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.

Toxoplasma gondii in animals used for human consumption.

Food-borne toxoplasmosis in humans may result from exposure to different stages of Toxoplasma gondii, in particular from the ingestion of tissue cysts or tachyzoites contained in meat, primary offal (viscera) or meat-derived products of many different animals, or the ingestion of sporulated oocysts that are contained in the environment and may contaminate food and water. Although the potential for transmission of the parasite to humans via food has been known for several decades, it is not known which routes are most important from a public health point of view. It is likely that transmission of the parasite to humans is influenced not only by the potential contamination of various food sources, but also by the individual behaviour of consumers in different ethnic groups and geographical regions. Most current methods for detection of T. gondii in meat-producing animals, in products of animal origin, or in the environment are insufficient because they do not allow quantification of infectious stages. Hence, most studies report only qualitative data from which it is difficult to assess the true risk of infection in individual cases. There is a need for quantitative data so that efficient strategies to reduce food-borne transmission of T. gondii to humans can be developed.

The conceptual basis for a new classification of the coccidia.

At the joint meeting of the 8th International Coccidiosis Conference and the Annual Scientific Meeting of the Australian Society for Parasitology in Palm Cove, Australia, in July 2001, a Controversial Roundtable was held on 'New classification of coccidia'. The aim of this Roundtable was to stimulate and encourage discussion and debate on current classification schemes for the group of parasitic protozoa known as the eimeriid coccidia. In the past, such classifications have been based only on phenotypic characters such as morphology, ultrastructure, life cycles, and host specificity. However, over the past 10-15 years, molecular phylogenetic studies on taxa of the eimeriid coccidia have revealed that several of the families, subfamilies, and genera that have been erected based on non-molecular characters are paraphyletic. Therefore, this Roundtable was an important forum for initial discussions on how a new and more comprehensive classification of the eimeriid coccidia, which takes into consideration both phenotypic and molecular characters, can be devised. The stimulus came from invited speakers who gave introductions into selected areas of taxonomy and classification. Following these introductions, a more general discussion with the audience addressed potential steps that may be taken in future work. This review is the immediate outcome of the Roundtable. It describes advantages and disadvantages of the use of phenotypic or molecular characters as the base for taxonomic schemes for eimeriid coccidia. It gives specific examples for drawbacks of current classifications based only on phenotypic characters as well as potential pitfalls associated with the use of only molecular phylogenies. It addresses current controversies as well as rules of taxonomy and nomenclature relevant for the eimeriid coccidia. Finally, it recommends the establishment of an international group of scientists to meet on a regular basis, stimulate further discussions, and give direction on how the final goal, i.e. a proposal for a revised, and widely accepted, classification of the eimeriid coccidia, may be achieved.

Molecular and bioassay-based detection of Toxoplasma gondii oocyst uptake by mussels (Mytilus galloprovincialis).

Toxoplasma gondii is associated with morbidity and mortality in a variety of marine mammals, including fatal meningoencephalitis in the southern sea otter (Enhydra lutris nereis). The source(s) of T. gondii infection and routes of transmission in the marine environment are unknown. We hypothesise that filter-feeding marine bivalve shellfish serve as paratenic hosts by assimilation and concentration of infective T. gondii oocysts and their subsequent predation by southern sea otters is a source of infection for these animals. We developed a TaqMan PCR assay for detection of T. gondii ssrRNA and evaluated its usefulness for the detection of T. gondii in experimentally exposed mussels (Mytilus galloprovincialis) under laboratory conditions. Toxoplasma gondii-specific ssrRNA was detected in mussels as long as 21 days post-exposure to T. gondii oocysts. Parasite ssrRNA was most often detected in digestive gland homogenate (31 of 35, i.e. 89%) compared with haemolymph or gill homogenates. Parasite infectivity was confirmed using a mouse bioassay. Infections were detected in mice inoculated with any one of the mussel sample preparations (haemolymph, gill, or digestive gland), but only digestive gland samples remained bioassay-positive for at least 3 days post-exposure. For each time point, the total proportion of mice inoculated with each of the different tissues from T. gondii-exposed mussels was similar to the proportion of exposed mussels from the same treatment groups that were positive via TaqMan PCR. The TaqMan PCR assay described here is now being tested in field sampling of free-living invertebrate prey species from high-risk coastal locations where T. gondii infections are prevalent in southern sea otters.

Toxoplasma gondii within skeletal muscle cells: a critical interplay for food-borne parasite transmission.

Toxoplasma gondii infects virtually any nucleated cell type of warm-blooded animals and humans including skeletal muscle cells (SkMCs). Infection of SkMCs by T. gondii, differentiation from the highly replicative tachyzoites to dormant bradyzoites and tissue cyst formation are crucial for parasite persistence in muscle tissue. These processes are also prerequisites for one of the major routes of transmission to humans via undercooked or cured meat products. Evidence obtained in vitro and in vivo indicates that SkMCs are indeed a preferred cell type for tissue cyst formation and long-term persistence of T. gondii. This raises intriguing questions about what makes SkMCs a suitable environment for parasite persistence and how the SkMC-T. gondii interaction is regulated. Recent data from our laboratory show that differentiation of SkMCs from myoblasts to syncytial myotubes, rather than the cell type itself, is critical for parasite growth, bradyzoite formation and tissue cyst maturation. Myotube formation is accompanied by a permanent withdrawal from the cell cycle, and the negative cell cycle regulator cell division autoantigen (CDA)-1 directly or indirectly promotes T. gondii stage conversion in SkMCs. Moreover, host cell cycle regulators are specifically modulated in mature myotubes, but not myoblasts, following infection. Myotubes also up-regulate the expression of various pro-inflammatory cytokines and chemokines after T. gondii infection and they respond to IFN-γ by exerting potent anti-parasitic activity. This highlights that mature myotubes are active participants rather than passive targets of the local immune response to T. gondii which may also govern the interaction between SkMCs and the parasite.

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.

First case of human babesiosis in Germany - Clinical presentation and molecular characterisation of the pathogen.

Babesiosis is a common infection of animals and is gaining increasing attention as an emerging tick-borne zoonosis of humans in Europe. Here we report on the first case of human babesiosis in Germany in a 63-year-old splenectomised German patient with a relapse of nodular lymphocyte-predominant Hodgkin's lymphoma. After treatment with a chimeric anti-CD20 antibody preparation (Rituximab), the patient was hospitalised because of anaemia and dark urine from haemoglobinuria. Presumptive diagnosis of babesiosis was made based on piriform parasitic erythrocytic inclusions in peripheral blood smears and confirmed by Babesia-specific 18S rDNA PCR. Sequence analysis revealed a >99% homology of the amplicon with the recently described EU1 organism clustering within the Babesia divergens/Babesia odocoilei complex. Despite treatment with quinine and clindamycin the patient relapsed and developed chronic parasitaemia requiring re-treatment and long-term maintenance therapy with atovaquone before he eventually seroconverted and the parasite was cleared. Our findings suggest that human babesiosis occurs in Germany and can take a chronic course in immunocompromised individuals.

Toxoplasma gondii in animals used for human consumption

Food-borne toxoplasmosis in humans may result from exposure to different stages of Toxoplasma gondii, in particular from the ingestion of tissue cysts or tachyzoites contained in meat, primary offal (viscera) or meat-derived products of many different animals, or the ingestion of sporulated oocysts that are contained in the environment and may contaminate food and water. Although the potential for transmission of the parasite to humans via food has been known for several decades, it is not known which routes are most important from a public health point of view. It is likely that transmission of the parasite to humans is influenced not only by the potential contamination of various food sources, but also by the individual behaviour of consumers in different ethnic groups and geographical regions. Most current methods for detection of T. gondii in meat-producing animals, in products of animal origin, or in the environment are insufficient because they do not allow quantification of infectious stages. Hence, most studies report only qualitative data from which it is difficult to assess the true risk of infection in individual cases. There is a need for quantitative data so that efficient strategies to reduce food-borne transmission of T. gondii to humans can be developed.

Seroprevalence of Babesia infections in humans exposed to ticks in midwestern Germany.

Babesiosis is considered to be an emerging tick-borne disease in humans worldwide. However, most studies on the epidemiology of human babesiosis to date have been carried out in North America, and there is little knowledge on the prevalence of infection and frequency of disease in other areas. The aim of this study was to investigate the prevalence of Babesia infections in a human population in Germany. A total of 467 sera collected between May and October 1999 from individuals living in the Rhein-Main area were tested for the presence of immunoglobulin G (IgG) and IgM antibodies to antigens of Babesia microti and Babesia divergens by indirect fluorescent-antibody (IFA) tests. These sera were derived from 84 Lyme borreliosis patients suffering from erythema migrans, 60 asymptomatic individuals with positive borreliosis serology, and 81 individuals with a history of tick bite. Cutoff values for discrimination between seronegative and seropositive results in the IFA tests were determined using sera from 120 healthy blood donors and 122 patients suffering from conditions other than tick-borne diseases (malaria, n = 40; toxoplasmosis, n = 22; syphilis, n = 20; Epstein-Barr virus infection, n = 20; and presence of antinuclear antibodies, n = 20). The overall specificities of the IFA tests for B. microti and B. divergens were estimated to be >or=97.5%. Positive IgG reactivity against B. microti antigen (titer, >or=1:64) or B. divergens antigen (titer, >or=1:128) was detected significantly more often (P < 0.05) in the group of patients exposed to ticks (26 of 225 individuals; 11.5%) than in the group of healthy blood donors (2 of 120 individuals; 1.7%). IgG antibody titers of >or=1:256 against at least one of the babesial antigens were found significantly more often (P < 0.05) in patients exposed to ticks (9 of 225) than in the control groups (1 of 242). In the human population investigated here, the overall seroprevalences for B. microti and B. divergens were 5.4% (25 of 467) and 3.6% (17 of 467), respectively. The results obtained here provide evidence for concurrent infections with Borrelia burgdorferi and Babesia species in humans exposed to ticks in midwestern Germany. They also suggest that infections with Babesia species in the German human population are more frequent than believed previously and should be considered in the differential diagnosis of febrile illness occurring after exposure to ticks or blood transfusions, in particular in immunocompromised patients.