Phytohormones regulate asexual Toxoplasma gondii replication.

The protozoan Toxoplasma gondii (T. gondii) is a zoonotic disease agent causing systemic infection in warm-blooded intermediate hosts including humans. During the acute infection, the parasite infects host cells and multiplies intracellularly in the asexual tachyzoite stage. In this stage of the life cycle, invasion, multiplication, and egress are the most critical events in parasite replication. T. gondii features diverse cell organelles to support these processes, including the apicoplast, an endosymbiont-derived vestigial plastid originating from an alga ancestor. Previous studies have highlighted that phytohormones can modify the calcium-mediated secretion, e.g., of adhesins involved in parasite movement and cell invasion processes. The present study aimed to elucidate the influence of different plant hormones on the replication of asexual tachyzoites in a human foreskin fibroblast (HFF) host cell culture. T. gondii replication was measured by the determination of T. gondii DNA copies via qPCR. Three selected phytohormones, namely abscisic acid (ABA), gibberellic acid (GIBB), and kinetin (KIN) as representatives of different plant hormone groups were tested. Moreover, the influence of typical cell culture media components on the phytohormone effects was assessed. Our results indicate that ABA is able to induce a significant increase of T. gondii DNA copies in a typical supplemented cell culture medium when applied in concentrations of 20 ng/µl or 2 ng/µl, respectively. In contrast, depending on the culture medium composition, GIBB may potentially serve as T. gondii growth inhibitor and may be further investigated as a potential treatment for toxoplasmosis.

Prevalence and molecular characterization of Toxoplasma gondii in different types of poultry in Greece, associated risk factors and co-existence with Eimeria spp.

Toxoplasma gondii is a protozoan parasite of public health importance, infecting all warm-blooded animals, including chickens. Undercooked chicken meat or relevant products such as sausages could lead to human infections. In free-range, organic and slow-growth farming systems where the susceptibility period for chickens is extended, more knowledge about potential risk factors is essential. This study is the first seroepidemiological survey in different regions and types of chicken farms in Greece, using a major tachyzoite surface antigen-based ELISA (TgSAG1), combined with magnetic-capture PCR (mc-PCR) and bioassay for the isolation of strains from the chickens' tissues. Potential risk factors for T. gondii infection in these hosts were also investigated. Additionally, the co-existence of T. gondii and Eimeria spp. infections was assessed to elucidate epidemiological links between these two protozoan infections. Overall T. gondii seroprevalence was 9.5%. Of the backyard chickens sampled, 41.2% were seropositive and 70% of the organic and free-range layer farms had at least one T. gondii seropositive hen. No serologically positive broilers were found, although mc-PCR revealed a positive sample, highlighting the importance of accurate early-infection direct detection of T. gondii infections to ensure public health. T. gondii isolates obtained by mouse bioassay were genotyped. All belonged to type II (ToxoDB#3) as confirmed also by microsatellite typing. Production system, type of nutrition, and feeding system automation were identified as the most significant risk factors, while no association was found between the presence of cats and T. gondii seropositivity as calculated on both a farm level and per individual bird sampled.

Eimeria infections in domestic and wild ruminants with reference to control options in domestic ruminants.

Eimeria infections are commonly seen in a variety of mammalian hosts. This genus of unicellular sporozoan parasites causes significant disease (coccidiosis) in different livestock species leading to economic losses for agricultural producers. Especially the production of cattle, sheep, and goat is strongly dependent on efficient coccidiosis control. However, many other livestock hosts like, e.g., camelids, bison, rabbits, and guinea pigs may benefit from reduced parasite transmission and targeted control measures as well. Besides livestock, also wildlife and pet animals may be affected by Eimeria infections resulting in clinical or subclinical coccidiosis. Wildlife herd health is crucial to conservation efforts, and Eimeria species are a prevalent pathogen in multiple mammalian wildlife species. This review aims to highlight the epidemiology of mammalian Eimeria infections in both wild and domestic ruminants, including host specificity, transmission, survival of environmental oocysts, occurrence, and risk factors for infection. Understanding general drivers of Eimeria infection may support adequate livestock and wildlife management. Furthermore, control options for livestock with reference to management factors, drug application, and alternative approaches are discussed. The goal of Eimeria control should be to reduce pathogen transmission in different host species and to improve sustainable livestock production. Controlling Eimeria infections in livestock is important considering both their animal welfare impact and their high economic relevance.

In vitro activity of selected natural products against Eimeria tenella sporozoites using reproduction inhibition assay.

Eimeria tenella is the causative agent of cecal coccidiosis in poultry characterized by weight loss, hemorrhagic diarrhea, and high mortality rates. Research into herbal candidates with possible anticoccidial activity has increased lately. As an alternative to animal experiments, an in vitro reproduction inhibition assay (RIA) was previously designed to determine the sensitivity of E. tenella isolates against ionophores. In this study, the RIA was used to test the anticoccidial activity of nutmeg oil, cinnamon oil, and glabridin. The concentration of nutmeg oil used in this study ranged between 1.1 and 139.1 µg/ml. Nutmeg oil exhibited a moderate in vitro inhibitory activity ranging from 35.5 to 49.5%. In contrast, no inhibitory effect was detected when incubating E. tenella sporozoites for 24 h with cinnamon oil at concentrations of 0.3 to 80.5 µg/ml. Glabridin (0.08-41.7 µg/ml) prevented the replication of sporozoites at a rate of 14.1 to 81.7% of inhibition. The calculated minimum concentrations of glabridin needed to inhibit parasite replication by 75%, 50%, and 30% (MIC75, MIC50, and MIC30) were 21.43 µg/ml, 5.28 µg/ml, and 0.96 µg/ml, respectively. Further studies to assess the in vitro efficacy of glabridin were performed by studying mRNA gene expression of stress-induced protein genes (HSP-70, NADPH, and EtPP5) after exposure of E. tenella sporozoites to glabridin at MIC75 for 0.5 h, 1 h, 2 h, and 4 h (a time-dependent experiment). Moreover, a dose-dependent experiment was performed using glabridin at a concentration matching MIC75, MIC50, and MIC30 for 24 h. In the time-dependent experiment, a significant (p < 0.05) increase of expression in NADPH and EtPP5 were detected after 4 h of incubation with glabridin at a concentration of 21.43 µg/ml. The dose-dependent experiment exhibited a gradual increase of expression in all studied genes, which indicates stress imposed on E. tenella sporozoites by glabridin. In our hands, RIA was suitable to assess the anticoccidial activity exhibited by the tested natural products as a precursor to in vivo studies which will help in the identification of novel anticoccidial candidates.

Prevalence and diversity of haemosporidians in a migratory high-elevation hummingbird in North America.

Hummingbirds (Trochilidae) are sensitive to environmental changes because of their extraordinary ecology, metabolism, and the highest red blood cell counts found in any vertebrate. These physiological attributes may render hummingbirds particularly susceptible to the effects of haemosporidian (blood parasite) infections. Much of the research on haemosporidians in hummingbirds has been conducted in South America; less is known about haemosporidian diversity and prevalence in North America. We sought to determine the prevalence and diversity of haemosporidians in a high-elevation species, the Broad-tailed Hummingbird (Selasphorus platycercus). Blood samples (N = 314) from 25 sites in Colorado and Wyoming were screened for haemosporidians using microscopy (n = 311) and PCR (n = 301). Both microscopy and sequencing diagnostic techniques detected haemosporidians in the same 5 hummingbirds, with an overall prevalence of 1.59%. Positive samples were sequenced at the cytochrome b gene and identified Haemoproteus archilochus and two Haemoproteus sp. not previously detected in North America. No parasites of the genera Plasmodium or Leucocytozoon were detected. Our study provides the first report of the prevalence and diversity of haemosporidians in Broad-tailed Hummingbirds in the Rocky Mountains.

Survey of coyotes, red foxes and wolves from Wyoming, USA, for Echinococcus granulosus s. l.

The paraphyletic group Echinococcus granulosus sensu lato is comprised of parasitic tapeworms of wild and domestic canids such as wolves (Canis lupus) and coyotes (Canis latrans), which serve as definitive hosts, and ungulates, which are the intermediate hosts. Members of this tapeworm group are characterized by both cosmopolitan distribution and zoonotic disease potential. This survey (conducted from 2012 through 2017) was designed to provide insight into the prevalence and distribution of this parasite in wild canids in Wyoming. Echinococcus sp. infections were documented in 14 of 22 gray wolves (63.6%), 1 of 182 coyotes (0.55%) and 0 of 5 red foxes (Vulpes fulva). Echinococcus granulosus s. l. was confirmed in 4 of these 14 specimens obtained from wolves with two parasite specimens corresponding morphologically with E. canadensis (G8/G10). These results suggest that wolves serve as the major definitive host of E. granulosus s. l. in Wyoming, while coyotes do not play an equivalent role. Limited sample size precludes evaluation of the importance of the red fox as a favorable definitive host. Whereas this study documents the occurrence of E. granulosus s. l. in Wyoming, the zoonotic disease risk does not appear to be high. Education remains the key to disease prevention, coupled with good hygienic practices by humans and anthelmintic treatment of domestic dogs exhibiting elevated risk of exposure.

Apicomplexan co-infections impair with phagocytic activity in avian macrophages.

Mixed infections of Toxoplasma gondii and Eimeria tenella are likely to occur frequently due to the high prevalence of both pathogens in free-ranging chickens. In this study, we investigated the co-occurrence of the two parasites in the same immune-competent host cell towards altered patterns of parasite-host interactions. Chicken blood monocyte-derived macrophages were co-infected with T. gondii RH tachyzoites and E. tenella Houghton sporozoites in vitro for 24 h. Through monitoring the uptake of pH-sensitive pHrodo™ Zymosan BioParticles ("Zymosan") by macrophages, we created a three-dimensional model and to analyze quantitatively phagocytosis using confocal laser scanning microscopy. Assessments of parasite populations were performed by qPCR at 2, 6, 12, and 24 h post-infection (hpi). At 6 hpi, phagocytosis was inhibited in the E. tenella-infected cultures while no inhibition of phagocytosis was observed due to T. gondii. Phagocytosis activity revealed more complex interactions during co-infection. At 12 and 24 hpi, phagocytosis response to "Zymosan" was distinctly weaker in co-infected cells than in all other groups except for cells mono-infected with high doses of E. tenella at 24 hpi. By qPCR, significantly reduced numbers of both intracellular parasites were recorded (10-fold) in all infected groups at 2 hpi. At 12 hpi, the T. gondii population reached lowest values but dramatically increased by 24 hpi. Our data confirm that macrophage phagocytosis is involved in the control of invasion by apicomplexan parasites in chicken which particularly applies to E. tenella infection and it was able to be altered by the co-existing parasites.

Cryptosporidium parvum alters glucose transport mechanisms in infected enterocytes.

The parasite Cryptosporidium parvum Tyzzer 1912 destroys parts of the intestinal brush border membrane which is important for the uptake of nutrients like glucose. In this study, glucose transport mechanisms of the host cells (IPEC-J2 cells) infected by C. parvum were investigated. The mRNA expression levels of glucose transporters (GLUT) 1 and 2 and Na+-coupled glucose transporter (SGLT) 1 were compared in infected and uninfected cells over an infection time of 24-96 h by RT-qPCR. Furthermore, the protein expression of SGLT 1 and GLUT 2 was quantified in western blot studies. While the protein expression of SGLT 1 was not altered in infected cells, mRNA expression of SGLT 1 and GLUT 1 was significantly increased 24 h p. i. and decreased 96 h p. i. The mRNA expression of GLUT 2 was significantly decreased 24 h, 72 h, and 96 h p. i. and also correlated significantly with the infection dose at 72 h p. i. In contrast to that, the protein expression of GLUT 2 was significantly increased 48 h p. i., associated with a significantly higher intracellular glucose level in infected cells compared with control cells at that time point of infection. This points to an adaptation of the host cells' glucose uptake taking place in the acute phase of the infection. A better understanding of these molecular mechanisms following a C. parvum infection may probably lead to an improvement of therapy strategies in the future.

Resistance towards monensin is proposed to be acquired in a Toxoplasma gondii model by reduced invasion and egress activities, in addition to increased intracellular replication.

Monensin (Mon) is an anticoccidial polyether ionophore widely used to control coccidiosis. The extensive use of polyether ionophores on poultry farms resulted in widespread resistance, but the underlying resistance mechanisms are unknown in detail. For analysing the mode of action by which resistance against polyether ionophores is obtained, we induced in vitro Mon resistance in Toxoplasma gondii-RH strain (MonR-RH) and compared it with the sensitive parental strain (Sen-RH). The proteome assessment of MonR-RH and Sen-RH strains was obtained after isotopic labelling using stable isotope labelling by amino acid in cell culture. Relative proteomic quantification between resistant and sensitive strains was performed using liquid chromatography-mass spectrometry/mass spectrometry. Overall, 1024 proteins were quantified and 52 proteins of them were regulated. The bioinformatic analysis revealed regulation of cytoskeletal and transmembrane proteins being involved in transport mechanisms, metal ion-binding and invasion. During invasion, actin and microneme protein 8 (MIC8) are seem to be important for conoid extrusion and forming moving junction with host cells, respectively. Actin was significantly upregulated, while MIC8 was downregulated, which indicate an invasion reduction in the resistant strain. Resistance against Mon is not a simple process but it involves reduced invasion and egress activity of T. gondii tachyzoites while intracellular replication is enhanced.

Characterisation of susceptibility of chicken macrophages to infection with Toxoplasma gondii of type II and III strains.

Toxoplasma gondii is known to be able to infect any nucleated cell including immune cells like macrophages. In addition, it is assumed that macrophages serve as trojan horse during distribution in hosts. The underlying causes of parasite host interaction remain yet not fully understood. The aim of the present study was to investigate susceptibility of chicken macrophages to infection with T. gondii and the process of infection in avian cells in comparison to cells of mammalian origin. Primary avian blood monocyte-derived macrophages were infected with tachyzoites of type II (ME49) and III (NED) strains. Long term observations of parasite replication in primary macrophages were compared to data obtained in an avian macrophage cell line (HD11) and a standard cultivation mammalian cell line (VERO). Furthermore, we assessed the immune response of the primary macrophages by long-term investigation of gene expression of IL-1 beta, IL-12p40, Lipopolysaccharide induced TNF-alpha factor (LITAF) and inducible nitric oxide synthase (iNOS) comparing viable and heat-inactivated tachyzoites of the ME49 strain. Albeit, we found no differences between both strains, replication of tachyzoites in avian primary macrophages was significantly different from immortalized cell lines HD11 and VERO. The crucial period of parasite replication was between 8 and 24 h post-infection coinciding with the upregulation of gene expression of cytokines and iNOS revealing an active macrophage response at this period. Gene expression in macrophages was higher after infection with viable tachyzoites than by exposure of cells to heat-inactivated tachyzoites. Hence, we conclude that the process of penetration is pivotal for host cell response to the parasite both in avian as in mammalian cells.

Replication of Toxoplasma gondii in chicken erythrocytes and thrombocytes compared to macrophages.

Toxoplasma (T.) gondii is able to infect various cell types in different hosts. The replication of this parasite within different peripheral mononuclear blood cell populations in chicken has not yet been fully understood. Aim of the present study was to investigate the impact of chicken erythrocytes and thrombocytes as potential host cells for T. gondii. Cultures of primary avian erythrocytes and thrombocytes were inoculated with tachyzoites of T. gondii type II strain ME49. Parasite replication was detected by a quantitative real-time PCR at different times postinoculation until 24 or 48 h, respectively, displaying long-term investigations for the chosen cultures. The parasite replication curve showed a continuous decrease of parasite stages in erythrocytes and thrombocytes. Observations by light microscopy showed massive destruction for both cell populations. Few macrophages in between the infected thrombocytes were viable during the investigation period and showed internalised tachyzoites by confocal laser scanning microscopy. These findings show that T. gondii is not capable of replication in chicken erythrocytes and thrombocytes; therefore, both cannot be considered as potential host cells. In further consequence, monocyte-derived macrophages seem to be the key to the dissemination mechanisms for T. gondii in chicken.

Quantitative proteomic studies in resistance mechanisms of Eimeria tenella against polyether ionophores.

Polyether ionophores are widely used to treat and control coccidiosis in chickens. Widespread use of anticoccidials resulted in worldwide resistance. Mechanisms of resistance development and expansion are complex and poorly understood. Relative proteomic quantification using LC-MS/MS was used to compare sensitive reference strains (Ref-1, Ref-2) with putatively resistant and moderately sensitive field strains (FS-R, FS-mS) of Eimeria tenella after isotopic labelling with tandem mass tags (TMT). Ninety-seven proteins were identified, and 25 of them were regulated. Actin was significantly upregulated in resistant strains in comparison with their sensitive counterparts. On the other hand, microneme protein (MIC4) was downregulated in resistant strains. Optimization of labelling E. tenella sporozoites by TMT might identify further proteins that play a role in the obvious complex mechanism leading to resistance against Monensin.

Molecular characterization of bovine Cryptosporidium isolated from diarrheic calves in the Sudan.

Cryptosporidiosis is a common protozoan infection causing morbidity and mortality in young cattle and may be zoonotically transmitted to humans. So far, there is no data available on the presence of Cryptosporidium spp. in the Sudan. The aim of this study was to isolate, identify, and genotype Cryptosporidium oocysts sampled from diarrheic calves housed at different farms in three different municipalities in Khartoum State (Khartoum, Khartoum North, Omdurman). A total of 149 fecal samples were evaluated microscopically for the presence of Cryptosporidium oocysts using the modified Ziehl-Neelsen staining method and 87 (58.3%) samples tested positive. Positive and negative samples were further analyzed by nested PCR targeting the SSU rRNA region. Positive samples were subjected to restriction enzyme analysis of PCR amplicons (PCR-RFLP). Nested PCR identified Cryptosporidium DNA in 53 samples (35.5%); restriction digestion of the PCR products revealed the presence of C. parvum (73.5%), C. ryanae (13.2%), C. andersoni (7.5%), and C. bovis (1.8%). Species distribution was clearly related to age with C. parvum being the predominant species in dysenteric pre-weaned calves. Sequencing of three genes (SSU rRNA, COWP, and GP60) for three C. parvum isolates originating from the three different municipalities showed that all belong to C. parvum subtype family IId. Based on data obtained by GP60, sequencing the two C. parvum isolates from Khartoum and Omdurman represent subtype IIdA18G1, whereas oocysts isolated in Khartoum North belong to subtype IIdA19G1. The observed genotypes are zoonotic and thus C. parvum in calves is potentially a health risk to humans in Khartoum State, Sudan. To the best of our knowledge, this is the first reported attempt to characterize Cryptosporidium isolated from cattle in the Sudan.

Quantitative detection of Toxoplasma gondii in tissues of experimentally infected turkeys and in retail turkey products by magnetic-capture PCR.

Magnetic-capture PCR was applied for the quantitative detection of Toxoplasma gondii in tissues of experimentally infected turkeys and retail turkey meat products. For experimental infection, three T. gondii strains (ME49, CZ-Tiger, NED), varying infectious doses in different matrices (organisms in single mouse brains or 10(3), 10(5), or 10(6) oocysts in buffer) were used. From all animals, breast, thigh, and drumstick muscle tissues and for CZ-Tiger-infected animals additionally brains and hearts were analyzed. Using the magnetic-capture PCR large volumes of up to 100 g were examined. Our results show that most T. gondii parasites are present in brain and heart tissue. Of the three skeletal muscle types, drumsticks were affected at the highest and breast at the lowest level. Type III strain (NED) seems to be less efficient in infecting turkeys compared to type II strains, because only few tissues of NED infected animals contained T. gondii DNA. Furthermore, the number of detected parasitic stages increased with the level of infectious dose. Infection mode by either oocyst or tissue cyst stage did not have an effect on the amount of T. gondii present in tissues. In retail turkey meat products T. gondii DNA was not detectable although a contact with the parasite was inferred by serology.

In vitro efficacy of allicin on chicken Eimeria tenella sporozoites.

Chicken coccidiosis is a major parasitic disease caused by Eimeria spp. It is controlled and treated using chemical anticoccidial agents. Development of partial or complete resistance toward these anticoccidials is considered a major problem in poultry industry. Allicin is an organosulfur compound produced as a result of the reaction between alliin and alliinase after hacking of garlic. In this study, tenfold dilution from 180 mg/ml to 1.8 ng/ml of allicin in distilled water was tested against E. tenella in vitro. The percent of inhibition in allicin was from 99.9 to 71.53% using 180 mg/ml and 180 ng/ml, respectively. The percent of inhibition was 56.24% using 1.8 ng/ml. We used allicin as a treatment from plants against chicken coccidiosis; however, in vivo study should be performed to confirm these results.

Combination of cell culture and qPCR to assess the efficacy of different anticoccidials on Eimeria tenella sporozoites.

Three in vitro studies were designed to develop an assay for anticoccidial efficacy by use of laboratory (Houghton) and field (T-376) Eimeria tenella strains. In study (1), minimum inhibitory concentrations (MICs) of monensin (Mon), maduramicin (Mad), salinomycin (Sal), and lasalocid (Las) were determined that are able to inhibit more than 50% of sporozoites in host cell (Madin-Darby bovine kidney (MDBK)) penetration and more than 95% of Houghton sporozoites development to mature merozoites (treatment time 24 h) using quantitative real-time PCR (qPCR). MICs were 0.5, 2.5, 1, and 0.5 µg/ml for Mon, Mad, Sal, and Las, respectively. Applying the previous MIC on T-376 strain revealed a different sensitivity profile. Mad reduced T-376 gene copies by only 89.3% after 96 h of infection. In study (2), Houghton strain sporozoites were incubated with MIC of the different tested ionophores for 2 and 4 h, respectively; afterwards, their ability to invade MDBK cells was determined using phase-contrast microscopy and qPCR. Treatment of sporozoites with ionophores for 4 h resulted in significant inhibition of invasion compared with non-treated parasites as assessed both by microscopy as well as qPCR. Inhibition rates for Mon, Mad, Sal, and Las were 90.2, 75.0, 88.3, and 82.6% using phase-contrast microscopy and 83.9, 81.4, 85.8, and 75.4% using qPCR, respectively. T-376 sporozoite invasion into MDBK cells was reduced to 48.9% by Mad. Study (3) was conducted to determine inhibition exerted by toltrazuril (Tol). Tol at 5 µg/ml reduced reproduction of Houghton strain by 95%, whereas T-376 was only reduced by 86.5%. The presented experiments indicate that infectivity inhibition of sporozoites incubated for 4 h with anticoccidials and development inhibition after 96 h of infection by qPCR are suitable means to assess sensitivity of E. tenella strains to anticoccidials.

Efficacy of an anticoccidial live vaccine in prevention of necrotic enteritis in chickens.

Necrotic enteritis (NE) is an important disease in poultry caused by Clostridium perfringens combined with predisposing factors, mainly eimeriosis. In the present study, we investigated the protective effect of a commercial attenuated anticoccidial live vaccine against NE in a clinical infection model using 60 day-old chicks. Vaccination was performed on study day (SD) 1 with natural booster-infections for 4 weeks from Eimeria spp. oocysts present in litter. On SD 28, five groups were formed (n=12): group V+/C-E- (vaccinated, uninfected), group V+/C-E+ (vaccinated, infected with Eimeria spp.), group V+/C+E+ (vaccinated, infected with clostridia and Eimeria spp.), group V-/C+E+ (unvaccinated, infected with clostridia and Eimeria spp.), and group NC (negative control). Efficacy was measured by clinical parameters, pathogen multiplication, and pathological parameters assessed during two necropsies on SD 34 and SD 40, respectively. Additionally, cytokine expression was measured in gut and spleen tissues at necropsy. Clinical signs of NE were observed only in the coinfected groups, mainly in group V-/C+E+. Accordingly, lowest body weight gain was observed in group V-/C+E+ (301.8 g from SD 28 to SD 40; group NC: 626.2 g). Oocyst excretion varied significantly (P<0.01) between all Eimeria spp. infected groups and was highest in group V-/C+E+, followed by V+/C+E+, and lowest in group V+/C-E+. NE typical intestinal lesions showed only in groups V+/C+E+ and V-/C+E+. The intestinal mucosa featured partly severe lesions in the jejunum, C. perfringens colonization was histologically visible. Upregulation of IFN-γ, was observed in the jejunal tissue of group V-/C+E+ (P<0.01 (SD 34) or P<0.05 (SD 40) compared to all other groups). IL-10 and IL-12 were upregulated in group V-/C+E+, IL-10 also in group V+/C+E+ (SD 40) while IL-2 expression remained unaltered. In conclusion, vaccination against coccidiosis was effective in preventing NE in a mixed infection comparable to field situations.

Humoral immune responses in chickens and turkeys after infection with Toxoplasma gondii by using recombinant antigens.

Toxoplasma gondii is a parasite which can be transmitted to humans via the consumption of contaminated meat products derived from different animal species, e.g., poultry. In Europe, the consumption rate of poultry meat is high and may pose a risk for humans. However, little is known about the prevalence and immune response against T. gondii in these animals. Based on these circumstances, we experimentally infected 18 turkeys and 16 chickens with the parasite. Turkeys were infected either with tachyzoites on different routes or with various amounts of oocysts. In contrast, chickens were only infected with different doses of oocysts. The immunoglobulin (Ig) Y humoral immune responses of these animals were investigated in a lineblot assay against the recombinant T. gondii antigens rGRA1, rGRA6, rGRA9, rSAG1, and rSUB1. By using the recombinant antigens rGRA6, rGRA9, and rSUB1 in the lineblot assay, we found a correlation between the humoral immune response and the parasite stage in turkeys. Thereby, an infection with oocysts induced a stronger, permanent long-lasting antibody response compared to tachyzoite-infected animals. Only a minor relation between the oocyst infection dose and the manifestation of the immune response in chickens was found 7 days post infection (dpi) by using rGRA1 and rGRA9. However, an inconstant detection of antigen-specific IgY antibodies in the lineblot assay seems not to be a sufficient method for the identification of a Toxoplasma infection in chickens. In contrast, the detection of anti-rGRA6, anti-rGRA9, and anti-rSUB1 IgY antibodies showed potential for the identification of an infection in turkeys.

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 10(9) colony-forming unit Clostridium perfringens. Chickens of group 1 were treated with 75 parts/10(6) 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.

Inactivation of exogenous endoparasite stages by chemical disinfectants: current state and perspectives.

Chemical disinfection is common practice and inevitable to achieve sufficient control over parasites particularly in intensive animal housing systems. To identify suitable chemicals, reliable data on antiparasitic efficacy of disinfectants are required. This review summarizes recently published experience with procedures applied to evaluate the viability of a variety of endoparasites following physical or chemical stress. It is concluded that laboratory models used to assess antiparasitic efficacy of e.g. commercial disinfectants should consider the most resistant stages of both helminths and protozoa, i.e. ascarid eggs and coccidia oocysts. To ensure reproducibility and transparency, standardized protocols are pivotal. Such protocols are established on a national level (e.g. DVG guidelines in Germany); however, internationally accepted certification procedures are currently lacking.