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.     

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.     

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.     

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.     

First isolation of Besnoitia besnoiti from a chronically infected cow in Spain

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.     

An outbreak of besnoitiosis in miniature donkeys

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.     

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.     

Examination of tissue cyst formation by Toxoplasma gondii in cell cultures using bradyzoites, tachyzoites, and sporozoites

Tissue cyst formation by a goat isolate (GT-1) of Toxoplasma gondii was examined in bovine monocyte, human fetal lung, and Madin-Darby bovine kidney cell cultures. Transmission electron microscopy (TEM) and cat feeding studies indicated that tissue cysts were present in all 3 cell lines examined. Tissue cysts were first seen 3 days postinoculation (PI) using TEM. Standard cell culture procedures were used and no additional condition was needed to induce tissue cyst formation. Cats fed cell cultures excreted T. gondii oocysts in their feces 5-7 days PI. These oocysts caused lethal infections in mice. Tissue cysts were produced in cell cultures regardless if the initiating inoculum consisted of bradyzoites, sporozoites, or a mixture of bradyzoites and tachyzoites. Tissue cyst formation has been followed through 40 subpassages of infected cells. By TEM tissue cysts still were present after 40 passages, but when 40th-passaged cultures were fed to cats, oocytsts were not excreted. This indicates that the parasite had become oocystless after repeated passage in vitro.     

Microtubule cytoskeleton behavior in the initial steps of host cell invasion by Besnoitia besnoiti

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.     

Molecular phylogeny of Besnoitia and the genetic relationships among Besnoitia of cattle, wildebeest and goats

Knowledge on parasites of the genus Besnoitia is sparse, which are classified in the subfamily Toxoplasmatinae of the phylum Apicomplexa. This arrangement hypotheses that Besnoitia represents the sister group to species such as Toxoplasma gondii and Hammondia hammondi. In order to test this hypothesis, phylogenetic analyses of 18S ribosomal DNA (rDNA) from Besnoitia, Hammondia, Isospora, Frenkelia, Eimeria, Neospora, Sarcocystis and Toxoplasma were performed. The 18S rDNA of Besnoitia besnoiti, Besnoitia jellisoni and Eimeria alabamensis were amplified by PCR and sequenced. Phylogenetic analyses by parsimony and maximum-likelihood methods showed Besnoitia to be reproducibly the sister group to a clade containing Hammondia, Neospora and Toxoplasma. Furthermore, Besnoitia of cattle, wildebeest and goats had identical ITS1 rDNA sequences, which questions the use of the taxon Besnoitia caprae to describe the Besnoitia found in goats.     

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.     

Autofluorescence of Toxoplasma gondii and Neospora caninum cysts in vitro

Autofluorescence of Toxoplasma gondii and Neospora caninum was studied by fluorescence microscopy during their differentiation from tachyzoites to bradyzoites in vitro using Vero as host cells. Stage conversion into bradyzoites and cysts was confirmed by immunofluorescent microscopy and Western blot analysis using SAG1- and BAG1-specific antibody, respectively. From day 4 postinfection (PI), pale blue autofluorescence of the bradyzoites and tissue cysts was observed with UV light at 330-385 nm, which coincided with the onset of cyst development. This autofluorescence under UV light of bradyzoites and tissue cysts increased in intensity from days 8 to 10 PI. In contrast to the autofluorescence shown by bradyzoites and cysts, tachyzoites and parasitophorous vacuoles containing tachyzoites never autofluoresced at any time examined. Autofluorescence of the cystic stages was of sufficient intensity and duration to allow the detection of cysts and bradyzoites of T. gondii and N. caninum. In this study, we describe for the first time the autofluorescence properties of in vitro-induced bradyzoites and cysts of T. gondii and N. caninum.     

Biologic, morphologic, and molecular characterisation of Neospora caninum isolates from littermate dogs

Isolation and biologic and molecular attributes of Neospora caninum from three littermate dogs are described. Tissue cysts were confined to the brain and striated muscles. N. caninum was isolated (isolates NC-6, NC-7, and NC-8) in rodents and cell culture that had been inoculated with brain tissue from the dogs. Schizont-like stages reactive with N. caninum antibodies were seen in cell cultures seeded with bradyzoites released from Percoll-isolated N. caninum tissue cysts from the brain of one dog. Tissue cysts were infective orally to mice and gerbils, but not to cats and dogs. The isolates were also identified as N. caninum by PCR and sequence analysis.     

Bovine besnoitiosis and its effect on the male reproductive system

Besnoitia besnoiti and several species of the genus have been reported in a variety of domestic and wild animals in many parts of the world (1, 2, 3, 4, 5). Cutaneous besnoitiosis in cattle has been described as a serious skin condition characterized by painful swellings, alopecia and thickening of the skin (6). It is a chronic, debilitating and occasionally fatal disease with both cutaneous and systemic manifestations (6, 7, 8). Besnoitia cysts and lesions have been observed in the testes, epididymes and blood vessels of bulls (9). The possibility of abortion in cows and sterility in bulls with besnoitiosis has been reported (6). Spontaneous clinical besnoitiosis in cattle with accompanying systemic manifestations has not been fully documented, and the effect of scrotal besnoitiosis on spermatogenesis in bulls has not been previously reported in Nigeria. Case report : Outbreaks of babesiosis, heartwater and suspected cutaneous besnoitiosis were observed in a government farm in the Kano State of Nigeria in March, 1979. The cattle population was over 1500, consisting of exotic (Bos taurus ) and indigenous (Bos indicus ) breeds and their crosses. Clinical Examination : All stages of clinical besnoitiosis were initially observed in 14 bulls and 1 cow in a period of 4 months. The farm was visited bi-weekly for 1 year, during which time 58 bulls and 2 cows showed the lesions. Most of the animals affected with this disease were 3-way crosses of Australian Draughtmaster x Rahaji x Niger Azwak (DM x Rh x AZ). Table 1 summarises the distribution of clinicial cases of besnoitiosis.     

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.     

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.     

Hematological and serum biochemical analyses in experimental caprine besnoitiosis

This study was undertaken to investigate the hematological and biochemical changes in experimentally infected goats with Besnoitia caprae from the time of infection till 360 days post-infection (PI). Six male goats were inoculated subcutaneously with 13 × 10(7) bradyzoites of B. caprae, and blood samples were collected from the jugular vein. The total erythrocyte and total leukocyte counts, hematocrit value, and differential leukocyte counts were determined. Serum biochemical analysis, including the total protein, albumin, total globulin, cholesterol, triglyceride, chloride, testosterone, calcium (Ca(2+)), inorganic phosphorus, sodium (Na(+)), potassium (K(+)), iron (Fe(2+)), glucose, serum amyloid A (SAA), haptoglobin (Hp), fibrinogen, ceruloplasmin, aspartate aminotransferase, alanine aminotransferase, creatine kinase, lactate dehydrogenase, and alkaline phosphatase, was undertaken. Skin biopsy from the limbs were collected at weekly intervals and histologically examined for Besnoitia cysts. Cysts were present in the skin biopsies of the leg of the infected goats from day 28 PI. There were variations in hematological analyses, but no significant difference was seen. From day 30 to 360 PI, results showed that SAA, Hp, fibrinogen, and ceruloplasmin concentrations increased, whereas testosterone concentrations decreased. Infected goats exhibited decrease of albumin and increase of serum total protein and globulin concentrations. By contrast, there were no significant differences in the remained analyses concentrations.     

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.     

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.     

Immunohistochemical and ultrastructural evidence for Neospora caninum tissue cysts in skeletal muscles of naturally infected dogs and cattle.

Protozoan stages were detected in the skeletal muscles of four dogs suffering from neosporosis and two neonatal calves with confirmed Neospora caninum- infection which could be immunohistochemically labelled by an antiserum against the bradyzoite-specific antigen BAG-5. In one calf, a tissue cyst was labelled by an antiserum against the N. caninum isolate NC-1. Ultrastructurally, a 0.3-1 microm-thick cyst wall surrounded the labelled parasites. The cysts were located within myofibres and contained varying numbers of bradyzoites each measuring 5.2(+/-0.6) x 1.6(+/-0.3) microm. The encysted stages showed typical ultrastructural features of N. caninum bradyzoites with subterminal nuclei, electron-dense rhoptries, and micronemes that were orientated perpendicular to the zoite pellicle. Immunohistochemistry and serology did not reveal any evidence for co-infection with Toxoplasma gondii. The detection of tissue cysts in skeletal muscle of N. caninum-infected intermediate hosts is of major epidemiological importance for the understanding of how definitive or intermediate carnivorous hosts become infected with N. caninum.