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.

First report of Demodex bovis infestation in bovine besnoitiosis co-infected dairy cattle in Italy.

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.

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.

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.

Dissection of the in vitro developmental program of Hammondia hammondi reveals a link between stress sensitivity and life cycle flexibility in Toxoplasma gondii.

Most eukaryotic parasites are obligately heteroxenous, requiring sequential infection of different host species in order to survive. Toxoplasma gondii is a rare exception to this rule, having a uniquely facultative heteroxenous life cycle. To understand the origins of this phenomenon, we compared development and stress responses in T. gondii to those of its its obligately heteroxenous relative, Hammondia hammondi and have identified multiple H. hammondi growth states that are distinct from those in T. gondii. Of these, the most dramatic difference was that H. hammondi was refractory to stressors that robustly induce cyst formation in T. gondii, and this was reflected most dramatically in its unchanging transcriptome after stress exposure. We also found that H. hammondi could be propagated in vitro for up to 8 days post-excystation, and we exploited this to generate the first ever transgenic H. hammondi line. Overall our data show that H. hammondi zoites grow as stringently regulated, unique life stages that are distinct from T. gondii tachyzoites, and implicate stress sensitivity as a potential developmental innovation that increased the flexibility of the T. gondii life cycle.

A new species of Cystoisospora Frenkel, 1977 (Apicomplexa: Sarcocystidae) from Oecomys mamorae Thomas (Rodentia: Cricetidae) in the Brazilian Pantanal.

Despite the great diversity of coccidians, to our knowledge, no coccidian infections have been described in Oecomys spp. In this context, we examined Oecomys mamorae Thomas (Rodentia: Cricetidae) from the Brazilian Pantanal for infections with enteric coccidia. Nine individuals were sampled, and one was found to be infected. The oöcysts were recovered through centrifugal flotation in sugar solution. Using morphological and morphometric features, we described a new species of Cystoisospora Frenkel, 1977. Sporulated oöcysts were ovoidal 20.0-29.1 × 16.4-23.2 (26.7 × 21.2) µm and contained two sporocysts, 12.9-19.1 × 9.4-13.9 (16.4 × 12.4) µm, each with four banana-shaped sporozoites. Polar granule and oöcyst residuum were both absent. We documented the developmental forms in the small intestine and described the histopathological lesions in the enteric tract. Our results indicate that the prevalence of Cystoisospora mamorae n. sp. in O. mamorae is low, and tissue damage in the enteric tract is mild, even in the presence of coccidian developmental stages.

Bobcats (Lynx rufus) are natural definitive host of Besnoitia darlingi.

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±0.2µm; 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.

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.

Chronic Cystoisospora belli infection in an immunocompetent Myanmar refugee - microscopy is not sensitive enough.

BACKGROUND: Cystoisosporiasis is an opportunistic infection seen more commonly in patients with acquired immunodeficiency syndrome. Although uncommon, Cystoisospora infection can occur in immunocompetent individuals but tend to be benign and self-limiting. Chronic infection however, has been described but diagnosis can often be challenging and requires a high clinical index of suspicion. CASE PRESENTATION: We present a case of delayed diagnosis of Cystoisospora belli (C. belli) in an immunocompetent 28-year-old refugee from Myanmar. She had a history of chronic diarrhea where exhaustive investigations over many years failed to reveal a diagnosis. Cystoisospora belli cysts were finally detected in stool 4 years after investigation commenced, and PCR testing on stored colon biopsies amplified a molecular product with 99 % sequence homology to C. belli. The patient improved promptly with trimethoprim-sulfamethoxazole treatment. CONCLUSION: In the appropriate clinical context we suggest molecular testing for C. belli or an empirical therapeutic trial.

Naturally acquired bovine besnoitiosis: Differential distribution of parasites in the skin of chronically infected cattle.

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 MedianCtindv 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.

Experimental infections of rabbits with proliferative and latent stages of Besnoitia besnoiti.

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 µg 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 °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.

Experimental transmission of Cystoisospora felis-like coccidium from bobcat (Lynx rufus) to the domestic cat (Felis catus).

Cystoisospora felis is an ubiquitous coccidian of cats. The domestic cat (Felis catus) is its definitive host and several mammalian and avian species are its optional intermediate/transport hosts. Nothing is known if it is transmissible to wild felids. In the present study C. felis-like oocysts were found in two naturally infected bobcats (Lynx rufus) from Pennsylvania. To study transmission of C. felis-like parasite from bobcats to domestic cats, sporulated oocysts of C. felis-like from one bobcat were orally inoculated into interferon gamma gene knockout (KO) mice, and 56 days later tissues of KO mice were fed to two coccidian-free cats; two littermate cats were uninoculated controls. The inoculated cats and controls were euthanized five and seven days later, and their small intestines were studied histologically. One inoculated cat excreted C. felis-like oocysts seven days post inoculation (p.i.) and was immediately euthanized. Mature schizonts, mature male and female gamonts, and unsporulated oocysts were found in the lamina propria of small intestine; these stages were morphologically similar to C. felis of domestic cats. No parasites were seen in histological sections of small intestines of the remaining three cats. The experiment was terminated at seven days p.i. (minimum prepatent period for C. felis) to minimize spread of this highly infectious parasite to other cats. Although oocysts of the parasite in bobcats were morphologically similar to C. felis of domestic cats, the endogenous stages differed in their location of development. The bobcat derived parasite was located in the lamina propria of ileum whereas all endogenous stages of C. felis of domestic cats are always located in enterocytes of intestinal epithelium. Characterization of DNA isolated from C. felis-like oocysts from the donor bobcat revealed that sequences of the ITS1 region was only 87% similar to the ITS1 region of C. felis from domestic cats. These results indicate that the parasite in bobcat is likely different than C. felis of cats.

Life Cycle of Hammondia hammondi (Apicomplexa: Sarcocystidae) in Cats.

Hammondia hammondi and Toxoplasma gondii are feline coccidians that are morphologically, antigenically, and phylogenitically related. Both parasites multiply asexually and sexually in feline intestinal enterocytes, but H. hammondi remains confined to enterocytes whereas T. gondii also parasitizes extra-intestinal tissues of the cat. Here, we studied multiplication of H. hammondi in feline intestine and compared with T. gondii cycle. Five parasite-free cats were inoculated orally with tissue cysts and free bradyzoites from skeletal muscles of gamma interferon gene knockout mice and killed at 1, 3, 4, 6, and 7 d later. At 1 and 3 d post inoculation (DPI), numerous individual intracellular bradyzoites were detected in histological sections of small intestine. At 4 DPI only schizonts were found and they were located in enterocyte cytoplasm above the host cell nucleus. At 6 and 7 DPI both schizonts and gamonts were seen and they were located in enterocytes. Ultrastucturally, schizogonic and gametogonic development of H. hammondi was similar to T. gondii. However, in H. hammondi merozoites rhoptries were longer, and coiled and contained more micronemes than in T. gondii. Ultrastructural development is illustrated in detail.

Dynamics of Besnoitia besnoiti infection in cattle.

Bovine besnoitiosis is caused by the cyst-forming apicomplexan parasite Besnoitia besnoiti. This disease progresses in two sequential phases: a febrile acute phase with oedemas and respiratory disorders, and a chronic phase characterized by the presence of subcutaneous tissue cysts and skin lesions. Serious consequences of the infection are poor body condition, sterility in bulls and eventual death. The role of host/parasite-dependent factors, which play a major role in the pathogenesis of the disease, is not yet fully elucidated. Isolate/strain virulence, parasite stage, dose and the route of parasite inoculation were studied under different experimental conditions, which make it difficult to compare the results. Data on host-dependent factors obtained from naturally infected cattle showed that (i) the seroprevalence of infection is similar in both sexes; (ii) seropositivity increases with age; (iii) both beef and dairy cattle are susceptible to the infection; and (iv) the cell-mediated immune response is likely to play a major role because a T cell response has been observed around several tissue cysts. Whether colostral antibodies are protective and to what extent the humoral immune response might reflect the disease/protection status require further research. Thus, a well-established experimental bovine model could help to clarify these important questions. The dynamics of B. besnoiti infection in cattle and available knowledge on relevant factors in the pathogenesis of the infection are reviewed in the present work.

Cystoisospora canis (Apicomplexa: Sarcocystidae): development of monozoic tissue cysts in human cells, demonstration of egress of zoites from tissue cysts, and demonstration of repeat monozoic tissue cyst formation by zoites.

Sporozoites of Cystoisospora canis penetrated and developed to monozoic tissue cysts in 4 human, 1 monkey, 1 bovine and 2 canine cell lines. No asexual division was documented although multiple infection of a single cell was observed. Examination of cultures using transmission electron microscopy demonstrated that they were monozoic tissue cysts and contained a single sporozoite. The appearance of monozoic tissue cysts in all cell lines was similar but the parasitophorous vacuole surrounding some sporozoites in DH82 dog macrophages was swollen. Monozoic tissue cysts were observed for up to 127 days in human pigmented retinal epithelial cells. Treatment of cell cultures containing monozoic tissue cysts with 0.75 sodium taurocholic acid and 0.25% trypsin stimulated egress of zoites (former sporozoites) from tissue cysts. Zoites collected from monozoic tissue cysts were able to penetrate and develop to monozoic tissue cysts in new host cells. Monozoic tissue cysts survived exposure to acid pepsin solution indicating that they would be orally infectious. The tissue cyst wall surrounding zoites did not autofluoresce as did oocyst and sporocyst walls exposed to UV light. We believe that C. canis can be used as a model system to study extra-intestinal monozoic tissue cysts stages of Cystoisospora belli of humans.

A century of bovine besnoitiosis: an unknown disease re-emerging in Europe.

Bovine besnoitiosis, which is caused by the cyst-forming apicomplexan parasite Besnoitia besnoiti, is a chronic and debilitating vector-borne disease characterized by both cutaneous and systemic manifestations. In Europe, this parasitic disease appeared in a few restricted areas in France and Portugal since the first recorded cases in the beginning of the 20th century. However, at present, the disease is considered to be re-emerging by the European Food Safety Authority due to an increased number of cases and the geographic expansion of besnoitiosis into cattle herds in several European countries. In this review, we will provide an update of the epidemiology and impact of B. besnoiti infection. Strategies to control this parasitic disease will also be discussed.

Development of early tissue cysts and associated pathology of Besnoitia besnoiti in a naturally infected bull (Bos taurus) from South Africa.

Besnoitia besnoiti is an apicomplexan that causes serious economic loss in cattle in many countries and the disease is now spreading in Europe. At least 2 phases of bovine besnoitiosis are recognized clinically. An acute febrile phase characterized by anasarca and necrosis of skin is associated with multiplication of tachyzoites in vascular endothelium; this phase is short-lived and rarely diagnosed. Chronic besnoitiosis characterized by dermal lesions is associated with the presence of macroscopic tissue cysts and is easily diagnosed. Here we report the development of early B. besnoiti tissue cysts in a naturally infected Hugenoot bull from South Africa. Tissue cysts were 10-70 µm in diameter, contained 1-12 bradyzoites, and were most numerous in the dermis, testicles, and pampiniform venous plexus. Amylopectin granules in bradyzoites stained red with periodic acid Schiff (PAS) reaction. Bradyzoites varied in size and in the intensity of PAS reaction (some were PAS-negative), some were plump, and others were slender. With immunohistochemical staining with Besnoitia oryctofelisi and bradyzoite-specific antibodies (BAG-1 made against Toxoplasma gondii bradyzoites), the staining was confined to parasites, and all intracystic organisms were BAG-1 positive. With Gomori's silver stain only bradyzoites were stained very faintly whereas the rest of the tissue cyst was unstained. Ultrastructurally many tissue cysts contained dead bradyzoites and some appeared empty. Unlike bradyzoites from mature cysts, bradyzoites in the present case contained few or no micronemes. These findings are of diagnostic significance. Ultrastructually host cyst cells had features of myofibroblasts, and immunohistochemistry using antibodies against MAC387, lysozyme, vimentin, Von Willebrand factor, and smooth muscle actin confirmed this. Polymerase chain reaction on DNA extracted from lymph node of the bull confirmed B. besnoiti diagnosis. Associated clinical findings, lesions, and histopathology are briefly presented. The bull died of nephrotic syndrome; anasarca in acute besnoitiosis due to protein-losing glomerulopathy is a finding not previously reported in cattle.

Evidence for bovine besnoitiosis being endemic in Italy--first in vitro isolation of Besnoitia besnoiti from cattle born in Italy.

Until 2009, bovine besnoitiosis had never been considered endemic in Italy and the only report on the disease in this country referred to animals imported from France shortly before. However, recently, an autochthonous outbreak of bovine besnoitiosis was reported in four herds located at the intersection of the borders between Emilia-Romagna, Toscana and Marche (Northern Apennine Mountains), which has led to an increased awareness concerning this disease. The present study describes a further outbreak of bovine besnoitiosis in Italy. The afflicted herd was a dairy herd with no evidence for contact with cattle from regions known to be endemic for bovine besnoitiosis. The farm investigation was initiated after a three-year old Holstein Friesian dairy cow with generalized thickening and lichenification of the skin was diagnosed with bovine besnoitiosis. The clinical diagnosis was confirmed by gross pathology, histopathology, serology and PCR. Bradyzoites released from tissue cysts obtained from the skin of this animal enabled the first in vitro isolation of Besnoitia besnoiti in Italy. This isolate was named Bb-Italy1. Sequencing of a 2118 bp spanning region including the complete internal transcribed spacer 1 and parts of the 18S and the 5.8S rRNA gene from DNA extracted from skin-derived zoites revealed a 99.9% identity to sequences known for other B. besnoiti isolated from cattle in Europe. Two GKO mice which had been inoculated intraperitoneally with bovine skin-derived bradyzoites became ill 7 days post inoculation. Parasitophorous vacuoles with multiplying zoites were observed in the cell culture inoculated with peritoneal fluids of these mice and a B. besnoiti infection in the mice and in the cell culture could be confirmed by real-time PCR. A serological investigation in the afflicted herd using immunoblots and an immunofluorescent antibody test (IFAT) revealed an overall herd seroprevalence of 9.7% (31/321), whereas within the female animals older than 2 years 17.0% (29/171) of the dams were tested positive. With one exception, an imported cow from Germany, all the seropositive animals were born in Italy. In connection with previously described autochthonous cases of bovine besnoitiosis the case described herein suggests that bovine besnoitiosis should be considered endemic in Italy.

Bovine besnoitiosis: epidemiological and clinical aspects.

Bovine besnoitiosis, due to the cyst-forming apicomplexan Besnoitia besnoiti, is widespread in Africa, in Asia and in the south of Europe. This infection leads to major economic losses in cattle production with a decrease in milk production, transient of definitive sterility of bulls and mortality. Since its emergence in Europe (Germany, Spain and Centre of France), more attention has been given to this infection during the last 10 years, including the recent development of serological methods for diagnosis. The life cycle of B. besnoiti remains a mystery: the definitive host is unknown and the transmission routes are poorly understood. B. besnoiti is thought to be transmitted mainly by hematophagous insects. This paper presents a review of what is currently known of the epidemiology of bovine besnoitiosis and the key manifestations of the disease. It highlights the urgent need for research programs to improve efficient and sustainable control methods.

Besnoitia oryctofelisi n. sp. (Protozoa: Apicomplexa) from domestic rabbits.

A species of Besnoitia from naturally infected rabbits from Argentina was propagated experimentally in mice, gerbils, rabbits, cats, and cell cultures. Cats fed tissue cysts from rabbits shed oocysts with a prepatent period of nine to 13 days. Sporulated oocysts were infective to gerbils, rabbits, outbred Swiss Webster and interferon gamma gene knockout mice. Bradyzoites were infective orally to gerbils and cats. Tachyzoites were successfully cultivated and maintained in vitro in bovine monocytes and African green monkey kidney cells. Schizonts were seen in the lamina propria of the small intestine of cats fed tissue cysts; the largest ones measured 52 x 45 microm. Schizonts were also present in mesenteric lymph nodes, livers, and other extra-intestinal organs of cats fed tissue cysts. Oocysts were 10-14 x 10-13 microm in size. This rabbit-derived species of Besnoitia resembled B. darlingi of the North American opossum, Didelphis virginiana with an opossum-cat cycle, but it was not transmissible to D. virginiana, and B. darlingi of opossums was not transmissible to rabbits. Based on biological, serological, antigenic, and molecular differences between the rabbit and the opossum Besnoitia, a new name, B. oryctofelisi is proposed for the parasite from domestic rabbits from Argentina.