Berit Bangoura

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

Abstract 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.

Prevalence of Eimeria bovis and Eimeria zuernii in German cattle herds and factors influencing oocyst excretion

AbstractThe present study was designed to investigate the prevalence of the pathogenic coccidia species E. bovis and E. zuernii in shed-reared animals in German dairy and fattening facilities.Samples were obtained from 65 cattle farms distributed randomly across all the regions of Germany, regardless of the occurrence of clinical problems. The samples were obtained rectally. Faecal consistency and the total number of oocysts per gram of faeces (OPG) were determined for Eimeria spp., along with the separate OPG values for Eimeria (E.) bovis and E. zuernii. A questionnaire was completed for each farm to record information about herd size and management together with individual animal data. Eimeria oocysts, regardless of the kind of Eimeria spp., were detected in 62 of these farms, which gives a prevalence of 95.4 %. The farm prevalence of the pathogenic species was 76.9 % for E. bovis and 83.1 % for E. zuernii. The average oocyst excretion level was 2,950 OPG in terms of total Eimeria spp. oocyst excretion, 700 OPG for E. bovis and 1,500 OPG for E. zuernii.The number of oocysts excreted could not be correlated significantly with farm type or farm management but depended on the floor type which influences the infection pressure, on the age of the calves and the time after rehousing. In general, higher oocyst excretion rates were found in calves kept on litter compared to rearing on slatted floor. Younger calves and calves sampled early after housing shed higher amounts of oocysts than older calves and calves stabled a longer period before sampling, respectively. Furthermore, there was a positive correlation between OPG and the observation of diarrhoea, defined as observation of a loose to liquid faecal consistency. Excretion of E. zuernii oocysts was more closely linked to the occurrence of diarrhoea than E. bovis oocyst excretion. This study confirms that the pathogenic coccidia E. bovis and E. zuernii are ubiquitous in German cattle populations and a significant cause of diarrhoeal disease in calf rearing.

Study of the Comparative Efficacy of Toltrazuril and Diclazuril against Ovine Coccidiosis in Housed Lambs

A blinded, controlled and randomised field study was conducted on a sheep farm with a known history of coccidiosis and a high prevalence mainly of the pathogenic coccidium Eimeria ovinoidalis. The efficacy of treatment with toltrazuril (Baycox® 5 % suspension) against natural infections with Eimeria crandallis and/or Eimeria ovinoidalis in housed lambs was investigated in comparison with diclazuril and untreated controls. Both drugs were administered either metaphylactically (i.e., in the prepatency of Eimeria spp.) or therapeutically (after onset of oocyst excretion). A total of 145 animals aged 1 to 5 days at the start of the study were included. Examination of faecal samples was performed every second day between days 13 and 49 of the study. The assessment of treatment efficacy was based mainly on total oocyst excretion and the number of E. crandallis and E. ovinoidalis oocysts (OPG) shed throughout the study. Oocyst excretion was reduced significantly in both groups treated with toltrazuril compared with the untreated control group and with both diclazuril-treated groups. The most prevalent and most severe diarrhoea was observed in the untreated control group. In this study, toltrazuril proved to be highly effective in controlling ovine coccidiosis both metaphylactically and therapeutically. The efficacy of toltrazuril was significantly higher than the efficacy of the control substance with regard to the duration and amount of oocyst excretion, both for the comparison of metaphylactic as well as therapeutic treatment.

Prevalence of specific IgG-antibodies against Toxoplasma gondii in domestic turkeys determined by kinetic ELISA based on recombinant GRA7 and GRA8.

The protozoan parasite Toxoplasma (T.) gondii is one of the most common zoonotic infectious agents worldwide. Besides its sexual reproduction in cats, T. gondii can also infect a wide spectrum of other warm-blooded animals. These include animals used for human consumption such as pigs or chickens. Nevertheless, the role of turkeys for the epidemiology of T. gondii infections has not been studied thoroughly. We have established a kinetic ELISA (KELA) for the detection of T. gondii-specific IgG antibodies in turkey serum samples. The test is based on the recombinant dense granule antigens GRA7 and GRA8. These proteins were used as an antigen mixture at a concentration of 0.13 μg per well. The overall sensitivity of the assay was between 92.6% and 100% and the specificity ranged from 78.1% to 100%, depending on the method used to calculate these parameters. Using this KELA we examined 1913 turkey serum samples from 14 turkey farms from different areas of Germany. From these sera, 387 produced a signal in the KELA, corresponding to a true seroprevalence of up to 20.2%. The seropositivity rate in individual fattening cycles at individual farms ranged from 0.0% to 77.1%, whereas the rates were highly variable within the individual farms and individual fattening cycles. Consequently, conditions of animal husbandry could not be associated with particular seroprevalence rates. Although seropositivity cannot be linked directly to infectious tissue cysts in the muscle tissue of commercially produced turkey meat, we state that there is a potential risk of being infected by consuming turkey meat products that were not heat treated.

Efficacy of early treatment with toltrazuril in prevention of coccidiosis and necrotic enteritis in chickens

In the present study, efficacy of the toltrazuril treatment for prevention of coccidiosis and necrotic enteritis was tested. Ninety-six 14-day-old commercial broiler chickens were caged and divided into eight groups (n=12), designated groups 1 to 8. Chickens of groups 1 to 6 were inoculated orally at 18 days of age with 25,000 oocysts of Eimeria tenella and 75,000 oocysts of Eimeria brunetti. At 22 days of age, chickens of groups 1 to 6 were infected with 109 colony-forming unit Clostridium perfringens. Chickens of group 1 were treated with 75 parts/106 toltrazuril in drinking water for 8 h on two consecutive days up to 12 h before Eimeria infection, while chickens of groups 2 to 5 were treated with the same dose of toltrazuril at 12 h, 36 h, 60 h and 84 h after Eimeria infection, respectively. The non-treated group 6 served as a positive control. Chickens in group 7 were treated with toltrazuril at 17 and 18 days of age, and those of group 8 remained uninfected and non-treated as a negative control. The feed conversion ratio was higher in the positive control compared with other groups. The mortality rates were 16.8% and 41.7% in the late toltrazuril-treated (at 84 h) and infected non-treated chickens, respectively. Lesions scores of necrotic enteritis or coccidiosis in infected, non-treated chickens were significantly more severe compared with negative controls (P<0.01) and late toltrazuril-treated (at 84 h) chickens (P<0.05). In conclusion, application of toltrazuril before Eimeria challenge protected chickens from coccidiosis and indirectly from successive necrotic enteritis caused by C. perfringens infection.

Quantitative comparison of different purification and detection methods for Cryptosporidium parvum oocysts.

UNLABELLED Cryptosporidium parvum (C. parvum) is the causal agent of cryptosporidiosis in many animals, mainly cattle, and possesses a high zoonotic potential. It occurs worldwide and ubiquitously. Detection of C. parvum is mainly performed directly but purification of the oocysts is useful to increase sensitivity and to obtain oocyst material for further use. The study was designed to compare (a) three different direct diagnostic methods, namely modified Ziehl-Neelsen staining, carbol fuchsin staining and conventional PCR, and (b) three routine oocyst purification methods, in particular flotation with saturated sodium chloride solution, Sheathers sucrose solution and a Percoll(®) gradient. During comparison of purification methods, special regard was paid to the ability to separate morphologically intact oocysts from the morphologically degenerated fraction or viable from non-viable oocysts, respectively. RESULTS (a) DIAGNOSTIC METHODS Most effective in C. parvum oocysts detection in calf faeces was PCR; carbol fuchsin and modified Ziehl-Neelsen stainings achieved comparable results. (b) Purification methods: Oocyst flotation using sodium chloride solution showed to be superior to Percoll(®) gradient centrifugation and sugar flotation in terms of purification quality, recovery efficacy (yield) and reduction of the proportion of degenerated or non-viable oocysts.

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.

Comparative efficacy of conventional primer sets in detection of Cryptosporidium parvum for diagnostic use

In this study, the sensitivity and specificity of different previously described primer sets for Cryptosporidium parvum detection by polymerase chain reaction (PCR) was evaluated. For this purpose, the primer sets defined by Cacciò et al. (FEMS Microbiol Lett 170(1):173–179, 1999) (tub), Widmer et al. (Appl Environ Microbiol 64(11):4477–4481, 1998) (btub) and Rochelle et al. (Appl Environ Microbiol 63:2029–2037, 1997) (cphsp), respectively, were used. Deoxyribonucleic acid (DNA) was isolated from three different sample materials: (1) from the faeces of an experimentally C. parvum-infected calf, (2) from purified C. parvum oocysts, and (3) from C. parvum-infected HCT-8 cell cultures. The DNA samples were subjected to PCR reactions with each of the three given primer sets to investigate sensitivity and suitability for routine use. The primers described by Cacciò et al. (FEMS Microbiol Lett 170(1):173–179, 1999) (TUB) were superior regarding sensitivity and specificity in terms of detection of C. parvum in faeces, in purified oocysts and also in cell culture, and may thus be applied for routine diagnostic use in common sample materials.

Anticoccidial efficacy testing: In vitro Eimeria tenella assays as replacement for animal experiments

SCOPE Availability of an accurate in vitro assay is a crucial demand to determine sensitivity of Eimeria spp. field strains toward anticoccidials routinely. In this study we tested in vitro models of Eimeria tenella using various polyether ionophores (monensin, salinomycin, maduramicin, and lasalocid) and toltrazuril. Minimum inhibitory concentrations (MIC95, MIC50/95) for the tested anticoccidials were defined based on a susceptible reference (Houghton strain), Ref-1. In vitro sporozoite invasion inhibition assay (SIA) and reproduction inhibition assay (RIA) were applied on sensitive laboratory (Ref-1 and Ref-2) and field (FS-1, FS-2, and FS-3) strains to calculate percent of inhibition under exposure of these strains to the various anticoccidials (%ISIA and%IRIA, respectively). The in vitro data were related to oocyst excretion, lesion scores, performance, and global resistance indices (GI) assessed in experimentally infected chickens. RESULTS Polyether ionophores applied in the RIA were highly effective at MIC95 against Ref-1 and Ref-2 (%IRIA≥95%). In contrast, all tested field strains displayed reduced to low efficacy (%IRIA<95%).%IRIA values significantly correlated with oocyst excretion determined in the animal model (p<0.01) for polyether ionophores. However, this relationship could not be demonstrated for toltrazuril due to unexpected lack of in vitro sensitivity in Ref-2 (%IRIA=56.1%). In infected chickens, toltrazuril was generally effective (GI>89%) against all strains used in this study. However, adjusted GI (GIadj) for toltrazuril-treated groups exhibited differences between reference and field strains which might indicate varying sensitivity. CONCLUSION RIA is a suitable in vitro tool to detect sensitivity of E. tenella towards polyether ionophores, and may thus help to reduce, replace, or refine use of animal experimentation for in vivo sensitivity assays.