Molecular analysis suggests that Namibian cheetahs (Acinonyx jubatus) are definitive hosts of a so far undescribed Besnoitia species.

BACKGROUND: Besnoitia darlingi, B. neotomofelis and B. oryctofelisi are closely related coccidian parasites with felids as definitive hosts. These parasites use a variety of animal species as intermediate hosts. North American opossums (Didelphis virginiana), North American southern plains woodrats (Neotoma micropus) and South American domestic rabbits (Oryctolagus cuniculus) are intermediate hosts of B. darlingi, B. neotomofelis and B. oryctofelisi, respectively. Based on conserved regions in the internal transcribed spacer-1 (ITS1) sequence of the ribosomal DNA (rDNA), a real-time PCR for a sensitive detection of these Besnoitia spp. in tissues of intermediate hosts and faeces of definitive hosts has recently been established. Available sequence data suggest that species such as B. akodoni and B. jellisoni are also covered by this real-time PCR. It has been hypothesised that additional Besnoitia spp. exist worldwide that are closely related to B. darlingi or B. darlingi-like parasites (B. neotomofelis, B. oryctofelisi, B. akodoni or B. jellisoni). Also related, but not as closely, is B. besnoiti, the cause of bovine besnoitiosis. METHODS: Faecal samples from two free-ranging cheetahs (Acinonyx jubatus) from Namibia that had previously tested positive for coccidian parasites by coproscopy were used for this study. A conventional PCR verified the presence of coccidian parasite DNA. To clarify the identity of these coccidia, the faecal DNA samples were further characterised by species-specific PCRs and Sanger sequencing. RESULTS: One of the samples tested positive for B. darlingi or B. darlingi-like parasites by real-time PCR, while no other coccidian parasites, including Toxoplasma gondii, Hammondia hammondi, H. heydorni, B. besnoiti and Neospora caninum, were detected in the two samples. The rDNA of the B. darlingi-like parasite was amplified and partially sequenced. Comparison with existing sequences in GenBank revealed a close relationship to other Besnoitia spp., but also showed clear divergences. CONCLUSIONS: Our results suggest that a so far unknown Besnoitia species exists in Namibian wildlife, which is closely related to B. darlingi, B. neotomofelis, B. oryctofelisi, B. akodoni or B. jellisoni. The cheetah appears to be the definitive host of this newly discovered parasite, while prey species of the cheetah may act as intermediate hosts.

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.

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.

Besnoitia besnoiti infection alters both endogenous cholesterol de novo synthesis and exogenous LDL uptake in host endothelial cells.

Besnoitia besnoiti, an apicomplexan parasite of cattle being considered as emergent in Europe, replicates fast in host endothelial cells during acute infection and is in considerable need for energy, lipids and other building blocks for offspring formation. Apicomplexa are generally considered as defective in cholesterol synthesis and have to scavenge cholesterol from their host cells for successful replication. Therefore, we here analysed the influence of B. besnoiti on host cellular endogenous cholesterol synthesis and on sterol uptake from exogenous sources. GC-MS-based profiling of cholesterol-related sterols revealed enhanced cholesterol synthesis rates in B. besnoiti-infected cells. Accordingly, lovastatin and zaragozic acid treatments diminished tachyzoite production.  Moreover, increased lipid droplet contents and enhanced cholesterol esterification was detected and inhibition of the latter significantly blocked parasite proliferation. Furthermore, artificial increase of host cellular lipid droplet disposability boosted parasite proliferation. Interestingly, lectin-like oxidized low density lipoprotein receptor 1 expression was upregulated in infected endothelial hostcells, whilst low density lipoproteins (LDL) receptor was not affected by parasite infection. However, exogenous supplementations with non-modified and acetylated LDL both boosted B. besnoiti proliferation. Overall, current data show that B. besnoiti simultaneously exploits both, endogenous cholesterol biosynthesis and cholesterol uptake from exogenous sources, during asexual replication.

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.

In vitro treatment of Besnoitia besnoiti with the naphto-quinone buparvaquone results in marked inhibition of tachyzoite proliferation, mitochondrial alterations and rapid adaptation of tachyzoites to increased drug concentrations.

We here assessed the in vitro efficacy of the naptho-quinone buparvaquone (BPQ) against Besnoitia besnoiti tachyzoites in vitro. BPQ is currently licensed for the treatment of theileriosis in cattle in many countries, but not in the EU. In 4-day treatment assays, BPQ massively impaired tachyzoite proliferation with an IC50 of 10 ± 3 nm, and virtually complete inhibition was obtained in the presence of nm BPQ. Exposure to 1 µm BPQ leads to ultrastructural changes affecting initially the mitochondrial matrix and the cristae. After 96 h, most parasites were largely distorted, filled with cytoplasmic amylopectin granules and vacuoles containing components of unknown composition. Host cell mitochondria did not appear to be notably affected by the drug. However, upon prolonged exposure (14-16 days) to increased BPQ concentrations, B. besnoiti tachyzoites exhibited the capacity to adapt, and they resumed proliferation at dosages of up to 10 µm BPQ, albeit at a lower rate. These BPQ-adapted parasites maintained this lower susceptibility to BPQ treatment after freeze-thawing, and inspection by the transmission electron microscopy revealed that they underwent proliferation in the absence of structurally intact mitochondria.

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.

The role of wild ruminants as reservoirs of Besnoitia besnoiti infection in cattle.

Bovine besnoitiosis, a parasitic disease caused by Besnoitia besnoiti, has been reported mainly in beef cattle raised under extensive pastoral systems and is considered to be re-emerging in Western Europe. Horizontal transmission probably occurs either by means of blood sucking arthropods or as a consequence of direct contact between infected and non-infected cattle. However, the role that wild ruminants (e.g., red deer (Cervus elaphus) and roe deer (Capreolus capreolus)) may play in the parasite life cycle as putative reservoirs remains elusive. Thus, we investigated the presence of Besnoitia spp. infection in 2608 wild ruminants located in areas where bovine besnoitiosis is present and identified the Besnoitia species detected. First, a serosurvey was conducted in red deer (n=309), roe deer (n=417), Pyrenean chamois (Rupicapra p. pyrenaica, n=383) and Iberian wild goat (Capra pyrenaica hispanica, n=288) from two areas of Aragon, northeastern Spain, where bovine besnoitiosis is endemic. Second, red deer (n=820), roe deer (n=37), fallow deer (Dama dama, n=166), Iberian wild goat (n=86) and European mouflon (Ovis orientalis musimon, n=102) from southwestern Spain, where new outbreaks have recently been reported, were also sampled. The presence of Besnoitia spp.-specific antibodies was confirmed by western blot in one red deer and one roe deer from the Pyrenees, and Besnoitia spp. DNA was detected by ITS1-PCR in the seropositive red deer. Besnoitia genotyping based on 6 microsatellite (MS) analyses was carried out in red deer samples and compared with B. besnoiti genotypes from 7 in vitro isolates and 3 infected bovines, B. tarandi (1 isolate) and B. bennetti (from tissues of an infected donkey) for Besnoitia spp. assignation. Multilocus MS analysis of B. besnoiti, B. tarandi and B. bennetti showed specific genotypes for each species. A restricted genetic diversity with two genotypes by variation in a unique MS marker was revealed among the 7 B. besnoiti isolates. Incomplete Besnoitia spp. genotype of 3 MS markers from red deer samples entirely matched the B. besnoiti genotypes. Accordingly, this work gives clues for the presence of B. besnoiti infection in red deer from Western Europe. Further molecular genotyping is needed to confirm that red deer may act as an intermediate host of B. besnoiti, although the low prevalences that were found indicate that wild ruminant species do not pose a significant risk of transmitting the infection to cattle.

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.

Epidemiologic study on Besnoitia besnoiti infection in dairy herds in Jordan.

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

Secreted effectors in Toxoplasma gondii and related species: determinants of host range and pathogenesis?

Recent years have witnessed the discovery of a number of secreted proteins in Toxoplasma gondii that play important roles in host-pathogen interactions and parasite virulence, particularly in the mouse model. However, the role that these proteins play in driving the unique features of T. gondii compared to some of its nearest apicomplexan relatives (Hammondia hammondi and Neospora caninum) is unknown. These unique features include distinct dissemination characteristics in vivo and a vast host range. In this review we comprehensively survey what is known about disease outcome, the host response and host range for T. gondii, H. hammondi, and N. caninum. We then review what is presently known about recently identified secreted virulence effectors in these three genetically related, but phenotypically distinct, species. Finally we exploit the existence of genome sequences for these three organisms and discuss what is known about the presence, and functionality, of key T. gondii effectors in these three species.

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.