Portugal and to establish some epidemiological characteristics of besnoitiosis. A random blood sampling of 450 animals from a farm that keeps around 2,000 cattle head was performed and sera were submitted to an indirect immunofluorescent antibody test (IFAT). Data on breed, age, sex, and birthplace of the tested animals and their mothers were recorded. The overall prevalence of positive animals was 16.89%, with significant differences between under 1-year-old calves (4.8%) and adults (19.67%). A higher antibody prevalence was shown in animals 1-2 years and >7 years old, in Salers breed and in cows imported from France or whose mothers had come from this country. Calves under 1 year old and crossbreed animals with ancestry born in the current farm presented the lowest antibody prevalence. The most significant risk factors revealed were age (>7 years old) and breed (Salers). Genetic studies should be carried out in order to confirm whether indeed there is a breed susceptibility to bovine besnoitiosis. We suggest that similar studies should be performed across southern Europe to establish strong epidemiologic data that would allow a rigorous transnational control program to be launched.

Validation of a commercial version of a competitive enzyme linked immunosorbent assay for the detection of antibodies to Besnoitia besnoiti

BackgroundSeveral reports suggest a further spread of besnoitiosis to countries in which Besnoitia besnoiti-infected bovine herds have not been noticed yet. Cattle infected without clinical signs may represent reservoirs. Serological analyses in affected herds or animals from endemic regions are necessary to identify subclinical or inapparent infections and stop transmission to naïve animals or herds. The Monoscreen AbELISA Besnoitia besnoiti (BIO K 466) is based on a previously published in-house competitive ELISA, the Bb-cELISA1, but has a different test architecture. The present study aimed to use sera from a previous evaluation of Bb-cELISA1 to assess whether BIO K466 shows identical results. In addition, further well-characterized positive and negative samples were analysed to estimate diagnostic sensitivity and specificity.MethodsA first set of sera consisted of a total of 305 bovine sera, collected from German herds infected by B. besnoiti, Neospora caninum or Sarcocystis spp. Sera had been characterized by reference serological tests (i.e. immunoblot, immunofluorescence antibody test and an in-house indirect ELISA). A second set consisted of 200 confirmed B. besnoiti-positive sera from French herds. Negative cattle sera (n = 624) originated from Norway and The Netherlands, countries in which bovine besnoitiosis has not been reported yet.ResultsUsing the first set of sera, the BIO K466 showed an estimated diagnostic sensitivity of 97.9% (95% CI: 91.9%–99.6) and a diagnostic specificity of 99.5% (95% CI: 96.9%–100%) relative to reference serological tests. A direct comparison of the results revealed an almost perfect agreement between the results of the in-house Bb-cELISA1 and the commercialized version (kappa 0.98; 95% CI: 0.95–1). The validation using positive bovine sera from France and negative sera from other European countries revealed a diagnostic sensitivity of 97.5% (95% CI: 93.9%–99.1%) and specificity of 99.5% (95% CI: 98.5%–99.9%).ConclusionIn conclusion, BIO K 466 appears to be a suitable tool to diagnose bovine besnoitiosis, but needs further validation especially in cases of inconclusive, suspected false-positive or -negative results in other serological tests.Graphic abstract

Absence of anti-Besnoitia spp. specific antibodies in European wild lagomorphs from the Iberian Peninsula.

Besnoitia besnoiti is an apicomplexan parasite whose life cycle is not completely understood. It is assumed that this parasite might have an indirect life cycle with a carnivore as a definitive host able to shed oocysts after the ingestion of mature cysts in tissues of an infected intermediate host. Cattle and wild cervids on the Iberian Peninsula can act as intermediate hosts of B. besnoiti, and exposure to the parasite has been demonstrated in equids. In this study, we aimed to assess the presence of members of the genera Besnoitia in wild lagomorphs from the Iberian Peninsula and the potential role of these host species in the life cycle of B. besnoiti, as all the animals were sampled from 19 regions of the Iberian Peninsula where cases of bovine besnoitiosis have been previously detected. Serum samples (Oryctolagus cuniculus: n = 552; Lepus europaeus: n = 122) were first analysed by ELISA and subsequently confirmed by Western blot (WB). Specific antibodies against B. besnoiti were not found in any sampled animal by WB. In addition, lung samples from a subset of wild rabbits (n = 16) were tested by PCR and Besnoitia spp. DNA was not detected. These results suggest that Besnoitia spp. are unlikely to circulate in wild lagomorphs in the Iberian Peninsula. Thus, lagomorphs are not expected to play a key role in the biological cycle of B. besnoiti. Further studies are necessary to assess whether different micromammal species, such as rodents, can serve as natural reservoirs of Besnoitia spp. in other European regions.

First Expert Elicitation of Knowledge on Drivers of Emergence of Bovine Besnoitiosis in Europe.

Bovine besnoitiosis (BB) is a chronic and debilitating parasitic disease in cattle caused by the protozoan parasite Besnoitia besnoiti. South European countries are affected and have reported clinical cases of BB. However, BB is considered as emerging in other countries/regions of central, eastern and northern Europe. Yet, data on drivers of emergence of BB in Europe are scarce. In this study, fifty possible drivers of emergence of BB in cattle were identified. A scoring system was developed per driver. Then, the scoring was elicited from eleven recognized European experts to: (i) allocate a score to each driver, (ii) weight the score of drivers within each domain and (iii) weight the different domains among themselves. An overall weighted score was calculated per driver, and drivers were ranked in decreasing order of importance. Regression tree analysis was used to group drivers with comparable likelihoods to play a role in the emergence of BB in cattle in Europe. Finally, robustness testing of expert elicitation was performed for the seven drivers having the highest probability to play a key role in the emergence of BB: i.e., (i) legal/illegal movements of live animals from neighbouring/European Union member states or (ii) from third countries, (iii) risk of showing no clinical sign and silent spread during infection and post infection, (iv) as a consequence, difficulty to detect the emergence, (v) existence of vectors and their potential spread, (vi) European geographical proximity of the pathogen/disease to the country, and (vii) animal density of farms. Provided the limited scientific knowledge on the topic, expert elicitation of knowledge, multi-criteria decision analysis, cluster and sensitivity analyses are very important to prioritize future studies, e.g., the need for quantitative import risk assessment and estimation of the burden of BB to evidence and influence policymaking towards changing (or not) its status as a reportable disease, with prevention and control activities targeting, firstly, the top seven drivers. The present methodology could be applied to other emerging animal diseases.

Impact of Endemic Besnoitiosis on the Performance of a Dairy Cattle Herd.

Simple SummaryBovine besnoitiosis, caused by the Apicomplexa parasite Besnoitia besnoiti, has been emerging in Europe as a disease of economic concern to the cattle industry. It is a chronic and debilitating disease mostly reported in beef cattle. However, in Europe, bovine besnoitiosis is increasingly common in dairy cattle; therefore, there is a need to assess the impact of this disease on milk production. To study the effect of B. besnoiti infection on dairy production and reproduction, a serological screening was performed on a dairy herd in an endemic area. The results showed that the herd was endemically infected, with high seroprevalence and low clinical prevalence, and the time on herd represented a risk factor to acquire the infection. Seropositive animals and cows with chronic skin lesions revealed higher milk somatic cell counts, and no negative impact on reproductive performance was found.This study aimed to assess the effect of Besnoitia besnoiti infection on the reproductive and productive performance of a dairy cattle herd. A serological screening was performed by indirect fluorescent antibody test (IFAT) on every animal aged over one year (n = 262). Subsequently, 211 animals were clinically examined, with 96 of those being screened for detection of sclerocysts. The overall seroprevalence was 62.9% (CI95%: 56.1–69.5%). On clinical examination, 7.6% (16/211) of the animals presented chronic skin lesions, and 47.9% (46/96) had sclerocysts. Multivariate logistic regression showed that the time on herd represented a risk factor, and the odds of acquiring the infection increased 1.683× per additional year on herd, ranging from less than a year to 8 years. Seropositivity and the presence of sclerocysts revealed an association with a higher milk somatic cell count, which may have a considerable economic impact on dairy production. Regarding reproductive indicators, no negative impact could be associated with clinical besnoitiosis or positive serological results. In conclusion, our study highlights the need to thoroughly evaluate the economic impact of this emerging disease in dairy herd production to help with decision making at both herd and regional levels, particularly in endemic areas.

An investigation into bovine besnoitiosis (Besnoitia besnoiti) in an Irish pedigree Aberdeen Angus herd

AbstractThis case report describes the investigation of the first outbreak of bovine besnoitiosis in a beef/suckler herd in Ireland. Infection with Besnoitia besnoiti was suspected by the attending veterinarian following clinical examination of a chronically affected cow with areas of alopecia and marked skin thickening. Diagnosis of besnoitiosis was confirmed by Cork Regional Veterinary Laboratory based on histopathology of skin biopsies. Subsequent serological investigation using enzyme‐linked immunosorbent assay testing revealed a 17.8% seroprevalence within the herd, with 33% of adult cows seropositive. A clinical whole‐herd investigation found an overall clinical prevalence of besnoitiosis of 38.8%, based on the presence of sclerocysts and/or vulval cysts. There was poor agreement between serological and clinical findings. In contrast to serological findings, 83.8% of 18‐month‐old heifers and bulls had sclerocysts. Due to the high prevalence of besnoitiosis and the challenges of controlling spread, this pedigree herd was discontinued by the farmer.

Investigation of an Autochthonous Outbreak of Bovine Besnoitiosis in Northwestern Sicily.

Bovine besnoitiosis is a parasitic disease caused by the protozoan Besnoitia besnoiti, leading to infertility in bulls and abortions in cows. In Italy, it is considered an emerging disease, recently introduced by the importation of animals from Spain and France. In the last decade, many outbreaks have been reported and confirmed in native cattle, mostly in northern and central Italy. This study reports on an autochthonous outbreak of bovine besnoitiosis in two nearby farms located in northwestern Sicily. A 15-month-old Limousine bull born on the farm showed typical clinical signs of the chronic disease phase, such as edema of the scrotum with subsequent hyperkeratosis associated with eschars and thickening of the skin. A histopathological examination revealed the presence of Besnoitia tissue cysts containing bradyzoites in the eyes, tendons, testicles, dermis, and nictitating membrane. A serological investigation using a commercial ELISA kit revealed a high seroprevalence of the antibody anti-B. besnoiti (79.2% for the farms in this study). Clinical disease showed low prevalence (1.5%) despite the high seroprevalence of specific antibodies in the herd, confirming that bovine besnoitiosis is an emergent endemic pathogen in Sicily, but its clinical behavior still remains sporadic.

Seroprevalence of Besnoitia besnoiti in Assiut Governorate, Egypt.

Bovine besnoitiosis is a widespread disease caused by Besnoitia besnoiti with significant economic losses in cattle production. There is a lack of knowledge about it in Egypt. This study was conducted to detect the seroprevalence of B. besnoiti in cattle and to find out the presence of the disease and the most important symptoms of besnoitiosis in cattle in Assiut Governorate, Egypt. A total of 190 cattle from Assiut city and its different rural centers were examined clinically and serologically for the presence of B. besnoiti. The serological examination was carried out by using the indirect enzyme-linked immunosorbent assay (ELISA) kit in serum (ID.Vet Innovative Diagnostics Louis Pasteur. Grabeis, France). The results were analyzed statistically using the chi-square test to assess the association between seroprevalence and different parameters (age, sex, season, housing, and health status). Thirteen cattle were seropositive for B. besnoiti by ELISA and showed symptoms of besnoitiosis. Acute symptoms included fever, tachycardia, edematous swellings of intermandibular space and limbs with polyarthritis, diarrhea, ruminal atony, and enlarged lymph nodes. The chronic symptoms included cough, mastitis, exophthalmia, cysts on the sclera and conjunctiva, nodules in the skin, and alopecia associated with tick infestation. The overall seroprevalence of B. besnoiti was 22.1%. Regarding sex, the seroprevalence was higher for females 34.6% than for males 6.97%. While, according to age susceptibility, the seroprevalence was highest (50.9%) with age ≥5 years, followed by age >1 to <5 years (14.6%), and only one animal of age ≤1 year was recorded at 2.2%. Concerning seasonal variations, the seroprevalence was highest in spring 42.9%, followed by autumn 29.3%, winter 13.6%, and summer 7.5%. Whereas, according to the housing system, it was 60% and 8.6% in farm and household rearing, respectively. Depending on the health status, the seroprevalence was 21.6% of clinically healthy and 23.2% of clinically diseased cattle. The existence of B. besnoiti antibodies has been demonstrated in clinical and subclinical infected cattle in Assiut Governorate, Egypt. The ELISA test is considered to be a good diagnostic method for detecting infection. Furthermore, additional studies are essential to minimize and prevent the spread of infection.

Emergence of Besnoitia besnoiti in Belgium.

Bovine besnoitiosis is a cattle disease caused by a protozoan parasite called Besnoitia besnoiti. It is of serious economic concern to the cattle industry and also compromises animal welfare. For several years, it has been considered an emerging disease in some countries and regions located in the north of Europe far away from the known endemic areas in the south. This study describes the situation in the southern part of Belgium, where the parasite was recently introduced through imports of animals coming from departments of France where the disease was present. It details the detection of clinical cases as well as disease transmission features related to contacts during grazing and sales of infected cattle. A tracking and monitoring system was quickly set up and detected twelve outbreaks. Several cattle were controlled, but the lack of appropriate regulations weakens disease-management efforts. Hopefully, this predictable and silent introduction triggers the awareness of decision-makers about the need for an appropriate prevention and control policy, law enforcement, and the implementation of necessary measures to avoid bovine besnoitiosis becoming endemic in Belgium or other non-endemic countries. In addition, more proactive surveillance is required from authorities through threat analysis in the context of the risk of emergence or re-emergence of infectious animal diseases.

Changes in serum biomarkers of inflammation in bovine besnoitiosis

BackgroundAcute and chronic besnoitiosis in extensive natural-service herds can have relevant effects in the health of bulls and negative consequences in their productive performance. Recent progress has been made in order to elucidate the pathogenesis of this disease. In this context, the study of biomarkers of inflammation in serum would contribute to gaining knowledge about the physiopathology of bovine besnoitiosis. Serological biomarkers could help in early diagnosis and prognosis, as seropositive bulls may have mild or severe testicular lesions.MethodsHerein, we have investigated the diagnostic and/or prognostic value of a panel of serum (serological) biomarkers related to inflammation, including total protein, globulin and albumin, haptoglobin (Hp), adenosine deaminase (ADA) paraoxonase-1 (PON-1) and acetylcholinesterase (AChE) in naturally and experimentally B. besnoiti-infected males classified according to different clinical phases of the disease (acute, chronic and subclinical besnoitiosis).ResultsResults showed a similar response pattern in these biomarkers for naturally and experimentally infected cattle, with a few relevant variations. Most significant changes occurred during the acute phase of infection, although significant changes in a few biomarkers were also observed during the chronic infection. Haptoglobin, albumin, PON-1 and ADA were identified as the biomarkers that showed changes of higher magnitude in the acute phase of the infection, whereas high total protein and globulin values were found in chronically infected cattle. We have described the changes of a panel of inflammatory biomarkers of acute and chronic bovine besnoitiosis.ConclusionsIn summary, several biomarkers with promising diagnostic value have been identified. The biomarkers associated with acute infection are related to previously reported molecular biomarkers in testicular parenchyma of infected bulls and could help in the diagnosis of early infections and complement results from specific immunoglobulin M (IgM) detection.Graphical abstract

Bovine besnoitiosis in a cattle herd in Sicily: an isolated outbreak or the acknowledgment of an endemicity?

Bovine besnoitiosis is a debilitating infectious disease caused by Besnoitia besnoiti (Apicomplexa; Sarcocystidae). The disease is mainly characterized by cutaneous and systemic signs, infertility in bulls, and abortion in cows. The current study describes an autochthonous outbreak of bovine besnoitiosis in Sicily, Southern Italy, being the first report of B. besnoiti infection in the island so far. In a cattle farm located in Syracuse province, a 4-year-old Belgian blue bull born in Sicily displayed typical clinical signs of the sub-acute/chronic disease phase with thickening of the skin of the scrotum and testicular degeneration. Histological examination of scrotal biopsies revealed the presence of several tissue cysts of B. besnoiti. The serological analysis of the herd using a commercial ELISA revealed a high seroprevalence (45 out of 55; 82%) of antibodies against B. besnoiti. Few seropositive animals (5 out of 45; 11%) showed clinical signs, cysts in vestibulum vaginae (1 out of 31; 3.2%), and testicular degeneration (4 out of 14; 28.6%) assessed by ultrasonographic investigations. The paucity of clinical signs associated with the high seroprevalence in the farm led to hypothesize that bovine besnoitiosis is endemic in the area though further studies are needed. Local practitioners should be more aware of the disease to facilitate the early detection of cases, prevent the spread of infection, and avoid economic losses and animal health problem.

Identification of molecular biomarkers associated with disease progression in the testis of bulls infected with Besnoitia besnoiti

Breeding bulls infected with Besnoitia besnoiti may develop sterility during either acute or chronic infection. The aim of this study was to investigate the molecular pathogenesis of B. besnoiti infection with prognosis value in bull sterility. Accordingly, five well-characterized groups of naturally and experimentally infected males were selected for the study based on clinical signs and lesions compatible with B. besnoiti infection, serological results and parasite detection. A broad panel of molecular markers representative of endothelial activation and fibrosis was investigated and complemented with a histopathological approach that included conventional histology and immunohistochemistry. The results indicated the predominance of an intense inflammatory infiltrate composed mainly of resident and recruited circulating macrophages and to a lesser extent of CD3+ cells in infected bulls. In addition, a few biomarkers were associated with acute, chronic or subclinical bovine besnoitiosis. The testicular parenchyma showed a higher number of differentially expressed genes in natural infections (acute and chronic infections) versus scrotal skin in experimental infections (subclinical infection). In subclinical infections, most genes were downregulated except for the CCL24 and CXCL2 genes, which were upregulated. In contrast, the acute phase was mainly characterized by the upregulation of IL-1α, IL-6 and TIMP1, whereas in the chronic phase, the upregulation of ICAM and the downregulation of MMP13, PLAT and IL-1α were the most relevant findings. Macrophages could be responsible for the highest level of gene regulation in the testicular parenchyma of severely affected and sterile bulls, and all these genes could be prognostic markers of sterility.

Molecular survey of Besnoitia spp. (Apicomplexa) in faeces from European wild mesocarnivores in Spain.

Numerous studies have unsuccessfully tried to unravel the definitive host of the coccidian parasite Besnoitia besnoiti. Cattle infections by B. besnoiti cause a chronic and debilitating condition called bovine besnoitiosis that has emerged in Europe during the last two decades, mainly due to limitations in its control associated with the absence of vaccines and therapeutical tools. Although the exact transmission pathways of B. besnoiti is currently unknown, it is assumed that the parasite might have an indirect life cycle with a carnivore as definitive host. Current lack of studies in wildlife might underestimate the importance of free-living species in the epidemiology of B. besnoiti. Thus, the aim of the present study is to assess the presence of Besnoitia spp. in free-ranging mesocarnivores in Spain. DNA was searched by PCR on faeces collected from wild carnivores as a first approach to determine which species could be considered as potential definitive host candidates in further research. For this purpose, a total of 352 faecal samples from 12 free-living wild carnivore species belonging to the Canidae, Felidae, Herpestidae, Mustelidae, Procyonidae and Viverridae families were collected in seven Spanish regions. PCR testing showed that Besnoitia spp. DNA was present in four faecal samples from red foxes collected in western Spain, an area with the greatest density of extensively reared cattle and associated with high incidence of bovine besnoitiosis in the country. To date, this is the first report of a B. besnoiti-like sequence (99.57% homology) from carnivore faeces in a worldwide context. Red foxes might contribute to the epidemiology of B. besnoiti, although further studies, mostly based on bioassay, would be needed to elucidate the accuracy and extent of these interesting findings.

Exploring alterations in hematological and biochemical parameters, enzyme activities and serum cortisol in Besnoitia besnoiti naturally infected dairy cattle

BackgroundBesnoitia besnoiti is an Apicomplexan protozoa causative of bovine besnoitiosis, a chronic and debilitating disease of cattle, with a variety of pathological findings that could alter some laboratory parameters. A study was conducted in a bovine besnoitiosis endemically infected dairy herd located in Italy characterized by high intra-herd seroprevalence and cattle with clinical signs of the disease. In the study, alterations in laboratory parameters, i.e. hematological and biochemical parameters, enzyme activities and serum cortisol levels, in Besnoitia besnoiti naturally infected cows were investigated in depth.MethodsLaboratory parameters in 107 cows, of which 61 were seronegative and 46 were 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.ResultsHematological analyses revealed that B. besnoiti infection determined a significant alteration to the leukocyte differential, with a higher percentage of granulocytes and a lower percentage of lymphocytes in seropositive and clinically affected animals (Mann–Whitney U-test, P = 0.022); erythrocyte and platelet counts did not show any difference between the considered groups of cows. Biochemistry tests evidenced that the parasite infection influenced serum protein values in seropositive cows and glutamate dehydrogenase values in clinically affected animals. No or only slight differences were revealed for all of the other biochemical and enzyme activity parameters in B. besnoiti-infected animals. In addition, despite the lack of statistical significance, seropositive and clinically affected cows evidenced higher concentrations of serum cortisol values compared to seronegative animals.ConclusionsAlthough physiological, pathological and farm-related factors could have influenced the results in investigated animals, further studies involving more animals from different farms would be advisable to infer the role of B. besnoiti on these alterations, since laboratory parameters could help veterinarians in the diagnosis of bovine besnoitiosis in cattle.

Real-time PCR on skin biopsies for super-spreaders\u2019 detection in bovine besnoitiosis

BackgroundBovine besnoitiosis, an emerging disease in Europe that can be transmitted by vectors, is caused by the apicomplexan Besnoitia besnoiti. Bovine besnoitiosis is difficult to control due to the complexity of its diagnosis in the acute stage of the disease, poor treatment success and chronically asymptomatic cattle acting as parasite reservoirs. When serological prevalence is low, detection and specific culling of seropositive cattle is feasible; however, economic considerations preclude this approach when serological prevalence is high. The aims of this study were to evaluate the accuracy of detection of super-spreaders in highly infected herds and to test their selective elimination as a new control strategy for bovine besnoitiosis.MethodsPrevious real-time PCR analyses performed on skin tissues from 160 asymptomatic animals sampled at slaughterhouses showed that the tail base was the best location to evaluate the dermal parasite DNA load. All seropositive animals (n = 518) from eight dairy or beef cattle farms facing a high serological prevalence of besnoitiosis were sampled at the tail base and their skin sample analysed by real-time PCR. A recommendation of rapid and selective culling of super-spreaders was formulated and provided to the cattle breeders. Subsequent serological monitoring of naïve animals was used to evaluate the interest of this control strategy over time.ResultsAmong the 518 seropositive animals, a low proportion of individuals (14.5%) showed Cq values below 36, 17.8% had doubtful results (36 < Cq ≤ 40) and 67.8% had negative PCR results. These proportions were grossly similar on the eight farms, regardless of their production type (beef or dairy cattle), size, geographical location or history of besnoitiosis. Within two weeks of the biopsy, the rapid culling of super-spreaders was implemented on only three farms. The numbers of newly infected animals were lower on these farms compared to those where super-spreaders were maintained in the herd.ConclusionsReal-time PCR analyses performed on skin biopsies of seropositive cattle showed huge individual variabilities in parasite DNA load. The rapid culling of individuals considered as super-spreaders seems to be a new and encouraging strategy for bovine besnoitiosis control.

Intracellular Parasites Toxoplasma gondii and Besnoitia besnoiti, Unveiled in Single Host Cells Using AP-SMALDI MS Imaging

The obligate intracellular apicomplexan parasites Toxoplasma gondii and Besnoitia besnoiti are important causes of disease in both humans and cattle. To date, effective specific treatments are lacking for both infections. To counteract severe symptoms leading to, e.g., disabilities and even abortion in the case of human toxoplasmosis and bovine besnoitiosis, novel targets are required for development of drugs and vaccines. A promising emerging technique for molecular characterization of organisms is high-resolution atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) mass spectrometry imaging (MSI) which enables semiquantitative visualization of metabolite distributions. MSI was here used to trace and characterize lipid metabolites in primary bovine umbilical vein endothelial cells (BUVECs) upon infection with tachyzoites, an early and pathogenic fast-replicating life stage of T. gondii and B. besnoiti. A cell bulk, derived from noninfected controls and parasite-infected cell pellets, was analyzed by AP-SMALDI MSI in technical and biological triplicates. Multivariate statistical analysis including hierarchical clustering and principle component analysis revealed infection-specific metabolites in both positive- and negative-ion mode, identified by combining database search and LC-MS2 experiments. MSI analyses of host cell monolayers were conducted at 5 μm lateral resolution, allowing single apicomplexan-infected cells to be allocated. This is the first mass spectrometry imaging study on intracellular T. gondii and B. besnoiti infections and the first detailed metabolomic characterization of B. besnoiti tachyzoites. MSI was used here as an efficient tool to discriminate infected from noninfected cells at the single-cell level in vitro.

Besnoitia besnoiti natural infection in a crossbred cow in India

Besnoitia besnoiti is an obligate intracellular parasite that causesbesnoitiosis in cattle, goats, and equids.Acute besnoitiosis is characterized bypyrexia, enlargement of the lymph nodes, anorexia, ruminal atony, and necrosis of the epidermis. A crossbred Jersey cow was referred for respiratory distress, swollen lymph nodes, patchy dermis, pyrexia, and anorexia. Skin histopathology revealed Besnoitia cysts. The animal was treated with oxytetracycline (10 mg/kg body weight, IV) for a week and with ivermectin (200 μg/kg, SC) once a week for 75 days with supportive therapy. Chronic bovine besnoitiosis was diagnosed, and the author concluded that no therapeutic plan would be successful. This case is unusual because besnoitiosis is not commonly reported in India