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This is the first report on the development and characterization of eight monoclonal antibodies (MABs) generated against whole- and membrane-enriched tachyzoite extracts of the apicomplexan parasite Besnoitia besnoiti. Confocal laser scanning immunofluorescence microscopy was used to localize respective epitopes in B. besnoiti tachyzoites along the lytic cycle. A pattern compatible with dense granule staining was observed with MABs 2.A.12, 2.F.3 and 2.G.4, which could be confirmed by immunogold electron microscopy for MABs 2.A.12 and 2.F.3. In particular, MABs 2.F.3 and 2.G.4 were secreted during early invasion, proliferation and egress phases. MABs 3.10.8 and 5.5.11 labelled the tachyzoite surface, whilst MABs 1.17.8, 8.9.2 and 2.G.A recognized the apical tip, which is reminiscent for microneme localization. Besides, the epitopes recognized by the latter two (MABs 8.9.2 and 2.G.A) exhibited a redistribution from the anterior part across the parasite surface towards the posterior end during invasion. Most MABs developed were genus-specific. Indeed, the MABs cross-reacted neither with T. gondii nor with N. caninum tachyzoites. In summary, we have generated MABs that will be useful to study the key processes in the lytic cycle of the parasite and with additional promising diagnostic value. However, the molecular identity of the antigens recognized remains to be elucidated.

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.

Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a chronic and debilitating disease that causes systemic and skin manifestations and sterility in bulls. Neither treatments nor vaccines are currently available. In the search for therapeutic candidates, calcium-dependent protein kinases have arisen as promising drug targets in other apicomplexans (e.g. Neospora caninum, Toxoplasma gondii, Plasmodium spp. and Eimeria spp.) and are effectively targeted by bumped kinase inhibitors. In this study, we identified and cloned the gene coding for BbCDPK1. The impact of a library of nine bumped kinase inhibitor analogues on the activity of recombinant BbCDPK1 was assessed by luciferase assay. Afterwards, those were further screened for efficacy against Besnoitiabesnoiti tachyzoites grown in Marc-145 cells. Primary tests at 5µM revealed that eight compounds exhibited more than 90% inhibition of invasion and proliferation. The compounds BKI 1294, 1517, 1553 and 1571 were further characterised, and EC 99 (1294: 2.38µM; 1517: 2.20µM; 1553: 3.34µM; 1571: 2.78µM) were determined by quantitative real-time polymerase chain reaction in 3-day proliferation assays. Exposure of infected cultures with EC 99 concentrations of these drugs for up to 48h was not parasiticidal. The lack of parasiticidal action was confirmed by transmission electron microscopy, which showed that bumped kinase inhibitor treatment interfered with cell cycle regulation and non-disjunction of tachyzoites, resulting in the formation of large multi-nucleated complexes which co-existed with viable parasites within the parasitophorous vacuole. However, it is possible that, in the face of an active immune response, parasite clearance may occur. In summary, bumped kinase inhibitors may be effective drug candidates to control Besnoitiabesnoiti infection. Further in vivo experiments should be planned, as attainment and maintenance of therapeutic blood plasma levels in calves, without toxicity, has been demonstrated for BKIs 1294, 1517 and 1553., (Copyright © 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

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., (Published 2014. This article is a U.S. Government work and is in the public domain in the USA.)

Current knowledge on bovine besnoitiosis, caused by the emerging apicomplexan pathogen Besnoitia besnoiti, is still fragmentary. So far, studies dealing with ultrastructural pathology focused mainly on the easily accessible chronic stage, whereas ultrastructural investigations of tachyzoites were confined to in vitro studies. In the study presented here, the ultrastructural pathology of naturally B. besnoiti-infected cattle in the acute and chronic disease stages and experimentally B. besnoiti-infected mice was monitored. Further, the ultrastructure of tachyzoites and bradyzoites was investigated. Skin samples of two adult Limousin cows and one adult Limousin bull naturally infected with B. besnoiti and liver and skin samples of gamma-interferon knockout mice infected with B. besnoiti were examined in semithin sections stained with toluidine blue and safranin and in ultrathin sections contrasted with uranyl acetate and lead citrate. Samples of vessel walls of the bull and nasal mucosa of one cow were examined by scanning electron microscopy. Few tachyzoites-like endozoites were detected for the first time in bovine skin, and large numbers of tachyzoites were detected in murine skin and liver. Within tissue cysts in bovine skin, numerous bradyzoites were observed displaying signs of degeneration. Tachyzoites had apicomplexan endozoite ultrastructure. B. besnoiti tachyzoites and bradyzoites differed in shape and the number of amylopectin granules. Transmission and scanning electron microscopy confirmed the presence of two different cyst wall layers, and the present results on cyst wall ultrastructure were in accordance with those previously obtained by histological sections.

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.

The objective of this study was to establish a standardized protocol to monitor Besnoitia tarandi prevalence and intensity in barren-ground caribou (Rangifer tarandus) herds by: 1) calculating the relative sensitivity and specificity of the gross visual assessment of four anatomical sites compared with microscopic evaluation, and 2) determining which of four anatomical sampling sites was the most sensitive for detecting B. tarandi cysts by microscopy. Sampled tissues consisted of the conjunctiva of the left eye and skin sections from the rostrum, metatarsus, and thigh from 312 harvested caribou. Diagnosis of infection with B. tarandi was based on observation of at least one cyst by microscopic examination. For each tissue, the maximal density of cysts (number of B. tarandi cysts/mm(2) in the section examined) was calculated for a measured area consisting of the dermis extending from the epidermis of the skin to the base of the hair follicles and adnexal structures. For the conjunctiva, the entire submucosa was evaluated. Gross visual evaluation markedly underestimated B. tarandi prevalence in caribou with a relative sensitivity ranging from 0.29 in the conjunctiva to 0.13 in the skin section from the thigh, whereas relative specificities ranged from 0.98 to 1.00. The metatarsus and rostrum skin sections had the highest probabilities of cyst detection of all four anatomical sampling sites. The metatarsus harbored significantly higher densities of B. tarandi cysts than the rostrum, thigh, or conjunctiva. In conclusion, microscopic evaluation of a skin section from the anterior aspect of the mid-third portion of the metatarsal region could be used as a standardized comparative indicator of density of B. tarandi infection in Rangifer.

The distribution pattern and associated tissue reactions with progressive changes in Besnoitia caprae cysts were investigated in 6 experimentally infected 16- to 20-month-old male goats. Each goat was subcutaneously inoculated with approximately 13 × 10(8) B caprae bradyzoites. The animals were examined daily for development of clinical besnoitiosis, and skin biopsies from distal parts of the limbs were taken at weekly intervals. At 15, 30, 60, 120, 180, and 365 days postinfection (DPI), 1 goat was euthanized. Samples were collected at autopsy from various organs for histologic and ultrastructural studies. No cysts were seen in tissue sections on 15, 30, and 365 DPI, but large numbers were present at 60, 120, and 180 DPI in the skin of the distal limbs, scrotum, and ears, with fewer in the tongue, palate, sclera, testicles, and spermatic cord. No cysts were seen in the lungs, liver, kidneys, spleen, central nervous system, or lymph nodes. Cyst numbers peaked at 60 DPI, then declined from 120 to 180 DPI. Degenerated cysts were relatively rare at 60 DPI but more numerous at 180 compared with 120. A granulomatous reaction--predominantly characterized by macrophages, lymphocytes, and plasma cells--surrounded each degenerated cyst. All goats showed testicular tubular degeneration with little or no spermatogenic activity. The sizes of cysts and their wall thickness, with the size of bradyzoites and some of their organelles, exhibited progressive chronologic changes.

The in vitro effects of 4 arylimidamides (DB811, DB786, DB750 and DB766) against the proliferative tachyzoite stage of the apicomplexan parasite Besnoitia besnoiti were investigated. These four compounds had been shown earlier to exhibit in vitro activities in the nanomolar range against the related apicomplexans Neospora caninum and Toxoplasma gondii. Real-time-PCR was used to assess B. besnoiti intracellular proliferation in vitro. Preliminary assessment by light microscopy identified DB811 and DB750 as the most promising compounds, while DB786 and DB766 were much less effective. Three-day-growth assays and quantitative real-time PCR was used for IC50 determination of DB811 (0.079 μM) and DB750 (0.56 μM). Complete growth inhibition was observed at 1.6 μM for DB 811 and 1.7 μM for DB750. However, when infected cultures were treated for 14 days, proliferation of parasites occurred again in cultures treated with DB750 from day 4 onwards, while the proliferation of DB811-treated tachyzoites remained inhibited. Electron microscopy of B. besnoiti-infected fibroblast cultures fixed and processed at different time-points following the initiation of drug treatments revealed that DB811 exerted a much higher degree of ultrastructural alterations compared to DB750. These results show that arylimidamides such as DB811 could potentially become an important addition to the anti-parasitic arsenal for food animal production, especially in cattle.

Cystoisospora canis is a coccidial parasite of the intestinal tract that can cause severe disease in dogs. Clinical signs include watery diarrhea, vomiting, fever, and weight loss. Extraintestinal stages of Cystoisospora spp. have been demonstrated in the mesenteric lymph nodes of paratenic hosts. Information on the biology of extraintestinal stages of canine Cystoisospora species is limited. The current study examined the development of C. canis in 2 noncanine cell lines and the ultrastructure of the monozoic cysts that formed. Monolayers of bovine turbinate cells and African green monkey kidney cells were grown on coverslips and inoculated with excysted C. canis sporozoites. Coverslips were collected on various days and fixed and stained for light microscopy (LM) or transmission electron microscopy (TEM). A single, centrally located, slightly crescent-shaped sporozoite surrounded by a thick cyst wall within a parasitophorous vacuole was observed with the use of LM and TEM. No division and no multinucleated stages were observed with either LM or TEM. With TEM, typical organelles of sporozoites were observed, such as rhoptries, dense granules, a crystalloid body, polysaccharide granules, and a conoid. The structure and ultrastructure of C. canis monozoic cysts produced in vitro are similar to extraintestinal cysts of other Cystoisospora species in experimentally infected animals and those of Cystoisospora belli observed in immunocompromised humans. This is the first study that fully demonstrates in vitro the development of what structurally resemble extraintestinal cysts of a Cystoisospora spp.

Besnoitia besnoiti was isolated from a skin biopsy of a chronically infected cow from central Spain. Zoites released from macroscopic cysts were adapted to its culture in vitro on a MARC-145 cell monolayer. Tachyzoites produced in vitro were either cryopreserved or used for genomic DNA isolation. A 2206 nt sequence containing 18S ribosomal RNA gene, internal transcribed spacer 1 (ITS 1), and a partial sequence of 5.8S ribosomal RNA gene was amplified by PCR and sequenced. This sequence showed a 99-100% identity to 18S, ITS1, and 5.8S sequences of B. besnoiti published in databases. After analysis by transmission and scanning electron microscopy of isolated bradyzoites and tachyzoites, it was observed that their ultrastructural morphology coincided with B. besnoiti. The isolate characterized in this study was identified as B. besnoiti on the basis of the disease produced, molecular characteristics, and morphology. The B. besnoiti isolate was denoted as BbSpain-1; it is the first isolate obtained and characterized in Spain and one of the first European isolates adapted to grow in vitro. The isolation and in vitro production of this B. besnoiti isolate offers a good opportunity to study general aspects of bovine besnoitiosis, including epidemiology, pathogenesis, and diagnosis of this re-emergent disease.

The paper reports the first detection of besnoitiosis of cattle in Germany. Just 2 years after the first appearance of the African Bluetongue disease (BTD) of cattle in Central Europe, another African agent of disease has arrived in Germany. While it was proven that the BTD virus was transmitted (after its first appearance) by endemic midges of the genus Culicoides (C. obsoletus, C. pulicaris), nothing is known, how the infectious stages of Besnoitia besnoiti-a member of the so-called cyst-forming coccidia-found their way to a herd in Southern Germany. The infected animals showed all characteristic clinical symptoms of besnoitiosis such as hyposclerodermia, hyperkeratosis, alopecia, and whitish tissue cysts in subcutaneous tissues as well as in the cornea. These cysts had diameters of up to 3 mm and consisted of a dense outer layer (=secondary cyst wall), which surrounded a host cell, that had been enormously enlarged by an inner parasitophorous vacuole containing thousands of 7-9 x 2 mum sized, banana-shaped cyst merozoites (=cystozoites, bradyzoites).Their fine structure was identical to that of published stages of B. besnoiti. During cyst development, the nucleus of the host cell had been hypertrophied and had apparently undergone several divisions, since many flattened, but very large nuclei were seen in light and electron microscopy. Thus, this study proves the arrival of another serious agent of disease of ruminants in Central Europe-a fact which is especially important, since in this species, there is neither information on the way of transmission from animal to animal nor exists concrete information on an efficacious therapy or on the modalities of its import into Germany.

A disease with clinical manifestations of thickening and alopecia of the skin over the lower limbs, around the eyes, face, and nose, thickening and shrinkage of the scrotum, and presence of white granular cysts in the sclero-conjunctiva in goats in Kerman Province, were reported to the Pathology Department of Shiraz Veterinary School. Primary histopathological studies demonstrated an outbreak of caprine besnoitiosis in this region. To study the histopathological and ultrastructural features of the disease, samples were collected from various organs of the suspected slaughtered goats for further investigations. In histopathological studies, dermis and subcutaneous fascia covering lower portion of the limbs, skin over frontal sinus, ear tips, scrotum, eye lids as well as the eye's sclera, epididymal and testicular parenchyma, and their tunics were severely infected with Besnoitia cysts. Tongue, pharynx, prepuce and penis, deeper striated muscles, subcutaneous bone matrices, abomasum, esophagus, subcutaneous tendons, and periosteal surfaces of the limb bones showed lower rates of infection. Except the vagina and vestibule, no cyst was observed in other female urogenital organs, the central nervous system, intestines, heart, liver, spleen, and different lymph nodes. The host reaction to the cysts was variable, ranging from the absence of inflammatory cells around intact normal cysts up to infiltration of macrophages, lymphocytes, plasma cells, eosinophils, fibroblasts, and connective tissues around the degenerated cysts. Ultrastructural studies showed this coccidian parasite belonged to eukaryotic protozoa, and the cystic form had the typical feature of the Besnoitia spp. of the apicomplexa. This study showed that the organism demonstrated ultrastructurally minor differences with other Besnoitia species infecting other animal species.

Tachyzoites of Besnoitia darlingi Brumpt, 1913 were redescribed based on new materials isolated from Virginia opossums (Didelphis virginiana, Kerr) from Michigan, U.S.A. Tachyzoites of the MIBD1 strain were propagated in bovine turbinate cell culture for more than two years. A comparison with previously described tachyzoites of the B. darlingi OP1 strain from Mississippi, USA revealed some morphological differences despite the remarkable genetic homogeneity between the two B. darlingi strains. MIBD1 tachyzoites were distinguished from OP1 tachyzoites by having more rhoptries, and fewer and haphazardly distributed micronemes at the conoidal end. This morphological heterogeneity between tachyzoites of the two strains suggests the role of geographical isolation in the Michigan strain. New morphological features of B. darlingi tachyzoites were described.

Nitazoxanide (NTZ) and its deacetylated metabolite tizoxanide (TIZ) exhibit considerable in vitro activity against Besnoitia besnoiti tachyzoites grown in Vero cells. Real-time-PCR was used to assess B. besnoiti tachyzoite adhesion, invasion, and intracellular proliferation in vitro. A number of NTZ-derivatives, including Rm4822 and Rm4803, were generated, in which the thiazole-ring-associated nitro-group was replaced by a bromo-moiety. We here show that replacement of the nitro-group on the thiazole ring with a bromo (as it occurs in Rm4822) does not impair the efficacy of the drug, but methylation of the salicylate ring at the ortho-position in a bromo-derivative (Rm4803) results in complete abrogation of the antiparasitic activity. Treatment of extracellular B. besnoiti tachyzoites with NTZ has an inhibitory effect on host cell invasion, while treatments with TIZ, Rm4822 do not. TEM demonstrates that the effects of Rm4822 treatment upon the parasites are similar to the damage induced by NTZ. This includes increased vacuolization of the parasite cytoplasm, and loss of the structural integrity of the parasitophorous vacuole and its membrane. Thus, Rm4822, due to the absence of a potentially mutagenic nitro-group, may represent an important potential addition to the anti-parasitic arsenal for food animal production, especially in cattle.

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

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

Neospora caninum and Hammondia heydorni are morphologically and phylogenetically related coccidians that are found in dogs. Although there is serological evidence of N. caninum infection in the white-tailed deer (Odocoileus virginianus), the parasite has not been yet isolated from the tissues of this host. In an attempt to isolate N. caninum from deer, hearts from 4 deer with antibodies to N. caninum were fed to 2 dogs. One of these dogs shed unsporulated oocysts 12-14 microm in diameter. Sporulated oocysts were not infective to Mongolian gerbils (Meriones ungulatus), and DNA isolated from these oocysts was not amplified using N. caninum-specific primers. However, positive amplification with the H. heydorni-specific first internal transcribed spacer (ITS-1) primers and common toxoplasmatiid ITS-1 primers confirmed the presence of H. heydorni DNA in the samples. The oocysts were considered to be H. heydorni on the basis of their morphology, biology, and molecular characteristics. This is the first record of a H. heydorni-like parasite in the white-tailed deer.

Besnoitia tarandi tissue cysts were found in naturally-infected reindeer (Rangifer tarandus) from Finland. Infectivity of its tissue cysts, bradyzoites, and tachyzoites to animals and cell culture was studied. The bradyzoites and tissue cysts were not infectious to out-bred mice, rabbits or gerbils. When fed tissue cysts, neither cats nor dogs excreted oocysts. However, the parasite was lethal to interferon-gamma gene knock out mice irrespective of the route of inoculation. The parasite was grown successfully in African Green Monkey cells from tissues of two reindeer for the first time. Non-dividing, uninucleate tachyzoites from smears from cell cultures were 5.6 x 1.4 microm (4.5-7.4 x 1.0-1.9, n=50) in size. Longitudinally-cut bradyzoites in tissue sections measured 7.4 x 1.3 microm (6.5-7.8 x 1.0-1.6, n=30). Ultrastructurally, tachyzoites and bradyzoites were similar to those in other Besnoitia species, and in particular to parasites described from cattle (Besnoitia besnoiti) and equids (Besnoitia bennetti) in that their bradyzoites lacked enigmatic bodies. Based on comparative analysis of three portions of nuclear ribosomal DNA (the small and large subunits and the first internal transcribed spacer) B. tarandi was found to be more closely related to the other congeners described from ungulates. The parasite was formally redescribed and specimens deposited in the US National Parasite Collection.

Concurrent infections with coccidians Toxoplasma gondii, Sarcocystis spp., and a Hammondia heydorni-like parasite were identified in tissues of three littermate pups on a Kelpie dog breeding farm in Australia. In total, 20 pups in four litters had died following vaccination with an attenuated distemper virus vaccine. Toxoplasma gondii tachyzoites were identified immunohistochemically in tissues of two dogs. Sarcocystis sp. sporocysts were seen in the intestinal lamina propria of two dogs. Asexual and sexual stages of H. heydorni-like parasite were found in enterocytes of the small intestine of two dogs. Ultrastructural development of schizonts and gamonts of this parasite is described. None of the protozoa in these dogs reacted with antibodies to Neospora caninum. Feeding of uncooked tissue of sheep was considered to be the likely source of infection for these coccidians in dogs.

Development and structure of different life cycle stages of Besnoitia oryctofelisi which has a rabbit-cat life cycle was studied by light and transmission electron microscopy. For light microscopy, Besnoitia oryctofelisi-infected tissues were stained with haematoxylin-eosin, periodic acid Schiff (PAS) reagent, and immunohistochemically with rabbit anti-B. oryctofelisi polyclonal antibodies and anti-BAG-1 antibodies. In vitro and in vivo-derived tachyzoites were 5-6 microm long and they were found to divide by endodyogeny. In tachyzoites, the nucleus was often central, and micronemes were few and located anterior to the nucleus. Earliest tissue cysts were seen in gerbils starting 12 days p.i. Early tissue cysts had an outer PAS-positive cyst wall, a middle PAS-negative host cell layer, and an inner PAS-negative parasitophorous vacuolar membrane. Organisms in early tissue cysts were PAS-negative, did not stain with anti-BAG-1 antibodies, and amylopectin granules and enigmatic bodies were absent. Tissue cysts beginning 17 days p.i. contained organisms that became PAS-positive and reacted with anti-BAG-1 antibodies, indicating they were bradyzoites. Immunoreactivity with polyclonal anti-B. oryctofelisi antibodies suggested that Besnoitia species bradyzoites are encapsulated by the host cell. Bradyzoites (10 microm) were about twice the length of tachyzoites and contained enigmatic bodies characteristic of Besnoitia bradyzoites. Unlike tachyzoites and tissue cysts, schizonts were located intravascularly in the lamina propria of the small intestine of cats. Merozoites were 5-6 microm long, had few rhoptries and amylopectin granules, had numerous micronemes and had a terminal nucleus.

Besnoitia besnoiti is an economically important tissue cyst-forming apicomplexan of cattle in Africa and Israel. Tissue cysts and bradyzoites of B. besnoiti from the skin of a naturally infected bull were studied by transmission electron microscopy. Tissue cysts enclosed host cell and bradyzoites. Bradyzoites were 6-7.5 x 1.9-2.3 microm in size and contained organelles found in coccidian merozoites including numerous micronemes, rhoptries, amylopectin granules, and a posteriorly located nucleus. Enigmatic bodies, characteristically found in Besnoitia sp. bradyzoites, were not observed. Therefore, enigmatic bodies should be removed as a generic character of the bradyzoites of Besnoitia species.

The diversity among coccidian parasites of the genus Besnoitia is incompletely known. Of the eight currently described members of the genus, only B. jellisoni is known to parasitize a rodent host. Here, we propose a new name, Besnoitia akodoni, for the species initially isolated form the rodent Akodon montensis in Brazil. The tissue cysts of B. akodoni were up to 442 microm in diameter and bradyzoites were 8.4 x 1.4 microm in size. The bradyzoites contained enigmatic bodies, micronemes and rhoptries. Tachyzoites were 5.8 x 1.5 microm in size and they could be grown in vitro in bovine monocytes and African Green monkey cells where they divided by endodyogeny. Besnoitia akodoni was infective to laboratory-raised mice (Mus musculus) and gerbils (Meriones unguiculatus) but not to cats (Felis catus). Comparison of the conserved sequences of the small subunit rDNA clearly established the close relationship of B. akodoni with other members of the genus. However, sequences of the more variable first internal transcribed spacer portion of the ribosomal DNA repeat support its differentiation from the other species of the genus.

Besnoitia darlingi from naturally infected opossums (Didelphis virginiana) from Mississippi, USA, was propagated experimentally in mice, cats, and cell culture and was characterised according to ultrastructural, genetic, and life-history characteristics. Cats fed tissue cysts from opossums shed oocysts with a prepatent period of nine or 11 days. Oocysts, bradyzoites, or tachyzoites were infective to outbred and interferon-gamma gene knockout mice. Tachyzoites were successfully cultivated and maintained in vitro in bovine monocytes and African green monkey cells and revived after an 18-month storage in liquid nitrogen. Schizonts were seen in the small intestinal lamina propria of cats fed experimentally-infected mouse tissues. These schizonts measured up to 45 x 25 microm and contained many merozoites. A few schizonts were present in mesenteric lymph nodes and livers of cats fed tissue cysts. Ultrastructurally, tachyzoites and bradyzoites of B. darlingi were similar to other species of Besnoitia. A close relationship to B. besnoiti and an even closer relationship to B. jellisoni was indicated for B. darlingi on the basis of the small subunit and ITS-1 portions of nuclear ribosomal DNA.

A Besnoitia species of the teiid lizard Ameiva ameiva (L.), from north Brazil was established in laboratory mice and hamster by the intraperitoneal inoculation of bradyzoites in the tissue cysts. In the lizards all the cyst wall layers were closely apposed. In the mice the layers of the wall were distinguishable, and ultrastructurally the inner cytoplasmic layer contained either a tight network of endoplasmic reticulum or packed mitochondria or both. These components were less frequent or sparse in the inner cytoplasmic layer of cysts in the lizard. The only animals available for experiments in attempts to indicate the definitive host of the parasite were 3 kittens of the domestic cat and a juvenile specimen of the snake Boa constrictor raised in captivity. No evidence of infection could be detected in these animals after feeding them with the tissues of mice harbouring cysts with very large number of bradyzoites.

'Isospora' lieberkuehni, an unusual isosporoid renal coccidium that parasitizes the European water frog was isolated from the edible frog, Rana kl. esculenta, in the Czech Republic. Sequencing of the small-subunit (SSU) rRNA gene showed that it belongs to the family Sarcocystidae, being closely related to a clade comprising members of the subfamily Toxoplasmatinae. The position within Sarcocystidae correlates with the mode of excystation via collapsible plates as postulated by previous authors. Phylogenetic, morphological and biological differences between 'Isospora' lieberkuehni and the other Stiedabody-lacking members of the genus Isospora justify separation of this coccidium on a generic level. Hyaloklossia Labbé, 1896 is the oldest available synonym and is herein re-erected. The original definition of the genus Hyaloklossia is emended based on recent observations.