Redescription of Besnoitia bennetti (Protozoa: Apicomplexa) from the donkey (Equus asinus)

Besnoitia bennetti tissue cysts were found in four naturally-infected donkeys (Equus asinus) from the USA. Infectivity of its bradyzoites and tachyzoites to animals and cell culture was studied. The bradyzoites were not infectious to out-bred Swiss Webster mice, rabbits or gerbils. When fed tissue cysts, cats did not excrete oocysts. However, the parasite was infectious to interferon-gamma gene knock out mice. The parasite from tissues of two donkeys was grown successfully in bovine monocyte monolayers for the first time. Non-dividing, uninucleate tachyzoites were approximately 6 x 1.5 microm in size. Longitudinally-cut bradyzoites in tissue sections measured 8.7 x 1.9 microm. Ultrastructurally, tachyzoites and bradyzoites were similar to those in other Besnoitia species, and in particular to parasites described from cattle (Besnoitia besnoiti) and reindeer (Besnoitia tarandi), 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. bennetti 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 Collections.     

Ultrastructure of Besnoitia besnoiti tissue cysts and bradyzoites

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.     

Besnoitiosis in a southern plains woodrat (Neotoma micropus) from Uvalde, Texas

Recently, Besnoitia neotomofelis was described from a southern plains woodrat (Neotoma micropus) from southern Texas. During May 2010, 1 of 55 southern plains woodrats trapped in Uvalde County, Texas, was diagnosed with besnoitiosis. Grossly, the woodrat had bilateral swellings of the cheeks, and numerous Besnoitia sp.-like cysts were observed in the tongue, facial region, musculature of the limbs, and subcutis of the dorsum and flanks. Little to no inflammation was noted around cysts. The cysts were morphologically similar to B. neotomofelis based on light and transmission electron microcopy. The sequence of the internal transcribed spacer region-1 was identical to the type isolate of B. neotomofelis. Attempts to isolate Besnoitia sp. in laboratory mice failed; however, Toxoplasma gondii was isolated in a Swiss Webster mouse. This represents the first report of besnoitiosis caused by B. neotomofelis in a southern plains woodrat and the first concurrent Besnoitia sp. and T. gondii infection in any host species.     

Redescription of Besnoitia tarandi (Protozoa: Apicomplexa) from the reindeer (Rangifer tarandus)

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.     

Myopathy associated with megaloschizonts of Haemoproteus meleagridis in a wild turkey from Florida

Necropsy of an emaciated adult wild turkey (Meleagris gallopavo osceola) that died in captivity soon after capture revealed numerous macroscopic 1-2 mm white cysts in the pectoral muscles. Microscopic, aseptate protozoan megaloschizonts, 50-150 microns in diameter, corresponded to the cysts in histological sections. The megaloschizonts were surrounded by a thick, hyaline wall and packed with spherical merozoites less than 1 micron in diameter. Muscle fibers surrounding most of the megaloschizonts exhibited early signs of dystrophic calcification. The fibers were swollen, pale and hyaline and contained scattered basophilic granules. The megaloschizonts were morphologically distinct from sarcocysts of Sarcocystis sp. and Besnoitia sp. and the thin-walled tissue cysts of Toxoplasma gondii. They were identical in structure and host reaction to the second-generation megaloschizonts of Haemoproteus meleagridis, reported previously from experimentally infected domestic turkeys. While the precise cause of death of the wild turkey could not be determined, the most prominent lesions were associated with the numerous intramuscular megaloschizonts.     

Besnoitia (Protozoa: Toxoplasmatinae) isolated from opossums Didelphis marsupilis in the Amazon region, Brazil

From a total of 224 Didelphis marsupialis examined, in 15 were found cysts of Besnoitia in muscles and viscera. It's the first time that this protozoan is isolated from naturally infected animals in Brazil. The experimental transmission to laboratory animals was done by the inoculum of tissue and cysts triturated.     

Development and ultrastructure of Besnoitia oryctofelisi tachyzoites,tissue cysts,bradyzoites, schizonts and merozoites

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 darlingi (Protozoa: Toxoplasmatinae): cyclic transmission by cats

Two of five opossums examined from the Kansas City area were found to be infected with Besnoitia darlingi. Inoculation of cysts resulted in acute lethal infections in mice and hamsters. Chronic infections with development of tissue cysts were obtained in mice by prophylaxis with sulfadiazine. Cysts were fed to cats and a dog which resulted in the shedding of isosporoid oocysts by cats. The prepatent period was 11 to 14 days. Unsporulated oocysts averaged 11.9 by 12.3 micrometer and sporulated in 48 to 72 hr. Mouse-to-mouse tramsmission was achieved by injecting triturated tissue containing cysts. This is the third species of Besonitia to be cyclically transmitted by cats.     

An epizootic of besnoitiosis in captive caribou (Rangifer tarandus caribou), reindeer (Rangifer tarandus tarandus) and mule deer (Odocoileus hemionus hemionus)

Besnoitia sp. was diagnosed in two caribou (Rangifer tarandus caribou) which died of pneumonia at the Assiniboine Park Zoo (Winnipeg, Manitoba, Canada) in 1983. During the following 3 yr besnoitiosis spread to an isolated herd of caribou, to mule deer (Odocoileus hemionus hemionus) and to reindeer (Rangifer tarandus tarandus). Reduction of exposure to biting insects appears to have reduced the transmission of besnoitiosis within the reindeer herd. The morbidity rate was approximately 82% in caribou and 67% in mule deer over the age of 2 mo. Most animals with clinical signs were euthanized; this precluded an estimation of the disease-related mortality rate. Twenty-eight caribou, 10 mule deer and three reindeer have been euthanized or died as a result of this epidemic. Attempts to artificially transmit the disease to potentially susceptible intermediate and definitive hosts were unsuccessful.     

Establishment of Besnoitia darlingi from opossums (Didelphis virginiana) in experimental intermediate and definitive hosts,propagation in cell culture,and description of ultrastructural and genetic characteristics

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.     

Biological and molecular characterization of Besnoitia akodoni n.sp. (Protozoa: Apicomplexa) from the rodent Akodon montensis in Brazil

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 sp. from goats in Kenya

The eyelids of goats in Kenya contained several, conspicuous white cysts which were up to 1.5 mm in size. By histological and electron microscopical studies it was confirmed that these cysts belong to the genus Besnoitia.     

A Hammondia-like parasite from the European fox (Vulpes vulpes) forms biologically viable tissue cysts in cell culture

Tissue cysts of parasites of the genus Hammondia are rarely described in naturally or experimentally infected intermediate hosts. However, ultrastructural examinations on tissue cyst stages of Hammondia sp. are needed, e.g. to compare these stages with those of Neospora caninum and other related parasites. We describe a cell culture system employed to examine the in vitro development of tissue cysts of a Hammondia sp.-like parasite (isolate FOX 2000/1) which uses the European fox as a definitive host. Cells of a diploid finite cell line from embryonal bovine heart (KH-R; CCLV, RIE 090) were infected by inoculation of sporozoites und cultivated for up to 3 months. Transmission electron microscopic examination of 17 day old cell culture material revealed the presence of cyst walls. Infected cell cultures cultivated for 2 months were used to feed a fox. Six to 13 days post infection the fox shed large numbers (n=1.2 x 10(7)) of Hammondia-sp. like oocysts which could not be distinguished from those used to infect the cell culture as determined by DNA sequencing of the internal transcribed spacer 1 and the D2/D3 domain of the large subunit ribosomal DNA. To find out the proportion of parasitophorous vacuoles that had developed into tissue cysts, the expression of bradyzoite markers was examined by probing infected cell cultures with mouse polyclonal antibodies against Toxoplasma gondii bradyzoite antigen 1 (anti-BAG1) and rat monoclonal antibodies against a cyst wall protein (mAbCC2). Nineteen and 90 days post infection all parasitophorous vacuoles in the cell cultures were positive with anti-BAG1 and mAbCC2. This shows that biologically viable (i.e. infectious) tissue cysts of a fox-derived Hammondia sp. isolate (FOX 2000/1) can be efficiently produced in this cell culture system. Since in vitro cystogenesis of dog-derived Hammondia heydorni has not been observed yet, in vitro cyst formation might be one trait to separate fox-derived Hammondia sp. from H. heydorni on a species level.     

Besnoitia darlingi (Apicomplexa,Sarcocystidae, Toxoplasmatinae): Transmission between Opossums and Cats1

ABSTRACT. Opossums (Didelphis marsupialis), act as intermediate hosts for Besnoitia darlingi and could be infected orally with sporozoites (oocysts) and bradyzoites (tissue cysts), or intraperitoneally (i.p.) with tachyzoites. Infections could presumably be transmitted through cannibalism. Cats (Felis catus), the definitive host, could be infected only with bradyzoites but not sporozoites. Oocysts shed by cats measure about 12 × 12 μm, resemble similarly sized oocysts of Toxoplasma gondii and Hammondia hammondi, and must be differentiated by the appearance of tissue cysts after experimental infection of intermediate hosts. Cats did not form tissue cysts of B. darlingi. Tachyzoites from the related B. jellisoni could be used in the Sabin-Feldman dye test to determine the development of antibody to B. darlingi in opossums after infection.     

Protoperidiniacean dinoflagellate cyst taxa from the Upper Miocene of ODP Leg 178, Antarctic Peninsula

Protoperidiniacean dinoflagellate cysts have been recovered from Upper Miocene sediments of Hole 1095, ODP Leg 178, drilled to the west of the Antarctic Peninsula. These cysts make up virtually the entire dinoflagellate cyst assemblages, and three new species are formally described herein as Selenopemphix bothrion sp. nov., Selenopemphix kepion sp. nov. and Selenopemphix minys sp. nov., together with one informal taxon and two species attributed to published taxa. The occurrence of Late Miocene protoperidiniacean-dominated assemblages in the vicinity of the Antarctic continent is of special interest. Their presence may indicate the initiation of the Antarctic Circumpolar Current and increased nutrient supply within the Southern Ocean as the Antarctic Divergence became established. A comparison with several other protoperidiniacean dinoflagellate assemblages of similar age in other parts of the world provides some preliminary evidence for possible concomitant oceanographic change at this time.     

Effects of Protostrongylus sp. and Pneumocystis sp. on the pulmonary tissue and the condition of mountain and brown hares from Finland

Mountain hares (Lepus timidus) and brown hares (Lepus europaeus) shot by hunters in several game management districts in southern and central Finland during the hunting season from September to the end of February 1998-2001 were examined for Protostrongylus sp. and Pneumocystis sp. Of the mountain hares, 96.5% (194/201) were infected with the lungworm Protostrongylus sp. and 16.9% (32/189) had cyst forms of the fungus Pneumocystis sp. in the lungs. The prevalence of the lungworm and fungus in brown hares was 60% (18/30) and 20.0% (6/30), respectively. The tissue changes associated with the lungworms were macroscopically and microscopically well demarcated. The majority and most severe histopathologic changes were seen at the distal part of the caudal lobes. Inflammatory cells, mainly eosinophils and macrophages, and in lesser degree neutrophils, lymphocytes, and plasma cells were typical findings in the worm-infected tissue. The condition and weight of the hare did not show any significant association with the intensity of the lungworm infection. All Pneumocystis-infected mountain hares were young, and their condition and weight correlated negatively with the intensity of the infection. The intensity of the Pneumocystis infection did not correlate with that of the lungworm infection. Within a tissue section, a slight but significant positive correlation was observed between presence of cysts and inflammatory cells.     

Morphological features and viability of Scrippsiella trochoidea cysts isolated from fecal pellets of the polychaete Capitella sp.

To investigate the impact of grazing by the polychaete Capitella sp. on the two cyst morphotypes of Scrippsiella trochoidea, the typical morphotype with short calcareous spines (spiny-type cyst) and artificially induced the transparent type without calcareous spines (naked-type cyst), we examined the morphological features and germination capability of the two cyst morphotypes isolated from fecal pellets of the polychaete Capitella sp. produced in a restricted habitat. The morphological destruction was observed in both spiny- and naked type cysts after passage through the gut of Capitella sp., and this seemed to occur rapidly for naked-type cysts. In addition, the germination of both spiny- and naked-type cysts isolated from fecal pellets on day 2 of harvesting was significantly reduced and subsequently completely abolished, in contrast to previous findings from ingestion studies. Our results indicate that continual grazing by Capitella sp. within a restricted habitat can compromise the survival of S. trochoidea cysts.     

Loss of Stages after Continuous Passage of Toxoplasma gondii and Besnoitia jellisoni*

SYNOPSIS. Toxoplasma gondii, passed from mouse to mouse in the tachyzoite stage for 30–35 generations, developed cysts, which, when fed to cats, failed to produce oocysts. Besnoitia jellisoni, passed similarly for 20 generations, lost the capacity to form cysts. These phenomena are explained by a loss of genomes or gene products during the rapid passage selecting for tachyzoites.     

Comparison of gross visual and microscopic assessment of four anatomic sites to monitor Besnoitia tarandi in barren-ground caribou (Rangifer tarandus)

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.     

Necropsy survey of gastric ulcers in a population of aged donkeys: prevalence, lesion description and risk factors

There is no information about the prevalence of gastric ulceration in donkeys or potential risk factors for its presence in donkeys. The donkey is a stoic, hardy animal that has not previously been thought to suffer from this disease. However, gastric ulceration was found to be a problem in a population of non-working UK donkeys resident at the Donkey Sanctuary and its prevalence was estimated by examining necropsy data over a 2-year period during 2005 to 2006. Associations with clinical and management factors were determined. In total, 426 donkeys were examined at necropsy to determine the presence of gastric ulceration. Lesions were described and scored according to a four-point scale. Management and clinical data from these donkeys were analysed to identify potential risk factors for the presence of gastric ulceration. Terminal blood samples were also studied to determine whether animals were exhibiting hyperlipaemia prior to death. Results showed that 41% (n = 174) of the donkeys studied had evidence of gastric ulceration at necropsy. Most (49%) of the ulcers were of a medium size (area of 2 cm2 - <10 cm2) and the most common site for ulcers was the margo plicatus. Of the donkeys examined, 18% had hyperlipaemia prior to or death or euthanasia and this was a risk factor for donkeys developing gastric ulceration; 62% of hyperlipaemia cases also displayed gastric ulceration (P < 0.001). Kidney disease was a potential risk factor (P = 0.02), with 74% of these animals having gastric ulceration. Donkeys that died or were euthanased due to respiratory disease were at a decreased risk of developing ulceration (P = 0.01) Donkeys fed a carbohydrate-based diet were more likely (P < 0.001) to have gastric ulceration than those fed a fibre-only diet, with 55% having gastric ulceration compared with 33% in the fibre-only group. This study has shown that gastric ulceration is commonly observed in donkeys at necropsy and may be extensive.