Exploring the life cycle of Besnoitia besnoiti - experimental infection of putative definitive and intermediate host species

The biology of Besnoitia besnoiti, the cause of bovine besnoitiosis, is poorly understood. Its definitive host is unknown, and information on potential intermediate hosts is scarce. In order to investigate potential definitive and intermediate hosts for European isolates of B. besnoiti, domestic dogs, cats, rabbits, guinea pigs (Cavia porcellus), gerbils (Meriones unguiculatus), common voles (Microtus arvalis) and NMRI-mice were inoculated with B. besnoiti isolated from naturally infected German cattle. Dogs and cats were fed 5×10(6)B. besnoiti tachyzoites (isolate Bb-GER1), or tissue cysts containing at least 2×10(7)B. besnoiti bradyzoites obtained from the skin of a naturally infected Limousin cow from the same herd where strain Bb-GER1 was isolated. Rodents and rabbits were subcutaneously inoculated with either 5×10(5) Bb-GER1 tachyzoites or 5×10(5) bradyzoites. Groups of 2-4 non-inoculated animals of each species were monitored as negative controls. Feces from all dogs and cats were daily examined by a sedimentation-flotation technique for at least 11 weeks after inoculation but no B. besnoiti oocysts were identified. Cats fed tachyzoites and dogs did not seroconvert, but specific antibodies to B. besnoiti tachyzoites were detected by IFAT (titer≥100) in 2 out of 3 cats fed tissue cysts since 5-7 weeks post infection. By immunoblot, these two cats exhibited a reaction pattern against tachyzoite antigens similar to that observed in naturally infected cattle. Antibodies against B. besnoiti tachyzoites were detected in all inoculated rodent species and rabbits by both, IFAT and immunoblot since 3 weeks post-inoculation. Rabbits and rodents, subcutaneously inoculated with same doses of inactivated bradyzoites remained serologically negative (IFAT titer<50). Clinical signs observed in the inoculated rabbits included fever, serous conjunctivitis and transient swelling of the testes. No clinical abnormalities were noticed in the other tested animal species. Voles developed pneumonia as observed by histological examination. B. besnoiti-DNA was detected by PCR in blood from rabbits, gerbils and voles at 9 days post-infection, and in skin, heart, lung, striated muscle and kidney tissues from voles at 19-21 weeks post-infection. Domestic dogs and cats could not be shown to be definitive hosts of B. besnoiti, but cats seroconverted after feeding on B. besnoiti tissue cysts indicating that B. besnoiti stages had invaded the cats' tissues. The molecular and serological results from this study indicate that European B. besnoiti isolates may infect cats, rabbits, guinea pigs, gerbils, mice and voles; however a persistence of the parasite could be demonstrated only in voles.     

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

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

A modified agglutination test for the diagnosis of Besnoitia besnoiti infection

Bovine besnoitiosis is caused by Besnoitia besnoiti, an obligate intracellular apicomplexan parasite. Affected animals present cutaneous and systemic manifestations and the disease may lead to considerable economic losses. Although generally associated to tropical and subtropical areas, bovine besnoitiosis is now considered an emergent disease in Europe, due to the increasing number of new cases and apparent geographical expansion. In this study we evaluated the performance of a modified agglutination test (B-MAT) in the serodiagnosis of bovine besnoitiosis in comparison to the indirect immunofluorescent-antibody test (IFAT). To establish optimal protocol conditions we used bovine sera with a known infection status for B. besnoiti infection. Positive animals (n=36) presented B. besnoiti dermal cysts and anti-B. besnoiti specific antibodies, as determined by the indirect immunofluorescence test (IFAT). Negative animals (n=103) were from non-endemic areas in Portugal and negative by the IFAT. From here, we evaluated the sensitivity and specificity of the B-MAT relative to the IFAT with a panel of sera from herds with history of bovine besnoitiosis in Portugal, Spain and France (n=402), using three serum dilutions (1:80, 1:160, 1:320). Considering the positive cut-off at 1:160 serum dilution, the B-MAT showed an almost perfect test agreement with the IFAT; (κ=0.968; 95% CI: 0.941-0.996) with a relative sensitivity of 97.2% (95% CI: 94.1-100%) and a relative specificity of 99.3% (95% CI: 98.4-100%). As a simple and inexpensive technique the B-MAT represents a valuable tool for the diagnosis and study of the epidemiology of bovine besnoitiosis.     

Quantitative real time polymerase chain reaction assays for the sensitive detection of Besnoitia besnoiti infection in cattle

Bovine besnoitiosis, an economically important disease in cattle in some countries of Africa and Asia, is emerging in Europe. The definitive host of Besnoitia besnoiti, the causative agent of bovine besnoitiosis, is unknown and the transmission of the parasite is not completely understood. Sensitive and quantitative DNA detection methods are needed to determine whether serologically positive animals are infectious and to examine the role of vectors (e.g. haematophagous insects) in the transmission of the parasite. To this end, we established two different 5'-nuclease quantitative assays to detect B. besnoiti infection in cattle and to estimate the parasite load in samples (BbRT1 and BbRT2). These PCRs are based on the sequence of the internal transcribed spacer region 1 (ITS-1) of the ribosomal RNA gene. Tests with serial dilutions of B. besnoiti genomic DNA in a buffer containing 100 ng/μl bovine DNA revealed a detection limit of 0.01 pg genomic B. besnoiti DNA. Reliable quantification was possible in samples containing ≥1 pg B. besnoiti genomic DNA with a coefficient of variation of ≤ 2%. To estimate the diagnostic sensitivity of the tests, skin biopsies and scrapings from the mucous membrane of the vestibulum vaginae (vaginal scrapings) were taken from cattle with clinical signs of chronic besnoitiosis. Regardless of the real time PCR assay used, 90.7% (39/43) of these animals were positive in at least one of two samples (skin or vaginal scrapings). Antibody titers, as determined by an immunofluorescent antibody test, and the threshold cycle values of the real time PCR obtained for skin samples and vaginal scrapings, were significantly correlated. The specificity of the PCRs was confirmed using genomic DNA from related parasites, including genomic DNA of Besnoitia spp., Neospora caninum, Toxoplasma gondii, Hammondia hammondi, Hammondia heydorni, Isospora spp., Sarcocystis spp., Eimeria bovis, Cryptosporidium parvum, and Trypanosoma brucei brucei. Since the sequence of the ITS-1 region of B. besnoiti is identical with that of Besnoitia species isolated from donkeys (Besnoitia bennetti), and reindeer (Besnoitia tarandi), both real time PCRs detected also DNA of these parasites. One of the B. besnoiti real time PCRs, BbRT1, but not BbRT2, cross-reacted with Besnoitia darlingi, Besnoitia oryctofelisi, and Besnoitia neotomofelis when large amounts of genomic DNA (10 ng) were used. The other B. besnoiti real time PCR assay (BbRT2) was specific for B. besnoiti, B. bennetti and B. tarandi, but did not react when 10 ng DNA of other related parasite species from the genus Besnoitia or other genera were subjected to analysis.     

Immunodiagnosis of Besnoitia besnoiti infection by ELISA and Western blot

Besnoitia besnoiti, an obligate intracellular apicomplexan protozoan parasite, is the causative agent of bovine besnoitiosis. This infection may dramatically affect body condition and lead to irreversible infertility in males, resulting in important economical losses in livestock production. Identification of serologically positive animals is of major relevance to elaborate appropriate measures of control. While identification of clinical cases is relatively easy to carry out, the finding of subclinical forms of infection is more difficult, thus serology is considered as an appropriate diagnostic tool. In view to improve and validate immunodiagnosis, we evaluated an enzyme-linked immunosorbent assay (ELISA), complemented with a Western blot (both using a somatic B. besnoiti-tachyzoite antigen) to detect anti-B. besnoiti antibodies in bovine sera. The comparative evaluation of the 2 methods, using 13 sera from animals affected by the chronic phase of besnoitiosis and 10 asymptomatic carriers, yielded a diagnostic sensitivity of 87% for ELISA and 91% for Western blot analyses. Specificity was tested with sera from animals with confirmed Toxoplasma gondii (n=5) and Neospora caninum (n=12) infection, and with 64 negative sera from either an endemic or a non-endemic area. The ELISA specificity ranged between 96.4% and 98%, the Western blot specificity between 96.4% and 100%. The present study demonstrated that ELISA and Western blot, using in vitro generated somatic B. besnoiti antigen, is a useful tool combination to reliably detect animals that have been exposed to B. besnoiti infection, including both asymptomatic and symptomatic courses of disease.     

A longitudinal study of Besnoitia besnoiti infections and seasonal abundance of Stomoxys calcitrans in a dairy cattle farm of southwest France

Bovine besnoitiosis, caused by the cyst-forming apicomplexan Besnoitia besnoiti, is commonly reported in some restricted regions of South-Western Europe, and in larger regions of Africa and Asia. This infection is thought to be transmitted by blood feeding insects and is responsible for major economic losses in cattle production. A recent emergence in Europe, notified in the Centre of France, Spain and Germany, has attracted more attention to this disease. Clinical signs could appear in some animals; however, many infected cattle remain asymptomatic or show scleral-conjunctival cysts (SCC) only. Recent development of serological methods allows carrying out seroepidemiological field studies. In this respect, a long-term investigation was performed in a dairy cattle farm localized in an enzootic area of besnoitiosis of South-western France between March 2008 and May 2009. The objective was to estimate the seasonal pattern of B. besnoiti infections based on the presence of SCC and serology (ELISA and Western blot). In parallel, an entomological survey was conducted to describe population dynamics of Stomoxys calcitrans and Tabanidae species. The seroprevalence determined by Western blot in a cohort of 57 animals continuously present during the whole survey increased from 30% in March 2008 to 89.5% in May 2009 and was always higher than the prevalence based on clinically assessed SCC. New positive B. besnoitia seroconversions occurred throughout the year with the highest number in spring. In addition, many seroconversions were reported in the two months before turn-out and could be associated with a high indoors activity of S. calcitrans during this period.     

A simple field diagnostic smear test for bovine besnoitiosis

A fast and inexpensive skin biopsy smear was used for confirming suspected clinical cases of bovine besnoitiosis. The technique was based on the demonstration of Besnoitia besnoiti bradyzoites (cystic stages), which appeared stumpy, each organism 6.2 microns by 3.1 microns in size, or banana-shaped, 7.7 microns by 1.5 micron in affected skin smears. A more rapid non-surgical technique, scleral conjunctival scraping, revealed similar bradyzoites, thus enhancing the diagnostic value of conjunctival cysts in more chronic infections. The technique will aid prompt management decisions to contain suspected outbreaks in herds not routinely served by tissue-processor-equipped diagnostic laboratories.     

Serodiagnosis of bovine besnoitiosis by ELISA and immunofluorescence tests

Sera from non-infected cattle and cattle infected with Anaplasma, Babesia, Theileria and Sarcocystis were tested for antibodies to Besnoitia in ELISA and immunofluorescence tests (IFT) with Besnoitia besnoiti of blue wildebeest origin as antigen. Only 2 out of 86 sera gave false positive reactions in ELISA and none in the IFT, indicating a high specificity for the tests. Three-hundred-and-three bovine sera from 3 farms in an area endemic for besnoitiosis were similarly tested and the results were correlated with clinical findings based on visual inspection for typical symptoms and the presence of cysts in the scleral conjunctiva. Most of the positive tests were observed in cattle older than 1 year. Of the cases with scleral cysts, 68,7% were positive in the ELISA and 81,74% in the IFT. However, 45,74% (ELISA) and 49,47% (IFT) of the clinically negative cattle were clinically positive, indicating a high incidence of clinically inapparent infection. These results indicate a relatively low sensitivity for these serological tests. An unexpected finding was that the ELISA remained negative for at least 60 days after experimental infection of the cattle, the maximum period for which tests were done, whereas the IFT became positive. No antibodies against B. besnoiti could be found in human sera. Besnoitia jellisoni antigen gave positive results with B. besnoiti antibodies in ELISA, but not in the IFT.     

Histopathologic and ultrastructural studies on experimental caprine besnoitiosis

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.     

Experimental transmission of Besnoitia caprae in goats

Experimental transmission of Besnoitia caprae from naturally chronically-infected goats to susceptible ones was achieved by intra-nasal instillation and intra-conjunctival inoculation of cystozoite-containing suspensions, subcutaneous implantation of fascia containing cysts and alternate needle pricking between the infected and non-infected goats. Typical chronic symptoms developed in the fascia-infected does. Cystozoite inoculation into the eyes and mouth did not result in infection. Kids born of dams with acute and chronic besnoitiosis did not contract the infection in utero, suggesting that intra-uterine transmission may not occur. In contrast to does with acute besnoitiosis, which occasionally aborted, the does with chronic besnoitiosis gave birth to healthy kids. Kids below the age of 4 months (pre-weaned period) born of both infected and non-infected does were susceptible to besnoitiosis but appeared to be more resistant than adult goats.     

Besnoitiosis in a miniature donkey

A 1-year-old male miniature donkey ( Equus asinus ) from a herd of eight was presented with a 9-month history of pruritic dermatitis, lethargy and anorexia. Physical examination revealed diffuse lichenification and scales involving the skin of the face, head and dorsum from the neck to the pelvis. The main histological alteration within the superficial and deep dermis was the presence of multiple large, spherical, thick walled, protozoal Besnoitia cysts. In addition, the inflammatory response consisted of a moderate, superficial and deep perivascular, mixed mononuclear cell infiltrate, with epidermal hyperplasia and compact orthokeratosis. Based upon the large size of the protozoal cysts and the ultrastructural features of the bradyzoites contained therein (conoid, polar ring, rhoptries, micronemes and microtubules), a diagnosis of cutaneous besnoitiosis was established. Treatment with trimethoprim-sulphamethoxazole resulted in significant clinical improvement. To our knowledge, this is the first reported case of besnoitiosis in a miniature donkey in North America.     

Natural Besnoitia sp. infection in domestic rabbits from Argentina

Besnoitia sp. are apicomplexan coccidian parasites affecting several species of mammals and cold-blooded animals in several countries. Besnoitia sp. tissue cysts were seen in several tissues of five rabbits from a rabbit breeder in La Plata, Argentina. Bradyzoites released from macroscopic tissue cysts were inoculated onto bovine monocytes, and into interferon gamma gene knockout (KO) mice. Besnoitia sp. tachyzoites were seen in the peritoneal exudate of KO mice on day 10 pi and these tachyzoites were infective to other KO mice. Tachyzoites grown in cell culture were infective to gerbils (Meriones unguiculatus). This is the first report of Besnoitia sp. infection in any host in Argentina.     

Influence of culture medium pH on internalization, growth and phenotypic plasticity of Neospora caninum

Neospora caninum, a strictly intracellular protozoan, is a major leading cause of parasite-induced abortion in cattle. A widely held view of N. caninum infection is that both cellular proliferation and stage interconversion (tachyzoite-bradyzoite transformation) are triggered, perhaps even modulated by, changes in cultural conditions. This study tested the hypothesis that exposure of N. caninum tachyzoites to different pH culture media affects the parasite's entry, proliferation and cyst formation in cultured cells. The endocytic pathway for N. caninum entry into the K562 cell line was found to be mediated by low pH of culture medium. Internalization of N. caninum by host cells was significantly increased in acidic and alkaline culture medium compared to cells maintained in neutral medium as revealed by transmission electron microscopy. Parasite proliferation within Vero cells was assessed by plaque formation assay and was found to be highest when pH level was optimum, paralleled by a decrease in the number of cysts. In contrast, parasite encystation increased when the pH level was alkaline or acidic, as evaluated by indirect immunofluorescence and immunocytochemical analyses. Acidic pH regardless of state of host cell infection suppressed the rate of host cell division. These findings suggest that culture medium pH has a determinable effect on the host cell-N. caninum interaction and support the hypothesis that pH of culture medium influence the entry, growth, and phenotypic plasticity of N. caninum in mammalian cells.     

Susceptibility of Djungarian hamsters (Phodopus sungorus) to Neospora caninum infection

Djungarian hamsters were examined for the susceptibility to Neospora caninum infection. After 29 Djungarian hamsters were intraperitoneally inoculated with 5 x 10(6) N. caninum tachyzoites of JPA1 strain, some animals showed symptoms such as ataxia, and many tissue cysts were detected in the brain and a cyst in the muscular tunics of stomach. Especially, more than 100 cysts per head were observed after 5 weeks post inoculation. It is suggested that the Djungarian hamster is a model useful to examine neosporosis.     

Preliminary findings from an experimental study of caprine besnoitiosis in Kenya

Inoculation of cystozoites obtained from natural, chronic cases of caprine besnoitiosis produced clinical disease in goats but not in rabbits, mice, guinea pigs, hamsters, rats or cattle. Histological examination of tissue sections from the experimental animals showedBesnoitia cysts only in goats. This, together with field observations that cattle reared together with goats having besnoitiosis do not contract the disease, suggests that theBesnoitia species that infects goats in Kenya is host-specific and is notBesnoitia besnoiti. We suggest that the nameBesnoitia caprae be adopted for the caprine pathogen.     

Ganglion cysts in a juvenile dog

Ganglion cysts were diagnosed in a 4-month-old male Afghan Hound. Grossly, the subcutaneous ovoid cysts around the caudal right elbow joint and left ischiatic tuberosity had abundant mucinous fluid and internal folding. The lesions recurred twice around the elbow joint after surgical removal. Neither cyst communicated with the joint cavity. Histologically, the cyst wall consisted of inner myxomatous and outer immature connective tissue. Some parts of the cyst wall had various stages of myxoid metaplasia of collagen tissue leading to new cyst formation. Ultrastructural study revealed that cells in the myxoid metaplastic lesion had well-developed cytoplasmic secretory elements, including abundant rough endoplasmic reticulum, Golgi apparatus, and many smooth-walled vesicles. These ganglion cysts apparently resulted from the metaplasia of fibroblasts to secreting cells.     

Comparative studies on hydatidosis in farm animals in Egypt.

A survey on the frequency of hydatid disease in animals slaughtered in Cairo abattoir revealed that the percentage in camels, sheep, pigs, cows and buffaloes attained 31.0%, 1.33%, 4.62%, 0.0% and 0.0% respectively. In camels and pigs the lungs were the main predilection sites of hydatid cysts, while in sheep the liver was the most infested organ. The wall thickness of the cysts was different according to the host species and the location of the cyst. The parasitic membrane of the cysts collected from camels consisted of two layers, an outer layer rich with mucopolysaccharides, and an inner germinal layer; some of its cells were rich with glycogen others were charged with lipids; the latter were mainly concentrated at the origin of the brood capsule. Hydatid cysts from camels showed a large number of calcareous bodies which stained deeply with both von Kossa stain and alcian blue stain. The majority of the examined cysts from sheep showed non-fertile germinal membranes. The most characteristic feature observed in the cysts collected from pigs was the presence of large amounts of fat droplets.     

Enzyme linked immunosorbent assay for detection of antibodies againstBesnoitia besnoiti in cattle

The use of the ELISA method for the detection of antibodies to B. besnoiti in cattle is described and compared to the IFAT technique. One hundred and twenty-one sera were examined, of which 61 were sera of calves experimentally infected with B. besnoiti, 52 sera from field animals and eight were sera with high titres of antibodies to other parasites. The specificity of both assays correlates but ELISA seemed to be more sensitive. The ELISA technique provides a rapid and reliable method for the screening of B. besnoiti infection in cattle.     

Neospora-like encephalomyelitis in a calf: pathology, ultrastructure, and immunoreactivity

Protozoal encephalomyelitis was diagnosed in a 3-day-old calf that was stunted, weak, and recumbent. Grossly, the calf had contracted tendons in the forelegs, a slightly doomed skull, a porencephalic cyst in the cerebellum, ulcerative esophagitis, and abomasitis. Histologically, there was a multifocal nonsuppurative encephalomyelitis with clusters of protozoal tachyzoites and numerous protozoal cysts. The porencephalic cyst and gastrointestional lesions appeared to be unrelated to the protozoal infection and were suggestive of a concurrent bovine virus diarrhea infection. A few groups of protozoal tachyzoites and numerous tissue cysts were found in neuropile, particularily in neurons of the spinal cord. By light microscopy, smaller tissue cysts were found in the brain (majority from 14 to 20 microns) than in the spinal cord (majority from 20 to 48 microns). The cyst walls ranged in thickness from less than 1 micron to a maximum of 2 microns wide. Bradyzoites contained PAS-positive slender bradyzoites (5-8 x 1-2 microns). Tissue cysts reacted positively to anti-Neospora caninum sera; but unlike N. caninum, they were positive to 2 of 4 antisera against Toxoplasma gondii and to antisera to H. hammondi. Ultrastructurally, tissue cysts closely resembled a Neospora-like organism, including the finding of interneuronal protozoal cysts, thick cyst walls, a lack of micropores in the bradyzoites, and the presence of numerous micronemes oriented perpendicular to the pellicle. Ultrastructural features in the calf protozoan that have not been reported for N. caninum in dogs included the presence of numerous tubulovesicular structures in the cyst ground substance and bradyzoite vesicles that contained small vesicular structures and short, flat membrane segments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Survival, immune responses and tissue cyst production in outbred (Swiss white) and inbred (CBA/Ca) strains of mice experimentally infected with Neospora caninum tachyzoites

The present work compared inbred (CBA/Ca) and outbred (Swiss white) strains of mice for their capacity to cope with a Neospora caninum infection and to consistently produce tissue cysts. In each experiment Swiss white and CBA/Ca mice were given three different doses of NC-1 tachyzoites. Lymphoproliferative and humoral responses as well as cytokine production were evaluated eight weeks after infection (PI) whereas tissue cyst production and histopathology were assessed 4, 6 and 10 weeks PI in immunosuppressed mice. Tissue cysts were observed 10 weeks after infection only in CBA/Ca mice receiving the two highest inoculum doses. Furthermore this strain showed the highest specific lymphoproliferative response. A mixed cytokine response with elevated IFN-gamma and fairly low IL-4 and IL-10 secretion was recorded. In both strains, no lesions were observed in the tissues of infected mice. This study indicates that CBA/Ca female mice infected with 5 x 10(6) NC-1 tachyzoites represent a useful model for the study of specific maternal immune responses in pregnant animals.