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

BACKGROUND: 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. METHODS AND RESULTS: 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 231 bp 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. CONCLUSIONS: Findings from this study will serve as baseline data in the epidemiology, prevention, and control of the protozoan among cattle in Iraq.

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.

Ezetimibe blocks Toxoplasma gondii-, Neospora caninum- and Besnoitia besnoiti-tachyzoite infectivity and replication in primary bovine endothelial host cells.

Coccidia are obligate apicomplexan parasites that affect humans and animals. In fast replicating species, in vitro merogony takes only 24­48 h. In this context, successful parasite proliferation requires nutrients and other building blocks. Coccidian parasites are auxotrophic for cholesterol, so they need to obtain this molecule from host cells. In humans, ezetimibe has been applied successfully as hypolipidaemic compound, since it reduces intestinal cholesterol absorption via blockage of Niemann−Pick C-1 like-1 protein (NPC1L1), a transmembrane protein expressed in enterocytes. To date, few data are available on its potential anti-parasitic effects in primary host cells infected with apicomplexan parasites of human and veterinary importance, such as Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti. Current inhibition experiments show that ezetimibe effectively blocks T. gondii, B. besnoiti and N. caninum tachyzoite infectivity and replication in primary bovine endothelial host cells. Thus, 20 µm ezetimibe blocked parasite proliferation by 73.1−99.2%, via marked reduction of the number of tachyzoites per meront, confirmed by 3D-holotomographic analyses. The effects were parasitostatic since withdrawal of the compound led to parasite recovery with resumed proliferation. Ezetimibe-glucuronide, the in vivo most effective metabolite, failed to affect parasite proliferation in vitro, thereby suggesting that ezetimibe effects might be NPC1L1-independent.

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.

Besnoitia besnoiti bradyzoite stages induce suicidal- and rapid vital-NETosis.

Besnoitia besnoiti is an obligate intracellular apicomplexan protozoan parasite, which causes bovine besnoitiosis. Recently increased emergence within Europe was responsible for significant economic losses in the cattle industry due to the significant reduction of productivity. However, still limited knowledge exists on interactions between B. besnoiti and host innate immune system. Here, B. besnoiti bradyzoites were successfully isolated from tissue cysts located in skin biopsies of a naturally infected animal, and we aimed to investigate for the first time reactions of polymorphonuclear neutrophils (PMN) exposed to these vital bradyzoites. Freshly isolated bovine PMN were confronted to B. besnoiti bradyzoites. Scanning electron microscopy (s.e.m.)- and immunofluorescence microscopy-analyses demonstrated fine extracellular networks released by exposed bovine PMN resembling suicidal NETosis. Classical NETosis components were confirmed via co-localization of extracellular DNA decorated with histone 3 (H3) and neutrophil elastase (NE). Live cell imaging by 3D holotomographic microscopy (Nanolive®) unveiled rapid vital NETosis against this parasite. A significant increase of autophagosomes visualized by specific-LC3B antibodies and confocal microscopy was observed in B. besnoiti-stimulated bovine PMN when compared to non-stimulated group. As such, a significant positive correlation (r = 0.37; P = 0.042) was found between B. besnoiti-triggered suicidal NETosis and autophagy. These findings suggest that vital- as well as suicidal-NETosis might play a role in early innate host defence mechanisms against released B. besnoiti bradyzoites from tissue cysts, and possibly hampering further parasitic replication. Our data generate first hints on autophagy being associated with B. besnoiti bradyzoite-induced suicidal NETosis and highlighting for first time occurrence of parasite-mediated vital NETosis.

Pathological findings in genital organs of bulls naturally infected with Besnoitia besnoiti.

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.

Metabolic requirements of Besnoitia besnoiti tachyzoite-triggered NETosis.

Besnoitia besnoiti is the causative agent of bovine besnoitiosis, a disease affecting both, animal welfare and cattle productivity. NETosis represents an important and early host innate effector mechanism of polymorphonuclear neutrophils (PMN) that also acts against B. besnoiti tachyzoites. So far, no data are available on metabolic requirements of B. besnoiti tachyzoite-triggered NETosis. Therefore, here we analyzed metabolic signatures of tachyzoite-exposed PMN and determined the relevance of distinct PMN-derived metabolic pathways via pharmacological inhibition experiments. Overall, tachyzoite exposure induced a significant increase in glucose and serine consumption as well as glutamate production in PMN. Moreover, tachyzoite-induced cell-free NETs were significantly diminished via PMN pre-treatments with oxamate and dichloroacetate which both induce an inhibition of lactate release as well as oxythiamine, which inhibits pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and transketolase, thereby indicating a key role of pyruvate- and lactate-mediated metabolic pathways for proper tachyzoite-mediated NETosis. Furthermore, NETosis was increased by enhanced pH conditions; however, inhibitors of MCT-lactate transporters (AR-C141900, AR-C151858) failed to influence NET formation. Moreover, a significant reduction of tachyzoite-induced NET formation was also achieved by treatments with oligomycin A (inhibitor of ATP synthase) and NF449 (purinergic receptor P2X1 antagonist) thereby suggesting a pivotal role of ATP availability for tachyzoite-mediated NETosis. In summary, the current data provide first evidence on carbohydrate-related metabolic pathways and energy supply to be involved in B. besnoiti tachyzoite-induced NETosis.

Besnoitia besnoiti-driven endothelial host cell cycle alteration.

Besnoitia besnoiti is an important obligate intracellular parasite of cattle which primarily infects host endothelial cells of blood vessels during the acute phase of infection. Similar to the closely related parasite Toxoplasma gondii, B. besnoiti has fast proliferating properties leading to rapid host cell lysis within 24-30 h p.i. in vitro. Some apicomplexan parasites were demonstrated to modulate the host cellular cell cycle to successfully perform their intracellular development. As such, we recently demonstrated that T. gondii tachyzoites induce G2/M arrest accompanied by chromosome missegregation, cell spindle alteration, formation of supernumerary centrosomes, and cytokinesis impairment when infecting primary bovine umbilical vein endothelial cells (BUVEC). Here, we follow a comparative approach by using the same host endothelial cell system for B. besnoiti infections. The current data showed that-in terms of host cell cycle modulation-infections of BUVEC by B. besnoiti tachyzoites indeed differ significantly from those by T. gondii. As such, cyclin expression patterns demonstrated a significant upregulation of cyclin E1 in B. besnoiti-infected BUVEC, thereby indicating parasite-driven host cell stasis at G1-to-S phase transition. In line, the mitotic phase of host cell cycle was not influenced since alterations of chromosome segregation, mitotic spindle formation, and cytokinesis were not observed. In contrast to respective T. gondii-related data, we furthermore found a significant upregulation of histone H3 (S10) phosphorylation in B. besnoiti-infected BUVEC, thereby indicating enhanced chromosome condensation to occur in these cells. In line to altered G1/S-transition, we here additionally showed that subcellular abundance of proliferating cell nuclear antigen (PCNA), a marker for G1 and S phase sub-stages, was affected by B. besnoiti since infected cells showed increased nuclear PCNA levels when compared with that of control cells.

Stomoxys calcitrans, mechanical vector of virulent Besnoitia besnoiti from chronically infected cattle to susceptible rabbit.

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.

Bovine besnoitiosis in an endemically infected dairy cattle herd in Italy: serological and clinical observations, risk factors, and effects on reproductive and productive performances.

Bovine besnoitiosis (Besnoitia besnoiti) is an emerging parasitic disease of cattle in Europe. This study reports a case of bovine besnoitiosis in a dairy farm housing 217 cattle in Italy. A serological screening was performed on the whole herd using the recommended approach of ELISA and confirmatory Western Blot. Seropositive animals were clinically examined to reveal symptoms and lesions of besnoitiosis. Risk factors and the effects of the parasite infection on reproductive and productive performances were evaluated. Histopathology and molecular analyses on tissues from a slaughtered cow affected by the chronic phase of the disease were carried out. An overall seroprevalence of 23.5%, which increased up to 43.5% considering only cows, was recorded. Clinical examination of 33 of the seropositive cows evidenced the presence of tissue cysts in at least one of the typical localizations (sclera, vulva, or skin) in 25 animals. Statistical analysis did not evidence any significative impact of the parasite infection on herd efficiency; however, a decrease of productive parameters was recorded in cows showing cutaneous cysts. Concerning the chronically affected cow, histopathology revealed B. besnoiti tissue cysts in the skin of the neck, rump, hind legs, eyelid and vulva, in the muzzle, in mucosal membranes of the upper respiratory tract, and in the lungs. Parasite DNA was detected also in masseter muscles, tonsils, mediastinal lymph nodes, liver, cardiac muscle, aorta wall, ovaries, uterus, and vulva. Bovine besnoitiosis continues to spread in the Italian cattle population. Breeders and veterinarians should be aware of this parasitic disease, and control programs should be developed based on surveillance through a diagnostic procedure including both clinical examination and laboratory tests.

Importance of serological cross-reactivity among Toxoplasma gondii, Hammondia spp., Neospora spp., Sarcocystis spp. and Besnoitia besnoiti.

Toxoplasma gondii, Neospora spp., Sarcocystis spp., Hammondia spp. and Besnoitia besnoiti are genetically related cyst-forming coccidia. Serology is frequently used for the identification of T. gondii, Neospora spp. and B. besnoiti-exposed individuals. Serologic cross-reactions occur in different tests among animals infected with T. gondii and H. hammondi, as well as among animals infected by T. gondii and N. caninum. Infections caused by N. caninum and N. hughesi are almost indistinguishable by serology. Neospora caninum, B. besnoiti and Sarcocystis spp. infections in cattle show some degree of serologic cross-reactivity. Antibody cross-reactivity between Neospora spp. and H. heydorni-infected animals is suspected, but not proven to occur. We review serologic cross-reactivity among animals and/or humans infected with T. gondii, Neospora spp., Sarcocystis spp., Hammondia spp. and B. besnoiti. Emphasis is laid upon antigens and serological methods for N. caninum diagnosis which were tested for cross-reactivity with related protozoa. Species-specific antigens, as well as stage-specific proteins have been identified in some of these parasites and have promising use for diagnosis and epidemiological surveys.

Serological dynamics and risk factors of Besnoitia besnoiti infection in breeding bulls from an endemically infected purebred beef herd.

Bovine besnoitiosis has been deemed a re-emerging disease in Western Europe and considered endemic in Spain, Portugal, France and in some areas of Northern Italy. This report refers to an infection outbreak in a purebred beef herd from Northern Italy involving a large number of bulls. In October 2013, 544 animals were serologically tested with an in-house ELISA followed by a confirmatory Western blot to evaluate Besnoitia besnoiti seroprevalence. A year later, 461 animals were then serologically re-tested together with imported animals (n = 268). Overall, 812 animals were involved in the study. Histology and immunohistochemistry were performed on skin biopsies of suspected animals and several tissue samples from a slaughtered bull. In the first sampling, 100 animals were seropositive (18.4%); in the second sampling, prevalence increased up to 36.5%, with incidence calculated at 39.6%. The risk factor analysis revealed that the infection was associated with age (OR = 1.007) and sex, with males presenting a greater risk (OR = 2.006). In fact, prevalence values in bulls increased from 29.6 to 56.7%, with an incidence of infection of 53.3%. Moreover, mating with a seropositive bull enhanced infection risk for a seronegative cow (OR = 1.678). Clinical signs typical of bovine besnoitiosis were found in seven seropositive animals, with confirmation of B. besnoiti through histology and immunohistochemistry. The study outcomes confirm that bovine besnoitiosis is a disease with serious economic impact on beef cattle breeding, particularly on bulls in service. Good management practises such as clinical monitoring and serological testing of imported animals should be implemented to control its occurrence.

Seroprevalence and risk factors of Besnoitia besnoiti infection in Korean cattle - short communication.

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.

Characterization of an outbreak of emerging bovine besnoitiosis in southwestern Spain.

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 Avileña 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.

Selected metabolic biochemical and enzyme activities associated with Besnoitia besnoiti infection in dairy cattle.

The main aim of the study was to explore, compare, and identify whether there is an association between Besnoitia besnoiti seropositivity in apparently healthy dairy cows with some biochemical parameters, enzyme activities, and beta-hydroxybutyrate (BHBA). A total of 98 dairy cows were included in the study, of which there was 50 seropositive and 48 seronegative cows. Analysis of serum antibodies against B. besnoiti antibodies was performed using an indirect enzyme-linked immunosorbent assay kit. Student's independent t test showed that there was a significant difference in BHBA, albumin, and lactate dehydrogenase (LDH) between the seropositive and seronegative groups. Univariable regression analysis showed no significant association between seropositivity status with any of the evaluated parameters except BHBA level, mastitis, and abomasum displacement. Multivariable logistic regression analysis showed that there was a strong association between seropositivity with BHBA level. The significant association between BHBA and B. besnoiti seropositivity represents preliminary finding that needs further exploration.

Naturally acquired bovine besnoitiosis: histological and immunohistochemical findings in acute, subacute, and chronic disease.

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.

Transcriptomics of Besnoitia besnoiti-Infected Fibroblasts Reveals Hallmarks of Early Fibrosis and Cancer Progression.

Endothelial injury, inflammatory infiltrate and fibrosis are the predominant lesions in the testis of bulls with besnoitiosis that may result in sterility. Moreover, fibroblasts, which are key players in fibrosis, are parasite target cells in a Besnoitia besnoiti chronic infection. This study aimed to decipher the molecular basis that underlies a drift toward fibrosis during the disease progression. Transcriptomic analysis was developed at two times post-infection (p.i.), representative of invasion (12 h p.i.) and intracellular proliferation (32 h p.i.), in primary bovine aorta fibroblasts infected with B. besnoiti tachyzoites. Once the enriched host pathways were identified, we studied the expression of selected differentially expressed genes (DEGs) in the scrotal skin of sterile infected bulls. Functional enrichment analyses of DEGs revealed shared hallmarks of cancer and early fibrosis. Biomarkers of inflammation, angiogenesis, cancer, and MAPK signaling stood out at 12 h p.i. At 32 h p.i., again MAPK and cancer pathways were enriched together with the PI3K-AKT pathway related to cell proliferation. Some DEGs were also regulated in the skin samples of naturally infected bulls (PLAUR, TGFß1, FOSB). We have identified potential biomarkers and host pathways regulated during fibrosis that may hold prognostic significance and could emerge as potential therapeutic targets.

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.

Transcriptional changes associated with apoptosis and type I IFN underlie the early interaction between Besnoitia besnoiti tachyzoites and monocyte-derived macrophages.

Besnoitia besnoiti-infected bulls may develop severe systemic clinical signs and orchitis that may ultimately cause sterility during the acute infection. Macrophages might play a relevant role in pathogenesis of the disease and the immune response raised against B. besnoiti infection. This study aimed to dissect the early interaction between B. besnoiti tachyzoites and primary bovine monocyte-derived macrophages in vitro. First, the B. besnoiti tachyzoite lytic cycle was characterized. Next, dual transcriptomic profiling of B. besnoiti tachyzoites and macrophages was conducted at early infection (4 and 8 h p.i.) by high-throughput RNA sequencing. Macrophages inoculated with heat-killed tachyzoites (MO-hkBb) and non-infected macrophages (MO) were used as controls. Besnoitia besnoiti was able to invade and proliferate in macrophages. Upon infection, macrophage activation was demonstrated by morphological and transcriptomic changes. Infected macrophages were smaller, round and lacked filopodial structures, which might be associated with a migratory phenotype demonstrated in other apicomplexan parasites. The number of differentially expressed genes (DEGs) increased substantially during infection. In B. besnoiti-infected macrophages (MO-Bb), apoptosis and mitogen-activated protein kinase (MAPK) pathways were regulated at 4 h p.i., and apoptosis was confirmed by TUNEL assay. The Herpes simplex virus 1 infection pathway was the only significantly enriched pathway in MO-Bb at 8 h p.i. Relevant DEGs of the Herpes simplex virus 1 infection (IFNα) and the apoptosis pathways (CHOP-2) were also significantly regulated in the testicular parenchyma of naturally infected bulls. Furthermore, the parasite transcriptomic analysis revealed DEGs mainly related to host cell invasion and metabolism. These results provide a deep overview of the earliest macrophage modulation by B. besnoiti that may favour parasite survival and proliferation in a specialized phagocytic immune cell. Putative parasite effectors were also identified.

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

Background: 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. Objectives: 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. Methods: 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. Results: 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. Discussion and conclusions: 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.