Prevalence and molecular detection of Eimeria species in different types of poultry in Greece and associated risk factors.

Coccidiosis is one of the most notable diseases in chickens having a high economic impact on the poultry industry worldwide. The present study is the first attempt to epidemiologically investigate Eimeria spp. distribution and associated risk factors under different housing and production systems in three major regions in Greece. Faecal samples were obtained from 42 operations (broilers, floor housed, free range and organic layers, backyard farms). A questionnaire was obtained from included operations to acquire additional information regarding farm management, location, production rate and diseases history. Positivity level was 85.7%. All seven Eimeria species were identified, and the most prevalent ones were E. acervulina (79.3%) and E. tenella (65.5%). Single-species and mixed infections were detected in 20.7% and 79.3% of the flocks, respectively. Flock size, type of outdoor area, production system and presence of respiratory disease proved significant risk factors. Flock size up to 10,000 birds correlated strongly (p = 0.02) with higher E. tenella quantities. A very strong correlation (p < 0.001) was found between the presence of respiratory disease and the average OPG level in broiler farms. Organic flocks showed higher prevalence of E. tenella (p = 0.023), while presence of vegetation at the outdoor area correlated strongly with E. brunetti (p < 0.001). Molecular analysis and correlation results in this survey give strong indications although more studies are needed to further understand the involvement of different Eimeria species in various husbandry, production and management systems, to gain more knowledge about the sustainable control of coccidia in poultry.

Central nervous signs, blindness and cerebral vermicosis in free-ranging peregrine falcons (Falco peregrinus) associated with aberrant larval migrations.

Four free-ranging peregrine falcons (Falco peregrinus) were submitted with a history of unilateral or bilateral blindness and central nervous signs to a veterinary clinic in Germany. There were no indications of trauma or ocular disease. Likewise, other differential diagnoses for CNS signs were ruled out within the diagnostic process. The clinical diagnostic panel in live falcons included general examination, radiography, endoscopy, hematology, ophthalmoscopy and parasitological examination of the feces, blood gas analysis and blood chemistry as well as computed tomography, and magnetic resonance imaging (MRI). A complete pathological and histopathological examination was performed post-mortem. The only common finding in all birds was an infection with the nematode parasite Serratospiculum tendo. The parasite was confirmed morphologically and via PCR. In two falcons intracerebral vermicoses was suspected in MRI and confirmed in subsequent histopathology, but molecular biological identification of the parasite species failed from brain tissue. Until today, S. tendo had been reported to affect the respiratory system, the liver and different parts of the gastrointestinal tract and to cause cachexia, inappetence, regurgitation, dyspnea and general signs of illness such as lethargy, poor plumage, and reduced reproduction. Our findings indicate that aberrant migration should be considered as cause for CNS signs in falcons. As S. tendo might be a possible cause for this, CNS signs might be included in the list of clinical signs of serratospiculiasis in falcons.

Host-pathogen interaction in Toxoplasma gondii-infected mixed chicken blood cell cultures.

Toxoplasma gondii has the ability to infect various nucleated cell types in different hosts. The aim of the present study was to investigate which chicken blood cells were targeted by T. gondii in a mixed blood cell culture similar to in vivo conditions and to evaluate parasite-host cell interactions. The study consisted of two subsequent experiments. In experiment 1, we applied T. gondii tachyzoites (ME49) at a multiplicity of infection of 1 tachyzoite per blood cell and examined parasite replication, cytokine, and inducible nitric oxide synthase (iNOS) mRNA expression between 1 h and 48 h post-infection (p.i.) by quantitative PCR. By using T. gondii RH-GFP tachyzoites expressing the green fluorescent protein (GFP) in experiment 2, we aimed for visualizing infected cells by confocal laser scanning microscopy (CLSM) and flow cytometric analysis at 24 h p.i. The parasite replication curve showed a massive decrease of parasite stages until 24 h p.i. followed by an approximately plateau phase. We observed mainly significantly increased iNOS mRNA expression levels in T. gondii-infected culture compared to uninfected cells. Flow cytometry and CLSM data confirmed monocytes/macrophages as main target cells for T. gondii. Moreover, different lymphocytes like B cells and cytotoxic T cells seem to be targeted to a low extent. Our findings indicate that monocytes/macrophages play a key role during T. gondii infection in chicken as host cells and triggering of immune response. To the best of our knowledge, this is the first report of a mixed chicken blood cell culture experimentally infected with T. gondii.

Toxoplasma gondii infections in chickens - performance of various antibody detection techniques in serum and meat juice relative to bioassay and DNA detection methods.

Chickens, especially if free-range, are frequently exposed to Toxoplasma gondii, and may represent an important reservoir for T. gondii. Poultry products may pose a risk to humans, when consumed undercooked. In addition, chickens are regarded as sensitive indicators for environmental contamination with T. gondii oocysts and have been used as sentinels. The aim of the present study was to determine the suitability of commonly used antibody detection methods, i.e. the modified agglutination test (MAT), IFAT and ELISA to detect T. gondii-infected chickens. Samples of experimentally and naturally infected chickens were used. The infection state of all chickens was determined by Magnetic-Capture (MC-) real-time PCR (RT PCR). Naturally exposed chickens were additionally examined by mouse bioassay and conventional RT PCR on acidic pepsin digests (PD-RT PCR). Blood serum and meat juice of various sources were tested for antibodies to T. gondii. In naturally infected chickens, there was substantial agreement between the mouse bioassay and MC-RT PCR or the mouse bioassay and conventional PD-RT PCR. PD-RT PCR was slightly more sensitive than MC-RT PCR, as all (26/26) bioassay-positive chickens also tested positive in at least one of the tissues tested (heart, drumstick). By MC-RT PCR, 92.3% (24/26) of the naturally infected bioassay-positive chickens were positive. The diagnostic sensitivity of MC-RT PCR was clearly related to the organ examined. Based on a quantitative assessment of the MC-RT PCR results in experimentally infected chickens, brain and heart tissues harbored an at least 100 times higher parasite concentration than breast, thigh or drumstick musculature. In naturally infected chickens, only three out of 24 birds, which were MC-RT PCR-positive in heart samples, also tested positive in drumstick musculature. Under experimental conditions, the agreement between MC-RT PCR and the serological techniques revealed 100% diagnostic sensitivity and specificity. Under field conditions, examinations of sera by ELISA, IFAT and MAT showed good performance in identifying chickens that were positive in either a mouse bioassay, MC-RT PCR, or PD-RT PCR as illustrated by diagnostic sensitivities of 87.5%, 87.5% and 65.2%, respectively, and diagnostic specificities of 86.2%, 82.8% and 100%, respectively. The examination of meat juice samples from breast, drumstick or heart musculature revealed similar or even better results in the ELISA. The results in the MAT with meat juice from breast musculature were less consistent than those of ELISA and IFAT because a number of negative chickens tested false-positive in the MAT. The MAT performed similar to ELISA and IFAT when applied to test meat juice samples collected from heart, thigh or drumstick musculature.

Experimental Toxoplasma gondii and Eimeria tenella co-infection in chickens.

The widespread apicomplexan parasites Toxoplasma gondii (T. gondii) and Eimeria tenella (E. tenella) are important pathogens with high prevalence in poultry. The aim of our study was the investigation of mutual influences in co-infected chickens, focusing on immune response and course of infection. Two separate trials were performed using in total 96 1-day-old chickens, divided into four study groups: group NC (negative control, uninfected), group PC-T (oral or intramuscular infection with T. gondii oocysts (trial 1) or tachyzoites (trial 2), respectively), group PC-E (oral infection with E. tenella (trial 1) or E. tenella and Eimeria acervulina (trial 2)), and group TE (co-infection). T. gondii and Eimeria infections were validated by different parameters, and cytokine expression in the gut and spleen was investigated. T. gondii-specific antibodies were detected earliest 4 days post infection (p.i.) by immunoblot and direct DNA detection was possible in 22.1% of all tissue samples from infected chickens. Eimeria spp. merogony seemed to be enhanced by co-infection with T. gondii, interestingly without marked differences in oocyst excretion between co-infected and Eimeria spp. mono-infected chickens. An increase of messenger RNA (mRNA) expression of Th1- (IFN-γ, IL-12, TNF-α) and Th2-related cytokines (IL-10) mainly in groups PC-E and TE was observed, however, without statistically significant differences between co-infection and single infection with Eimeria. In conclusion, most of the measurable immune response could be attributed to Eimeria infection. To the best of our knowledge, this is the first report on co-infection experiments of T. gondii with Eimeria spp. in chickens.

High seroprevalence of Toxoplasma gondii and probability of detecting tissue cysts in backyard laying hens compared with hens from large free-range farms.

Serological assays are commonly used to determine the prevalence of Toxoplasma gondii infection in livestock, but the predictive value of seropositivity with respect to the presence of infective tissue cysts is less clear. The present study aimed at the identification of seropositive and seronegative free-range laying hens from organic and backyard farms, and the relationship with the presence of viable tissue cysts. In addition, potential risk and protective factors on the selected farms were investigated. An in-house T. gondii surface antigen (TgSAG1, p30, SRS29B) ELISA was validated with sera from experimentally infected chickens and used to examine 470 serum samples collected from laying hens from large organic and small backyard farms at the end of their laying period. A total of 11.7% (55/470) of all chickens tested positive, and another 18.9% (89/470) of test results were inconclusive. The highest seroprevalences were observed on small backyard farms with 47.7% (41/86) of chickens being seropositive while another 20.9% (18/86) of test results were inconclusive. Twenty-nine seropositive, 20 seronegative and 12 laying hens which yielded inconclusive ELISA results, were selected for further examination. Hearts and limb muscles of these hens were examined for T. gondii tissue cysts in a bioassay with IFNÉ£-knockout or IFNÉ£-receptor-knockout mice. Viable T. gondii was isolated from 75.9% (22/29) of the seropositive, 25.0% (3/12) of the inconclusive, and 5.0% (1/20) of the seronegative chickens. All 26 chickens tested positive in heart samples, while drumstick muscles (i.e. limb muscles) tested positive only in three. Data on putative risk and protective factors were collected on the farms using a standard questionnaire. Generalised multilevel modelling revealed farm size, cat related factors ('cats on the premise', 'cats used for rodent control'), hen house/hall related factors ('size category of hen house/hall', 'frequency category of cleaning hen house/hall', 'service period') as significantly associated with seropositivity to T. gondii in hens. The final model, which included the age of the birds as an effect modifier and farm as a random effect variable, revealed that the use of cats for rodent control and an area available per hen in the chicken run of ≥10sqm were statistically significant risk factors for T. gondii seropositivity. Overall this study showed that exposure to T. gondii is common in small backyard farms but is rare on large organic farms with a high density of free-range hens, even when cats were present on the premises.

Co-infection of Trypanosoma pestanai and Anaplasma phagocytophilum in a dog from Germany.

Infections with arthropod-borne pathogens are an increasing threat world-wide that requires heightened vigilance from veterinary and medical practitioners, especially when they involve new or unusual organisms. A dog was presented to a local veterinary clinic in Germany with malaise, pale mucous membranes and stiff joints. Clinical assessment revealed pyrexia, leukopenia and thrombocytopenia. On suspicion of a tick-borne infection, blood samples were examined for clinical and biochemical parameters and subjected to a Anaplasma phagocytophilum-, Borrelia spp.- and Ehrlichia canis-specific real-time PCR. Additionally, a sample of the pre-therapeutic buffy coat was co-cultured with the Ixodes scapularis cell-line ISE6 for 20days. Only the PCR specific for A. phagocytophilum DNA yielded a positive result, and furthermore, Anaplasma morulae were visible in granulocytes and tick cells. After co-culturing, extracellular trypomastigote and epimastigote stages of Trypanosoma sp. with an average length of 29.7µm were observed, featuring a pointed posterior end. Sequence analysis of a 2080bp fragment of the 18S rRNA gene showed 99% identity to the 18S rRNA gene of Trypanosoma pestanai, previously described from a European badger (Meles meles) in France. The dog's condition improved rapidly in response to doxycycline treatment for three weeks. The clinical status normalized and clinical blood parameters were found to be within the reference ranges. To our knowledge this is the first description of T. pestanai infection in a dog, the first detection of T. pestanai in Germany and the first documented co-infection with these two pathogens. Co-infections with unusual opportunistic vector-borne pathogens should be considered, if acute canine granulocytic anaplasmosis is evident.

Persistence of Toxoplasma gondii tissue stages in poultry over a conventional fattening cycle.

Toxoplasma gondii is a widely spread protozoon in humans, mammals and poultry. Regarding the latter, nothing is known yet about the duration of T. gondii persistence and distribution over a conventional fattening cycle of turkeys and chickens. Twenty-four turkeys and 12 broiler chickens were infected intravenously with 1×10(6) T. gondii tachyzoites (strain NED). Serum antibody levels were determined weekly by ELISA (turkeys) or immunofluorescent antibody test (chickens). Turkeys were slaughtered at 4, 8, 12 and 16 weeks post-infection (p.i.), and chickens 5 or 10 weeks p.i. (n = 6 per group). Sixteen different tissue samples per bird were analysed for T. gondii by PCR. All infected animals showed seroconversion. In turkeys, 15.9% of all samples were tested positive for T.-gondii-DNA. Among the edible tissues (drumstick, thigh, breast muscle, heart, liver and gizzard) 7.8% tested positive. Among poultry slaughtered after different periods of time after infection no significant differences (P>0.05) regarding the number of positive samples were observed. Only 4 out of 192 samples (2.1%) from infected chickens contained detectable T. gondii DNA.The PCR findings suggested that T. gondii may persist in poultry. Particularly in turkey it was shown that edible tissues stay infected for at least 16 weeks p.i. which indicates a potential risk for consumers of undercooked turkey meat whereas chickens appear less susceptible to T. gondii infection.

Necrotic enteritis in chickens: development of a straightforward disease model system.

The interaction between Eimeria species and Clostridium perfringens was investigated in two different necrotic enteritis (NE) models: 120-day-old broilers were used in two separate experiments consisting of six groups (n=10) each. Besides controls, chickens were infected with coccidia on study day (SD) 18 (Eimeria maxima and Eimeria acervulina (experiment 1) or Eimeria tenella and Eimeria brunetti (experiment 2) and/or a NetB toxin positive C perfringens strain (both experiments: SD 14 or SD 22, respectively)). Body weight, feed intake, mortality rate, clinical disease, Eimeria species oocyst excretion and C perfringens counts were recorded. NE and coccidiosis specific lesion scores were assessed (SD 24 and SD 30). In coinfected groups, NE-typical clinical signs occurred. Coccidiosis-specific lesions were most severe in coinfected groups (significant for E tenella, P<0.05). Most pronounced NE lesions occurred in coinfected chickens compared with C perfringens monoinfected groups (experiment 2, C perfringens infections on SD 22: P<0.05). In experiment 2, E tenella antibody levels were (non-significantly) higher in coinfected groups than in Eimeria species monoinfected groups. Thus, infection with E tenella and Eimeria brunetti followed by C perfringens inoculation is regarded as an easy to handle and suitable model for investigations into NE of chickens.

Effects of pH, sodium chloride, and curing salt on the infectivity of Toxoplasma gondii tissue cysts.

Toxoplasma gondii is one of the most common zoonotic parasites in the world. The parasite causes no or mild symptoms in immunocompetent humans. However, a high potential hazard exists for seronegative pregnant women and immunocompromised patients. The consumption of meat containing tissue cysts or oocyst-contaminated vegetables and fruits or the handling of cat feces poses a high risk of infection with T. gondii. It is known that raw minced meat, raw fresh sausages, and locally produced raw meat products are possible causes of T. gondii infection. The infectivity of T. gondii tissue cysts in meat products depends, among other factors, on the pH and the salt concentration. Therefore, the impact of these two factors on the tissue cysts was examined. For this purpose, dissected musculature and brain from experimentally infected mice (donor mice) were placed in a cell culture medium (RPMI 1640). The medium was adjusted to different pH values (pH 5, 6, and 7) with lactic acid and to different salt concentrations (2.0, 2.5, and 3.0%) with sodium chloride (NaCl) or nitrite-enriched curing salt (NCS) for the various tests. After storage at 4°C for different time periods, the materials were fed to bioassay mice. Later, the brains were examined for presence of T. gondii to assess the infectivity. The data show that T. gondii tissue cysts have a high pH tolerance. Cysts were infectious in the muscle for up to 26 days (pH 5). In contrast to their tolerance to pH, cysts were very sensitive to salt. Muscle cysts survived at an NaCl concentration of up to 2.0% only, and for no longer than 8 days. At NaCl concentrations of 2.5 and 3.0%, the cysts lost their infectivity after 1 day. When NCS instead of NaCl was used under the same conditions, T. gondii muscle cysts retained infectivity for only 4 days at 2.0%. Consequently, NCS (NaCl plus 0.5% nitrite) has a stronger effect on T. gondii cysts than does common table salt. Sausages produced with low NaCl concentration and short contact times pose a potential risk for susceptible individuals.

Experimental Toxoplasma gondii oocyst infections in turkeys (Meleagris gallopavo).

Toxoplasma (T.) gondii is a protozoan parasite with a broad range of intermediate hosts. Humans are often infected by ingestion of tissue cysts in raw or undercooked meat or meat products. Turkeys as food-producing animals can also serve as intermediate hosts. The aim of the present study was to investigate occurrence and predilection sites of T. gondii infection in turkeys after oral infection with oocysts. Experimental infections with different doses of T. gondii oocysts were performed in 36 turkeys to mimic natural infection. Systemic distribution of parasitic stages was investigated by screening 14 different tissues including the edible tissues heart, liver, thigh, breast and drumstick muscle. Parasite detection was based on a conventional nested polymerase chain reaction (PCR). Animals were sacrificed 6-12 weeks after infection. Results demonstrated parasite spreading over the whole organism after oral infection by oocysts. Most frequently affected tissues were brain (47.2% of all brains were positive for T. gondii) and thigh muscle (25.0% positive samples). Other muscles were regularly T. gondii-positive, all other sampled tissues were positive at least once. Thus, edible tissues are one of the predilection sites of T. gondii in turkeys which renders raw or undercooked turkey meat a potential risk for parasite transmission to humans. Data were compared to results from previous parenteral turkey infections with tachyzoites. With the exception of brain, liver and breast muscle affection, no significant differences were observed between both infection routes. Both infection models could be used for research purposes with certain advantages and disadvantages.

Tissue tropism of Toxoplasma gondii in turkeys (Meleagris gallopavo) after parenteral infection.

Turkeys are known to be natural hosts for the zoonotic protozoan parasite Toxoplasma gondii. The objective of the present study was to gain further knowledge of possible predilection sites of T. gondii infection in this species after parenteral application of tachyzoites. A total of 38 turkeys were infected with different doses of T. gondii tachyzoites. Birds were killed either 6 to 8 or 10 to 12 weeks after the experimental infection. Fourteen different tissues per bird were investigated by a nested polymerase chain reaction (PCR) for the presence of the parasites' DNA. T. gondii DNA was found in any type of tissue analysed; in 86.1 % of all infected birds, at least one sample was tested positive. Over all intravenously infected birds, 15.4 % of all analysed samples contained T. gondii DNA. Most frequently affected tissues were liver (43.3 % positive samples), breast muscle (26.7 % positive samples) and heart (20.0 % positive samples), while the brain was less frequently positive (6.7 %). The number of positive tissues varied from zero to seven tissues per animal with at least one T. gondii-positive edible tissue sample in 80 % of all intravenously infected birds. Still, the results did not indicate defined target tissues or a cyst distribution pattern. Nonetheless, edible organs were most frequently parasitised. The number of positive findings did not differ between the early and the late examination time points. Therefore, a persistence of the tissue stages until the end of the study (12 weeks after infection) is concluded.

Influence of experimental Eimeria zuernii infection on clinical blood chemistry in calves.

Coccidiosis, often caused by Eimeria zuernii infection, is an important diarrhoeal disease in calves [Fitzgerald, P.R., 1980. The economic impact of coccidiosis in domestic animals. Adv. Vet. Sci. Comp. Med. 24, 121-143]. Infection trials were performed to investigate the effects of experimental E. zuernii coccidiosis on clinical blood chemistry in calves. Three groups of calves were formed: group 1 (n=14) served as uninfected control group, group 2 (n=11) was infected with 150,000 sporulated E. zuernii oocysts per calf, and group 3 (n=16) was infected with 250,000 sporulated E. zuernii oocysts per calf. Measurements throughout the prepatent and the patent period revealed a marked influence of E. zuernii infection on the following parameters: total protein, albumin, urea, bilirubin, creatine kinase, free fatty acid concentration, and cholesterol. Aberrances in these were most pronounced in group 3. No significant and/or distinct changes after infection could be detected in blood glucose concentration. E. zuernii infection impairs intestinal function and induces catabolic metabolism in affected calves. Bilirubin, urea and cholesterol concentration, and creatine kinase activity were particularly affected indicating catabolism of protein and lipids.

Influence of experimental Eimeria zuernii infection in calves on electrolyte concentrations, acid-base balance and blood gases.

Coccidiosis, often caused by Eimeria zuernii, is an important disease in calf rearing and is clinically mainly associated with diarrhoea (PR Fitzgerald in Adv Vet Sci Comp Med, 24:121-143, 1980). Calves were experimentally infected with E. zuernii oocysts to investigate the effects of artificial E. zuernii coccidiosis on electrolyte concentrations, acid-base balance and blood gases. Therefore, animals were assigned to three groups: group 1 (n = 14) served as uninfected control group, group 2 (n = 11) was infected with 150,000 sporulated E. zuernii oocysts per calf, and group 3 (n = 16) was infected with 250,000 sporulated E. zuernii oocysts per calf. Aberrances which were attributed to coccidiosis were observed in the following parameters: sodium and chloride concentrations, pH (only high-dose infected group 3), base excess, standard bicarbonate, total carbon dioxide and partial pressure of carbon dioxide. Alterations were most pronounced in the high-dose infected group 3. Anion gap and oxygen saturation did not show significant differences between the groups. Due to diarrhoea and malabsorption in coccidiosis-affected calves, there is a distinct loss not only of fluid and blood but also of electrolytes and alkaline buffer substances which provokes the development of an acidosis. This is counteracted by metabolism and respiration but cannot be compensated in severely affected and moribund calves.

Parasitological and clinical parameters of experimental Eimeria zuernii infection in calves and influence on weight gain and haemogram.

Infection trials were performed to characterize experimental Eimeria zuernii coccidiosis parasitologically and clinically and to investigate the effects on weight gain and haemotologic parameters in affected calves. Three groups of calves were formed: Group 1 (n = 14) served as uninfected control group, group 2 (n = 11) was infected with 150,000 sporulated E. zuernii oocysts per calf, and group 3 (n = 16) was infected with 250,000 sporulated E. zuernii oocysts per calf. All infected animals shed oocysts and showed diarrhoea; a positive correlation could be shown between quantified oocyst excretion and faecal consistency. Measurements throughout the prepatent and the patent period revealed a marked influence of E. zuernii infection on weight gain, leukocyte concentration, haemoglobin, haematocrit, and mean cellular volume. Aberrations in these parameters were most pronounced in the highly infected group. The results of this study confirm that acute sublethal E. zuernii coccidiosis causes distinct loss of fluid and blood via intestine. This dominates also the haematological picture of the disease, which is mainly characterized by haemoconcentration. Leukocyte concentration was depressed during the early patent period, whereas it increased markedly from day 24 after infection on.

Control of clinical coccidiosis of calves due to Eimeria bovis and Eimeria zuernii with toltrazuril under field conditions.

The efficacy of metaphylactic treatment with toltrazuril (Baycox 5% suspension) against natural infections with Eimeria bovis and/or Eimeria zuernii in calves was investigated. The study was conducted with 208 calves on five calf-rearing farms in Germany and the Czech Republic. All participating farms had a notable incidence of coccidiosis. Animals were treated 14 days after stabling in the respective facility. One group was treated with 15 mg toltrazuril/kg body weight, and a second group served as the sham-treated control. Assessment of efficacy was based on faecal consistency and oocyst excretion of E. bovis and E. zuernii, both investigated throughout the study. Duration and rate of oocyst excretion as well as number of scour days with E. bovis or E. zuernii oocyst shedding and the severity of diarrhoea were significantly lower in the toltrazuril-treated groups. It is concluded that a single metaphylactic treatment with toltrazuril controls coccidiosis of housed calves under various field conditions.

Pathology and treatment of Eimeria zuernii coccidiosis in calves: investigations in an infection model.

Two studies were conducted in the Eimeria zuernii infection model in order to investigate the pathology of E. zuernii coccidiosis and the efficacy of toltrazuril (Baycox 5% suspension) in this infection. For this purpose, a total of 30 calves were infected experimentally with E. zuernii oocysts and faecal samples taken regularly from the rectum and examined for faecal consistency and oocyst excretion. Six of the calves underwent pathological examination at various points in time after infection. Significant macroscopic and microscopic changes were demonstrated and parasitic stages were identified in the intestinal mucosa of infected calves during the late prepatent and patent period. Inflammatory reactions revealed by light microscopy were confirmed by electron microscopical investigations. Treatment of calves with toltrazuril during the late prepatent period resulted in significantly lower frequencies of diarrhoea and levels of oocyst excretion, and weight gain was significantly higher than in shamtreated animals.