Seroepizootiology and molecular diagnosis of Neospora caninum infection in cattle in Serbia

Инфекција паразитом Neospora caninum изучава се од 80-их година прошлог века, до када је због сличности агенса са Toxoplasma gondii била погрешно дијагностикована. Данас је неоспороза говеда широм света болест растућег значаја, која код репродуктивних категорија изазива побачаје, смртност телади и велике економске губитке ако се јави у епизоотској форми. Почетна истраживања преваленце инфекције N. caninum код говеда у Србији датирају од 2008. године, мећутим нема података о губицима које неоспороза потенцијално проузрокује у говедарству Србије. У овом раду испитали смо серопреваленцу ове инфекције на репрезентативном узорку крава са целе територије Србије и анализирали епизоотиолошке факторе који могу имати улогу у појави и ширењу инфекције код говеда у нашој земљи, што би могло да резултира предлогом развоја мера превенције и последичног унапређења сточарске производње. Такође, извршили смо оптимизацију метода молекуларне дијагностике инфекције N. caninum и сагледали могућности примене молекуларних метода у дијагностици неоспорозе. Серуми пореклом од укупно 1496 говеда испитани су компетитивним ELISA тестом на присуство N. caninum специфичних антитела. Показано је да серопреваленцa износи 7,2%, са ниским до умереним (од 2,2 до 12%) степеном прокужености у свих дванаест епизоотиолошких подручја Србије. Инфекција је установљена на 10,7% испитаних фарми. Резултати cELISA теста за серопозитивне јединке, тј. вредности степена инхибиције кретале су се од 40,3 – 91,8%, средње вредности 71,9±17,1%, и медијане 79,9%, што значи да је практично половина серопозитивних крава имала специфична антитела у високом титру, тј. високе вредности степена инхибиције од преко 80%. Испитивање епизоотиолошких фактора у смислу фактора ризика за инфекцију N. caninum урађено је применом логистичке регресионе анализе. Повезаност инфекције са потенцијално релевантним епизоотиолошким факторима (величина стада, доминантно заступљена раса, начин гајења, начин држања у штали, употреба силаже у исхрани, порекло грла за ремонт стада, присуство и број паса, појава побачаја у стаду, просечна годишња количина падавина, регион) прво је испитана униваријантном логистичком регресијом, и варијабле значајне на нивоу P≤0,25 укључене су у мултиваријантни регресиони модел...Neospora caninum is a relatively recently recognized protozoan parasite of animals. It has been studied only since 1988, until when it was misdiagnosed as Toxoplasma gondii, due to the morphological similarity of the agents. Today, neosposoris in cattle is known as a disease of growing importance worldwide, causing abortions and large economic losses if epidemic abortions occur. The first investigations of the prevalence of N. caninum infection in cattle in Serbia were not carried out before 2008, and no data exist on the potential damaging effects of neoporosis on cattle husbandry in Serbia. In this study we examined the seroprevalence of the infection in a representative sample of cows from the entire territory of Serbia as well as the epizootiological factors that may play a role in the emergence and spread of infection. Sera originating from a total of 1496 cattle were tested by competitive ELISA assay for the presence of N. caninum-specific antibodies. It was shown that the seroprevalence was 7.2%, ranging from a low to a moderate (2.2 to 12%) prevalence accross all twelve epizootiological areas of Serbia. At least one seropositive animal was detected on 10.7% of farms. Results of the cELISA, i.e. the degree of inhibition in seropositive individuals, ranged from 40.3 to 91.8%, mean = 71.9 ± 17.1%, median = 79.9%, which means that almost one half of the seropositive cows had specific antibodies in high titres, or high values of inhibition of more than 80%. Possible association of the infection with biologically plausible risk factors including herd size, breed, husbandry methods, feeding practices (use of silage), origin of replacement stock, presence and number of dogs, occurrence of abortions in the herd, average annual rainfall and region, was analyzed by univariate analysis, and variables significant at P≤0.25 were included in multivariate logistic regression models. The results showed that the single risk factor for infection in individual animals was keeping cows in loafing barns (OR=3.31, 95% CI=1.95-5.60, P<0.001), while risk factors for the presence of infection on farms were the use of loafing barns (OR=18.49, 95% CI=5.40-63.36, P100 animals (OR=24.08 95% CI=3.85-150.50, P=0.001). In addition to a comprehensive insight into the current state of N. caninum infection in cattle in Serbia, the results of this research on infection risk factors both at the individual and farm level in our country suggest preventive measures that can be applied to improve animal health and reduce economic losses, thereby improving livestock production..

Toxoplasmosis in Serbia: time for an action plan

Known for a century, Toxoplasma gondii has been studied in Serbia half this time, ever since the introduction of the Sabin-Feldman test at the Institute for Medical Research (IMR) in 1959. However, despite 50 years of continuous efforts, exact data on the frequency of acute clinical disease, acute infections in pregnancy and congenital infection in the offspring are still lacking, due to the vague regulatory provision that toxoplasmosis is subject to reporting "in case of epidemiological indications". It is, however, clear that the major Toxoplasma-induced public health issue in Serbia, like elsewhere in Europe, is congenital toxoplasmosis (CT). Continuous monitoring of particular patient groups showed a dramatic decrease in the prevalence of infection over the past two decades, and a consequently increased proportion of women susceptible to infection in pregnancy, suggesting a potential increase in the incidence of CT. Studies of risk factors for infection transmission have provided data to guide national health education campaigns. It is expected that the recent appointment of the National Reference Laboratory for Toxoplasmosis as the focal point for the collection of data from the primary level, will provide the means for accurate assessment of the measure of the problem, which is a prerequisite of an evidence-based nation-wide prevention program. In the meantime, health education of all pregnant women, focused at risk factors of major local significance, is advocated as a sound and financially sustainable option to reduce congenital toxoplasmosis

In Vivo and In Vitro Virulence Analysis of Four Genetically Distinct Toxoplasma gondii Lineage III Isolates

Toxoplasma gondii archetypes II and III are mildly virulent, yet virulence of variant strains is largely unknown. While lineage II dominates in humans in Europe, lineage III strains are present in various intermediate hosts. In Serbia, lineage III represents 24% of the population structure and occurs most frequently in domestic animals, implying a significant presence in the human food web. In this study, the virulence of four genetically distinct lineage III variants was assessed in vivo and in vitro. In vivo, two strains were shown to be intermediately virulent and two mildly virulent, with cumulative mortalities of 69.4%, 38.8%, 10.7%, and 6.8%, respectively. The strain with the highest mortality has previously been isolated in Europe and may be endemic; the strain with the lowest mortality matches ToxoDB#54, while the remaining two represent novel genotypes. Identical alleles were detected at ROP5, ROP16, ROP18, and GRA15. A set of in vitro analyses revealed proliferation and plaque formation as virulence factors. Higher levels of expression of ENO2 in intermediately virulent strains point to enhanced metabolism as the underlying mechanism. The results suggest that metabolic attenuation, and possibly stage conversion, may be delayed in virulent strains

Toxoplasma gondii genotypes circulating in domestic pigs in Serbia

Consumption of undercooked or raw pork is considered a significant risk factor for human infection with Toxoplasma gondii. In this study, we investigated the genetic structure of 18 T. gondii strains obtained from slaughter pigs from Northern Serbia (mainly Vojvodina). The examined samples originated from eight pigs from large commercial farms, six backyard pigs and four free-range Mangalica pigs, all found to be positive for either viable T. gondii or T. gondii DNA. Genotyping was attempted from both pig tissues and mouse brains from the bioassays using a multiplex multilocus nested polymerase chain reaction-restriction fragment length polymorphism (Mn-PCR-RFLP) method with seven markers (GRA6,.lt. SAG2, PK-1, BTUB, C22-8, CS3 and Apico). Identification was achieved for nine T. gondii isolates. Seven isolates were classified as type II and two as type III. These results are consistent with previous studies on animal isolates from Serbia as well as with previous reports that type III is more frequently found in samples from Southern Europe than in those from other parts of the continent

Toxoplasma gondii in pork and pigs in Serbia – a real food safety hazard

Infection with the apicomplexan protozoon Toxoplasma gondii is one of the most prevalent parasitic zoonotic infections globally, with existing seroprevalences varying between continents, countries, and even within countries and between individual communities. It is estimated that one third of the world's human population is infected with T. gondii, with many studies showing that the dominant mode of infection is consumption of undercooked meat harbouring T. gondii tissue cysts. Prevalences of infection in food animals in different countries range from 0 to 93%. Because of the absence of clinical symptoms in infected animals, and the unfeasibility of rapid and unequivocal detection of microscopic tissue cysts in pork, infected pigs remain unrecognized, and their meat becomes an essential source of infection for humans. The data on T. gondii infection in pigs in Serbia from several studies, as well as on the detection of the parasite in different food categories, from fresh pork to heat-treated products, are discussed

Risk factors for toxoplasma infection in pregnant women in FYR of Macedonia

The aim of the study was to identify risk factors for Toxoplasma gondii infection in pregnant women in FYR of Macedonia. Retrospective analysis of serological and epidemiological data in a series of 235 pregnant women from Macedonia, tested for Toxoplasma infection between January 2004 and December 2005, showed on overall prevalence of infection of 20.4 %. Exposure to transmission factors significantly increased the risk of infection (RR = 1.989, 95 % Cl = 1.041-3.800, p = 0.037). The single infection transmission factor that was a predictor of infection in the whole series was exposure to soil (RR = 1.946, 95 % Cl = 1.026-3.692, p = 0.041). Based on prevalence and the established risk factors for Toxoplasma infection in Macedonia, the health education programme as a sustainable measure for the prevention of congenital toxoplasmosis should focus on educating women of generative age to avoid contact with soil (forming, gardening), and/to adhere to strict hygienic practices afterwards

Trichinella spp. In wild mesocarnivores in an endemic setting

Human trichinellosis and Trichinella infection in pigs are both still endemic in the Balkans, including Serbia. Because of the flow between the sylvatic and the domestic cycle of Trichinella spp., monitoring wildlife has been recommended for the risk assessment of Trichinella spp. infection in swine. We have previously shown the presence of Trichinella infection in wild carnivores including the wolf and the golden jackal, and here we report on Trichinella infection in several other mesocarnivore species. From a total of 469 animals collected between 1994 and 2013, Trichinella larvae were detected in 29 (6.2%, 95% CI = 4.0-8.4) animals, including 14 red foxes (4.7%), 7 wild cats (35%), 5 beech martens (4.8%), 2 pine martens (16.7%), and 1 European badger (6.25%). No Trichinella larvae were detected in the examined specimens of European polecats, steppe polecats and European otters. Species identification of the Trichinella larvae performed for 18 positive samples revealed T. spiralis in 77.8% and T. britovi in 22.2% of the isolates. Both species were detected in red foxes and wild cats. The predominance of T. spiralis in wildlife in Serbia indicates the (past or present) spillover of this pathogen from domestic to wild animals

Comparative evaluation of three commercial Toxoplasma-specific IgG antibody avidity tests and significance in different clinical settings

Determination of the avidity of specific IgG antibodies has become a generally accepted diagnostic aid for dating Toxoplasma infection. In this study, the Labsystems, VIDAS and EUROIMMUN Toxoplasma IgG avidity assays were compared on a series of 133 Toxoplasma IgG- and IgM-positive sera from symptomatic patients (n=28), from pregnant (n=43) and nonpregnant (n=26) women, and on 18 IgG-positive and IgM-negative sera from chronically infected patients. The results showed excellent concordance between the Labsystems and VIDAS tests in both the IgM-positive (r=0.82, kappa=0.771) and IgM-negative (kappa=0.609) sera, whilst the agreement of the EUROIMMUN assay with both the Labsystems and VIDAS tests in the IgM-positive sera was moderate (kappa=0.575 and kappa=0.525, respectively) and in the IgM-negative sera was poor (kappa=0.000). Analysis of the kinetics of the maturation of avidity in 13 patients in whom follow-up sera were available showed that, despite a general trend of maturation, in two patients the avidity did not become high during 6 and 11 months of follow-up. In view of the clinical setting, in the symptomatic patients, despite one case of complete discrepancy and five cases of partial discrepancy, the Labsystems and VIDAS tests were in almost perfect agreement (kappa=0.812), whilst the agreement in pregnant and non-pregnant women was substantial (kappa=0.754 and kappa=0.708, respectively). In conclusion, the Labsystems and VIDAS tests are equally reliable for the measurement of Toxoplasma IgG avidity; the choice of test should depend on the laboratory setup. The EUROIMMUN test may be an acceptable alternative in resource-limited settings, but should be used prudently