Groups of animals including in serological testing
10. 1. Introduction
The health status of livestock largely reflects on the human population. Livestock is important in terms of production of safe foodstuffs or breeding purposes.
Infections caused by pathogenic protozoa give rise to frequent problems mainly in tropical and subtropical regions, where they are widespread. It is reported that up to 4000 protozoa live as parasites. Worldwide, the most prevalent protozoan infection is malaria, while the most prevalent infection in the Slovak Republic is toxoplasmosis, by which the 30% of population are infected on average.
Parasitic pathogenic protozoa largely parasitize intracellularly, the course of these infections is acute, often cause the death. On the other hand, they can progress subclinically. The latent respectively chronic stage can follow the acute form and infections can persist throughout the whole life of the host. The course of the disease mostly depends also on the pathological agents. They stimulate the innate and adaptive immune response of the host. In mostly protozoan infections the immune response is not so sufficiently effective for a complete destruction of the parasite. This situation ensures the survival of the parasite and it is the characteristic feature for mostly protozoan infections.
Since a total elimination of the influence of negative factors (including pathogens) in each animal species is impossible, in the case of an unexpected outbreak of disease the solution is in its rapid and reliable diagnostics. The detection of pathogens as infection agents is carried out in laboratories using multiple techniques. The direct proof of parasite is usually microscopically and it is clear confirmation of infection. In systematic infections where the direct proof of parasite is unlikely serological methods are carried out in diagnostics for detection of antigen or antibody present in the biological samples. Serological methods often don´ t solve the problems of diagnosis in the early stage of infection or in the case of latent infection. For diagnostics of these stages are required more sensitive laboratory methods. In At present molecular methods based on the detection of the nucleic acid are used in the laboratories. A polymerase chain reaction (PCR - standard or quantitative) has wide range of using in the detection of parasites.
Toxoplasmosis is an acute parasitic infection monitored based on the epidemiological situation in the country. Therefore it is necessary to interconnect an epidemiological monitoring of infection in humans and animals because of a zoonotic character of this infection.
We here review the information available on the seroprevalence of
2. Basic characteristics about
Toxoplasmosis may affect a number of organs, but it primarily affects lungs, the CNS (central nervous system) and eyes. Canine and feline toxoplasmosis is a multi-systemic disease; however a latent form of the disease usually develops. Dogs may act as a mechanical factor in transmitting toxoplasmosis to humans by rolling in foul-smelling substances and by ingesting fecal material. Just remember that 50% of stray dogs and dogs carry
. T. and the immune system
4. Non-specific immune response
In immuno-competent hosts this parasite activates asymptomatic chronic infection, what make possible its transmission and survival. The infection of
5. Specific immune response
The non-specific immune response has led to differentiation of macrophages and B-lymphocytes into antigen presenting cells. The effector cells are stimulated by dendritic cells presenting the antigen to T-lymphocytes. This mechanism requires a close interaction between the antigen presenting cell and the T-lymphocytes . The interaction of parasite with mechanisms of non-specific immune response is important to orientation of progress of specific immunity. The induction of IL-12 and then IFN-γ stimulate the progression of Th1 subpopulations so that polarize the immune response for behoof cellular immunity. After acute infection, the cells presenting antigens (macrophages) are exciting to produce IL-12 and initiate differentiation of immature CD4 T-lymphocytes to Th1. The cellular immunity initiates the production of IFN-γ. This cytokine effects as a major mediator of cellular immunity during toxoplasmosis. The key function in specific immune response plays T-lymphocytes. These effector cells, which are involved in resistance to
CD4+ and CD8+ T-lymphocytes are the main players involved in resistance of the host to
6. Humoral immune response
Antibodies play a minor role but remain the essential means for diagnosing toxoplasmosis. The production of specific IgG antibodies usually begins 4 weeks after the infection and can continue for several months while the dynamics of antibody production does not yield substantial change during the course of disease. IgG are the second immunoglobulins to appear in toxoplasmosis. They play a role in protection of the foetus because they are capable of crossing the placenta. The main target antigens of IgG are the surface antigens of the parasite .
IgM antibodies may appear earlier and decline more rapidly than IgG antibodies. The serum IgM only appears at the end of the first week following infection. These immunoglobulins are the best activators of the complement system. Due to their structure, they enable excellent agglutination and have a high level of cytotoxicity. This phenomenon is used especially in serological diagnosis techniques. Their persistence is subject to a high level of individual variation and can be as much as a year in most cases, thanks to the use of increasingly sensitive detection techniques .
IgA may be detected in sera of actuely infected adults and congenitally infected infants. In acquired toxoplasmosis, the appearance of IgA is not systematic. In immunodepressed subjects, IgA is thought to be an early marker in 50 % of cases. In congenital toxoplasmosis, the detection of IgA is valuable, since these can be detected in the absence of IgM. IgA (like IgM) do not cross the placenta and are actively involved in the diagnosis of congenital toxoplasmosis [2,13].
IgE antibodies are detectable in sera of actually infected adults, congenitally infected infants and children with congenital toxoplasmic chorioretinitis. The appearance on IgE in acute or congenital toxoplasmosis is random. The presence of this isotype is correlated with the beginning of complications, such as adenopathies, chorioretinitis, and
7. Acute and chronic infection
Cell-mediated immune responses are essential for host control of intracellular infections.
The T-lymphocytes, macrophages, and activity of interleukin IL-12 and IFN-γ is necessary for maintaining quiescence of chronic
8. Congenital infection
Congenital toxoplasmosis poses a public health problem, being capable of causing foetal death and ocular and neurological sequelae in congenitally infected children. Congenital infection occurs only when mothers first encounter
9. Diagnosis of toxoplasmosis
The diagnosis of
Histological examination of biological samples shows insufficient reliability if animals are infected by a few parasites. Mouse inoculation is the most reliable method even in the case if detection of cysts in mouse brain demands 40 days. Tachyzoites of virulant strains can be isolated from peritoneal exudate 3-4 days after inoculation. Inoculation of samples in cell cultures (VERO, human fibroblasts) demands specialised laboratories .
The most reliable method for prenatal diagnostics are PCR, mouse inoculation, cell techniques with usage of amniotic fluid, blood of fetus and peripheral maternal blood in pregnant serologically positive individuals. Utilization of quantitative PCR has developed sensitive, specific and rapid method for detection of
Diagnostics of acute, postnatally achieved primary toxoplasmosis will be based on serological methods. Acute infection caused by
We obtained data about seroprevalence of anti-
For obtaining serological data about seroprevalence we used two serological tests: complement fixation test (CFT) and ELISA. CFT was performed by the micromodified method after Zástěra
An enzyme-linked immunosorbent assay (ELISA) was carried out for the detection of IgG and IgM antibodies to
For molecular analysis total DNA was then purified from white blood cells by using the commercial kit QIAamp DNA Mini Kit (QIAGEN, Germany) according to the manufacturer´s instructions. Amplification of the isolated DNA was carried out by the standard PCR and real time PCR method from the
Standard PCR was executed in 25 μL reaction volume containing 0.2 μM of each primer (TGR1E-1, TGR1E-2), 0.2 mM of each dNTP, 1.5 mM MgCl2 and 2.5 U of
For quantitative real time cloned
The examined animals were divided into groups for better understanding of the relationship between the seroprevalence of toxoplasmosis and the age of animals. Each group of examined animals was divided into subgroups according to the age (sheep: female and male lambs up to 4 months of age, rams and ewes; goats: kids - young goats up to 4 months of age, adults - from 7 months of age; pigs: suckling piglets, sows; cattle: calves - up to 6 months of age, heifers, dairy cows; wild boars under 1 year old and adult; Table 1).
For PCR analysis (standard PCR and quantitative real time PCR) each sample was examined by ELISA for detection of IgM and IgG specific antibodies to
Fisher´s exact test was used to compare the success of real-time PCR depending on the presence of IgM or IgG antibodies.
During our study we obtained following data in serological analysis. By CFT 698 animals were examined for the presence of overall anti-
For comparison of CFT and ELISA 102 dog serum specimens were examined for the presence of antibodies to
In molecular analysis by standard PCR and quantitative PCR at first IgM antibodies which appear at the beginning of infection and which are characteristic for acute infection were detected in 45 of 256 (17.6%) by ELISA. IgG antibodies which corresponded with chronic infection were detected in 120 of 256 (46.8%). In 91of 256 (35.5%) animals neither IgM nor IgG were detected by ELISA. The occurrence of IgM or IgG antibodies in each species is summarized in Table 3.
According to the serological results animals were divided into three groups: animals with suspicion of acute (group I, n=45) or chronic toxoplasmosis (group II, n=120) and without infection (group III, n=91). For statistical analysis, we considered group I (acute infection, IgM positive) and group II (chronic infection, IgG positive).
By standard PCR the presence of DNA
Using quantitative real time PCR the presence of DNA
Standards with the known dilution of
|N||Real time PCR||P value|
|Group I (IgM+)||45||9||36||<0.0001*|
|Group II (IgG+)||120||1||119||-|
|Sample||Number of copies||Group|
|Sheep 1||5.92 x 104||IgM+|
|Lamb 1||1.49 x 105||IgM+|
|Sheep 3||3.67 x 104||IgM+|
|Sheep 4||5.75 x 102||IgM+|
|Sheep 5||3.89 x 104||IgM+|
|Sheep 6||2.56 x 103||IgM+|
|Wild boar||1.05 x 105||IgM-|
|Rabbit 1||1.07 x 102||IgM+|
|Rabbit 2||2.09 x 102||IgM+|
|Rabbit 3||3.17 x 102||IgM+|
The diagnosis of toxoplasmosis may be established by serological tests, polymerase chain reaction (PCR), histological demonstration of the parasite and/or its antigens (i.e. immunoperoxidase stain), or isolation of the organism. The serological tests are able to show the presence of IgM antibodies which can represent the acute infection as well as these IgM antibodies can be residual. Avidity tests may help in this setting by distinguishing between IgG of high and low affinity, corresponding to either chronic or acute toxoplasmosis. Results of serological tests don´t reflect the course of infection. The presence of specific serum antibodies is possible to detect in organism of each potential host who come in the contact with the pathogen. Based on the serological prevalence in the population is possible to suppose only the certain relationship between population and morbidity. The advantage of serological tests is that specific antibodies can be detected two week before histological proof of parasite in pathological lesions and 4 weeks before the molecular proof of parasites in infected tissues. The positive serological result is only indicative of infection, whereas direct detection of
These results suggest that
Among laboratory diagnostic techniques, a complement fixation test is one of the most frequently employed techniques for detecting antibodies to
Gene amplification methods (PCR, LCR, NASBA, etc.) are now used widely in the diagnosis of infectious diseases. Key advantages are their relative speed, the potential to detect very low numbers of pathogens (or, more precisely, specific nucleic acid sequences from pathogens) and the ability to discriminate accurately at the species or sub-species level. In the case of non-persistent pathogens that are cleared from the body, a positive PCR finding is usually significant. The diagnosis of toxoplasmosis by PCR, however, is complicated by the fact that the parasite persists (principally in heart, brain and skeletal muscle in the form of quiescent tissue cysts) for many years after active infection has ceased. Thus, the presence of
All mammals and birds that are consumed by humans may serve as intermediate hosts for
Tissue cysts of
The paper is a result of research work done within the frames of grant projects VEGA of the Ministry of Education of the Slovak Republic No. 1/0271/11
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