Ivana Klun

High prevalence of intestinal zoonotic parasites in dogs from Belgrade, Serbia--short communication.

To identify areas of risk for canine-related zoonoses in Serbia, the aim of this study was to provide baseline knowledge about intestinal parasites in 151 dogs (65 household pets, 75 stray and 11 military working dogs) from Belgrade. The following parasites, with their respective prevalences, were detected: Giardia duodenalis (14.6%), Ancylostomatidae (24.5%), Toxocara canis (30.5%), Trichuris vulpis (47.0%) and Taenia-type helminths (6.6%). Of all examined dogs, 75.5% (114/151) were found to harbour at least one parasite species. Of these, mixed infections with up to four species per dog occurred in 44.7% (51/114). Infections with all detected species were significantly higher (p < 0.05) in military working (100%) and stray dogs (93.3%) versus household pets (50.8%). Among all parasites, agents with zoonotic potential including Giardia, Ancylostomatidae and Toxocara were detected in 58.3% (88/151) of all examined dogs with a significant difference (p < 0.05) among the subgroups (100%, 62.7% and 46.2% for military working dogs, stray dogs and household pets, respectively). The high prevalence of zoonotic parasites registered in the dog population from a highly urban area in south-eastern Europe indicates a potential risk to human health. Thus, veterinarians should play an important role in helping to prevent or minimise zoonotic transmission.

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 non-pregnant (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 set-up. The EUROIMMUN test may be an acceptable alternative in resource-limited settings, but should be used prudently.

Kinetics of parasite burdens in blood and tissues during murine toxoplasmosis.

A sensitive real-time PCR technique was used to examine the distribution of Toxoplasma gondii in the blood and tissues of mice during acute and chronic infection. Groups of Swiss Albino mice, inoculated i.p. with 10(2) or 10(6) tachyzoites of the RH strain as a typical type-1 strain, or fed 10 cysts of the Me49 strain as a typical type-2 strain, were killed at different time points post-infection (p.i.), and blood and organs including the lungs, brain and liver were harvested for DNA extraction. Toxoplasma DNA was quantified by a real-time PCR targeted at the 529bp gene fragment, with a detection limit of a single parasite per g/ml of tissue. The results showed a strain- and dose-dependent spread of Toxoplasma. In infection with type-1 parasites, in case of a high infective dose, Toxoplasma DNA was detected within 24h p.i. in all analyzed tissues including the brain. Conversely, in case of a low infective dose, parasitaemia was undetectable early p.i., at a time when Toxoplasma DNA was detected in the tissues, but reached very high levels as infection progressed. With both infective doses, pre-death parasite burdens were higher in the blood than in the tissues, whereas the same loads in the lungs suggest that reaching these Toxoplasma burdens may be critical for survival. In infection with Me49 parasites, steady high parasite burdens were noted up to the end of the experiment at d42 only in the brain, parasitaemia was low but detectable throughout, and Toxoplasma DNA was completely cleared only from the liver. These data are important to better understand the pathogenesis of toxoplasmosis, and also as baseline data for the experimental evaluation of novel chemotherapeutics.

Toxoplasmosis in Naturally Infected Rodents in Belgrade, Serbia

To assess the role of synanthropic rodents in the epidemiology of urban toxoplasmosis, Toxoplasma gondii infection was examined in 144 rats (Rattus norvegicus) and 12 mice (Mus musculus) captured using live animal traps in three locations in Belgrade city characterized by poor housing and degraded environment. In rats, specific IgG antibodies were detected by modified agglutination test in 22 (27.5%) of the 80 blood samples available. Toxoplasma brain cysts were microscopically detected in 11 (7.6%), and Toxoplasma DNA by real-time polymerase chain reaction was demonstrated in 15 (10.4%) animals. Of these, both cysts and Toxoplasma DNA were detected in five (3.5%) rats. In mice, cysts were observed in 3 (25%), but Toxoplasma DNA was detected in even 10 (83.3%) animals, including all 3 with morphologically recognized cysts. Being a link in the chain of Toxoplasma infection, the existence of urban rodent reservoirs of infection represents a public health risk.

Kinetics of Toxoplasma infection in the Balkans.

ZusammenfassungZIEL: Die Kinetik der Toxoplasmose-Infektion am Balkan wurde untersucht. DATENQUELLE: In den letzten 30 Jahren in allen Balkanländern veröffentlichte Berichte über Toxoplasmoseinfektionen bei Frauen im gebärfähigen Alter. ERGEBNISSE: Das wesentliche Merkmal der Toxoplasmoseinfektion am Balkan ist ein mit der Zeit kontinuierlicher Rückgang der Infektionshäufigkeit. Das systematische Erfassen der Prävalenz der Toxoplasmoseinfektion in der Population gebärfähiger Frauen in Slowenien, Serbien und Griechenland hat in den letzten 30 Jahren einen signifikanten kontinuierlichen Rückgang in allen drei Ländern gezeigt. Auch in Montenegro und in der früheren jugoslawischen Republik Mazedonien (fjRM) wurde ein Abfall beobachtet, wobei in diesen Ländern aber nur in den letzten 10 Jahren eine systematische Erfassung vorliegt. Ein anderes überregionales Merkmal ist die Beobachtung, dass die Prävalenz der Infektion zurzeit nicht über 50 % liegt. Außerdem besteht im Osten der Balkanhalbinsel eine Abnahme der Prävalenz der Toxoplasmose von Norden nach Süden: von Süd Ungarn (als Nordgrenze des Balkans) über Serbien und fjRM nach Nord Griechenland. Weiters wurde im Westen Sloweniens und im Osten Serbiens eine Saisonabhängigkeit mit signifikant mehr Fällen der akuten Infektion im Winter als im Sommer festgestellt. Trotz eines allgemeinen Trends zur Abnahme der Infektion scheinen die Risikofaktoren der Übertragung in der Region verschieden zu sein: während ein Kontakt mit Katzen in Slowenien wichtig schien, wurde die Einnahme von nicht ausreichend gekochtem Fleisch als wichtigster Risikofaktor in Serbien und Albanien gefunden. In der fjRM und in Nord-Griechenland war der Kontakt mit Dünger der führende Risikofaktor. SCHLUSSFOLGERUNG: Ein Trend zur Abnahme der Prävalenz der Toxoplasmose im Balkan mit der Zeit ist Teil eines sich verändernden Musters der Toxoplasmose Infektion in ganz Europa. Strategien zur Vorbeugung einer kongenitalen Toxoplasmose sollten diese Dynamik der Infektion berücksichtigen.SummaryAIM: The kinetics of Toxoplasma infection in the Balkans were reviewed. SOURCE OF DATA: Published reports on Toxoplasma infection in women of childbearing age in the last 30 years for all Balkan countries. RESULTS: The dominant feature of Toxoplasma infection in the Balkans is a continuous decrease in the prevalence over time. Systematic monitoring of Toxoplasma infection prevalence in populations of women of childbearing age in Slovenia, Serbia and Greece over the last 30 years has shown a continuous significant decrease in all three countries. Moreover, a decrease has also been shown in Montenegro and Former Yugoslav Republic of Macedonia where Toxoplasma infection has been surveyed only during the past decade. Another region-wide feature is that the prevalence of infection currently does not surpass 50%. Furthermore, a decrease in Toxoplasma prevalence from the north to the south has been shown in the eastern part of the Balkan Peninsula, from southern Hungary (as a region neighbouring the Balkans at the north), over Serbia and FYRoM to northern Greece. Seasonality of infection, with significantly more cases of acute infection in the winter than in the summer, was observed in Slovenia in the west and Serbia in the east. Despite a common decreasing trend, different infection transmission risk factors seem predominant across the region; while contact with cats was discussed as important in Slovenia, consumption of undercooked meat was shown to be the leading risk factor in Serbia and Albania, and contact with soil in FYRoM and in northern Greece. CONCLUSION: A decreasing trend of Toxoplasma prevalence over time in the Balkans is part of a changing pattern of Toxoplasma infection throughout Europe. Strategies for the prevention of congenital toxoplasmosis should take the infection dynamics into account.

Seasonal Variations in Human Toxoplasma Infection in Serbia

BACKGROUND Seasonal variations in the occurrence of toxoplasmosis have been studied only sporadically. We thus examined the seasonal distribution of acute toxoplasmosis in Serbia during a 4-year period (2004-2008). MATERIALS AND METHODS A total of 391 consecutive symptomatic (recent lymphadenopathy) and 715 asymptomatic (women tested for obstetric reasons) patients were tested for Toxoplasma immunoglobulin G (IgG) (including IgG avidity) and IgM antibodies. The distribution of patients with acute infection, and of all patients from both groups, was analyzed for seasonality. In addition, factors (including undercooked meat consumption, contact with cats and with soil) possibly contributing to seasonality were analyzed in patients with acute infection (cases) matched by age, sex, and time (month and year) of infection (symptomatic) or presentation (asymptomatic) with seronegative patients (controls). RESULTS Acute toxoplasmosis was serologically (IgG avidity low, IgM positive) diagnosed in 39 (10.0%) symptomatic and 38 (5.3%) asymptomatic patients. In both groups, monthly distribution of acute infections showed significant (p < 0.0001) seasonality, which was related to the four seasons of the year (p < 0.0001). Importantly, the observed seasonality was not related to the distribution of all examined patients in either group (p < 0.001). In the symptomatic patients, acute infections occurred more often between October and March (p = 0.0486). Although more asymptomatic acute infections were diagnosed between February and July (p = 0.0037), low IgG avidity suggests that infection had occurred within the previous trimester (between November and April). Undercooked meat consumption was shown as a risk factor for symptomatic infection in the October-March period (odds ratio 7.67, 95% confidence interval 1.61-36.45). CONCLUSION Seasonality patterns should be taken into account in the health education guidelines for the prevention of toxoplasmosis in pregnant women.

Molecular Detection and Genotyping of Toxoplasma gondii from Clinical Samples

Over the past two decades, molecular diagnosis of toxoplasmosis, which is based on the detection of T. gondii DNA in clinical samples, became an indispensable laboratory test. This method is independent of the immune response, and depending on methodological approach, may facilitate more accurate diagnosis, especially in cases in which inadequacy of conventional methods is faced with deteriorating and potentially severe clinical outcome (congenital, ocular toxoplasmosis and cases of immunosuppression).

Human giardiasis in Serbia: asymptomatic vs symptomatic infection

Despite the public health importance of giardiasis in all of Europe, reliable data on the incidence and prevalence in Western Balkan Countries (Serbia, Bosnia and Herzegovina, Croatia, Montenegro and FYR Macedonia) are scarce, and the relative contribution of waterborne and food-borne, or person-to-person and/or animalto- person, transmission of human giardiasis is not yet clear. To provide baseline data for the estimation of the public health risk caused by Giardia, we here review the information available on the epidemiological characteristics of asymptomatic and symptomatic human infection in Serbia. Although asymptomatic cases of Giardia represent a major proportion of the total cases of infection, high rates of Giardia infection were found in both asymptomatic and symptomatic populations. No waterborne outbreaks of giardiasis have been reported, and it thus seems that giardiasis mostly occurs sporadically in our milieu. Under such circumstances, control measures to reduce the high prevalence of giardiasis in Serbia have focused on person-to-person transmission, encouraging proper hygiene, but for more targeted intervention measures, studies to identify other risk factors for asymptomatic and symptomatic infections are needed.

Toxoplasma gondii infection induces lipid metabolism alterations in the murine host

Host lipids have been implicated in the pathogenesis of Toxoplasma gondiiinfection. To determine if Toxoplasmainfection influences the lipid status in the normal host, we assessed serum lipids of Swiss-Webster mice during infection with the BGD-1 strain (type-2) at a series of time points. Mice were bled at days zero and 42 post-infection, and subgroups were additionally bled on alternating weeks (model 1), or sacrificed at days zero, 14 and 42 (model 2) for the measurement of total cholesterol (Chl), high density lipoproteins (HDL), low density lipoproteins (LDL) and triglycerides and adiponectin. At day 42, brains were harvested for cyst enumeration. A significant decrease (p = 0.02) in HDL and total Chl was first noted in infected vs. control mice at day 14 and persisted to day 42 (p = 0.013). Conversely, LDL was unaltered until day 42, when it increased (p = 0.043). Serum LDL levels at day 42 correlated only with cyst counts of above 300 (found in 44% mice), while the change in HDL between days zero and 42 correlated with both the overall mean cyst count (p = 0.041) and cyst counts above 300 (p = 0.044). Calculated per cyst, this decrease in HDL in individual animals ranged from 0.1-17 µmol/L, with a mean of 2.43 ± 4.14 µmol/L. Serum adiponectin levels remained similar between infected and control mice throughout the experiment.

A large-scale study of the Trichinella genus in the golden jackal (Canis aureus) population in Serbia.

Over the last decades the golden jackal (Canis aureus) has significantly expanded its range throughout Southeast and Central Europe, and the Balkan Peninsula is considered to be a core area of the species distribution in this part of the range. Due to its increasing number, ability of long distance movement through a wide range of landscapes and opportunistic feeding habits, the golden jackal may represent an important reservoir and transmitter of a variety of zoonotic agents, including parasites. The Balkans, Serbia included, remain an endemic area for various zoonotic parasites including Trichinella spp. Trichinella has recently been recorded in jackals in Serbia, which prompted us to carry out a large-scale survey of its prevalence, distribution and species identification in this host. In cooperation with local hunters, carcasses of a total of 738 legally hunted golden jackals were collected at 24 localities over an 11-year period (2003-2013). Analysis of tongue base tissue revealed Trichinella larvae in 122, indicating a prevalence of infection of 16.5%. No difference in the prevalence of infection was found between genders [16.2% in males and 16.9% in females (χ(2)=0.05, p=0.821)], or among the study years (G=7.22, p=0.705). Trichinella larvae were found in 13 out of the 24 examined localities. Molecular identification was performed for 90 isolates, and 64 (71.1%) larvae were identified as Trichinella spiralis and 25 (27.9%) as Trichinella britovi. Mixed infection (T. spiralis and T. britovi) was recorded in a single case. Although T. spiralis was more prevalent, T. britovi had a wider distribution, and was the only recorded species in jackal populations from the mountainous region of eastern Serbia. On the other hand, T. spiralis was dominant in jackals in the lowlands of central and northern Serbia, where domestic pigs are mostly reared. These results show that the golden jackal is involved in both the domestic and sylvatic cycle, and that it has emerged as a major host species in the sylvatic cycle of the Trichinella genus. Therefore, continued monitoring of Trichinella infection in golden jackals in Serbia and the whole of the Balkans is recommended in order to control transmission of this parasite to humans and domestic animals.