Marija Markovic

Atypical strain of Toxoplasma gondii causing fatal reactivation after hematopoietic stem cell transplantion in a patient with an underlying immunological deficiency.

ABSTRACT In immunocompromized patients, including hematopoietic stem cell transplant (HSCT) recipients, life-threatening toxoplasmosis may result from reactivation of previous infection. We report a case of severe disseminated toxoplasmosis that developed early after allogeneic HSCT for T-cell lymphoblastic leukemia/lymphoma in a 15-year-old Toxoplasma gondii-seropositive boy with Nijmegen breakage syndrome, a rare genetic DNA repair disorder associated with immunodeficiency. The donor was the patients HLA-identical brother. Prophylaxis with cotrimoxazole was discontinued a day before the HSCT procedure. Signs of lung infection appeared as early as day 14 post-HSCT. The presence of tachyzoite-like structures on Giemsa-stained bronchoalveolar lavage (BAL) fluid smears suggested toxoplasmosis. Real-time PCR targeted at the T. gondii AF146527 gene revealed extremely high parasite burdens in both blood and BAL fluid. Although immediate introduction of specific treatment resulted in a marked reduction of the parasite load and transient clinical improvement, the patient deteriorated and died of multiple organ failure on day 39 post-HSCT. Direct genotyping of T. gondii DNA from blood and BAL fluid with the PCR-restriction fragment length polymorphism method revealed type II alleles with SAG1, SAG2, and GRA6 markers but alleles of both type I and type II with GRA7. Additional analysis with 15 microsatellite markers showed that the T. gondii DNA was atypical and genetically divergent from that of the clonal type I, II, and III strains. This is the first report of increased clinical severity of toxoplasmosis associated with an atypical strain in the setting of immunosuppression, which emphasizes the need to diagnose and monitor toxoplasmosis by quantitative molecular methods in cases of reactivation risk.

Evidence for genetic diversity of Toxoplasma gondii in selected intermediate hosts in Serbia

To contribute to the insight into the worldwide population structure of Toxoplasma gondii, we genetically characterized a total of eight strains isolated from intermediate hosts including humans, sheep and pigeons in Serbia. Although parasite DNA was detected in 28.2% (60/213) of the human samples from 162 patients serologically suspected of active toxoplasmosis, as well as in 5/7 seropositive pigeons and in 2/12 seropositive sheep examined, multilocus PCR-RFLP genotyping, using SAG1, 5SAG2, 3SAG2, GRA6, 5GRA7 and 3GRA7 as markers, was successful in only four human isolates (of which one was isolated from both the bronchoalveolar lavage fluid and blood samples of a single patient), one sheep and three pigeons. Of the eight isolates, five were type II (62.5%), one was type III, one was atypical, and one had a type I allele at GRA6 as the single locus genotyped. Although type II, as elsewhere in Europe, predominated, these results may suggest a higher genetic diversity of T. gondii in Serbia, reflecting local environmental contamination and also the geographical position of the country in South-East Europe.

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.

The Wolf (Canis lupus) as an Indicator Species for the Sylvatic Trichinella Cycle in the Central Balkans

Abstract Wildlife is the most important reservoir of Trichinella spp. worldwide. Although the Balkans are a recognized European endemic region for Trichinella infections, data on wildlife are scarce. To monitor the circulation of these zoonotic parasites in the Central Balkan region, the wolf (Canis lupus) was selected because of its abundance (>2,000 individuals) and because it is at the top of the food chain. A total of 116 carcasses of wolves were collected in Serbia and in the neighboring areas of Macedonia and Bosnia and Herzegovina (B&H) between 2006 and 2013. Trichinella spp. larvae were found in 54 (46.5%) wolves. The great majority (90.5%) originated from Serbia, where 52 of the 105 examined animals were Trichinella positive (49.5%; 95% confidence interval u200a=u200a39.9–59.1). One positive animal each was found in B&H and Macedonia. All larvae were identified as Trichinella britovi. The high prevalence of Trichinella infection in wolves suggests that this carnivore can be a good indicator species for the risk assessment of the sylvatic Trichinella cycle in the Central Balkans.

Clinical significance of molecular methods in the diagnosis of imported malaria in returning travelers in Serbia

OBJECTIVESnThe goal of this study was to assess the clinical significance of conventional and PCR-based molecular diagnosis in patients with imported malaria in Serbia.nnnMETHODSnGiemsa microscopy, the rapid diagnostic test, and quantitative real-time PCR (qPCR) were used to detect Plasmodium species in 109 whole-blood samples from patients after their return from malaria endemic areas, including those clinically suspected for malaria (n=97) and healthy travelers (n=12) examined as part of epidemiological surveillance.nnnRESULTSnA total of 45 patients were diagnosed with malaria: 42 (93.3%) by microscopy and three (6.7%) additional ones by qPCR. The agreement between the results of species-specific qPCR and microscopy was 73.3%; it was as high as 90.6% for Plasmodium falciparum infections. Follow-up analysis demonstrated persistence of Plasmodium sp DNA for a mean 6 days after the disappearance of parasitemia on microscopy.nnnCONCLUSIONSnDue to its sensitivity and specificity, qPCR is a helpful method complementary to microscopy, particularly in cases of low parasitemia. In addition, it is superior to microscopy for species identification.

Effect of explant type, medium and soil mixture content on Dianthus deltoides L. rooting and acclimatization

The possibility of developing roots and shoots on Dianthus deltoides nodal n and apical cuttings on rooting media was investigated in vitro conditions. n The explants developed roots and shoots successfully, and the best results n were achieved on MS medium without plant hormones. Number of roots per shoot, n length of roots and ramification of roots were not significantly dependent on n the medium (NAA-naphthaleneacetic acid concentration), or on the type of n explant used (nodal or apical cuttings). The acclimatization rate of n microplants was influenced by root numbers and ramification. The transfer to n soil was successful, maximum survival rates (96%) were achieved on peat - n sand (4:1) mixture.

Cobalt impregnated pillared montmorillonite in the peroxymonosulfate induced catalytic oxidation of tartrazine

Aluminum pillared montmorillonite impregnated with cobalt (CoAP) was synthesized and characterized using chemical analysis, XRD and N2-physisorption. CoAP was tested as a catalyst in the peroxymonosulfate (Oxone®) induced catalytic degradation of tartrazine. The influence of Oxone®/catalyst ratio and temperature on CoAP catalytic performance was investigated. The UV–Vis spectra obtained after predetermined periods of time of reaction were analyzed in order for tartrazine solution composition to be monitored. The reaction was more efficient at 50xa0°C than at 30xa0°C and the presence of new peaks for the reaction at 50xa0°C was observed. The peaks were deconvoluted and further analyzed. The intensity of two characteristic peaks gradually decreased during the investigated reaction following the first order kinetics. Newly formed peaks indicated the formation of degradation products. The initial increase of the intensity of some of them was followed by certain decrease as the reaction proceeded. CoAP was found to be efficient catalyst in Oxone® induced catalytic decolorization of tartrazine. The degradation of different products formed in tartrazine oxidation was evidenced.

Micropropagation of Endangered and Decorative Species Dianthus pinifolius Sibth. et Sm.

The aim of this study was to establish an efficient protocol for the in vitro propagation of the endangered, endemic and decorative species Dianthus pinifolius Sibth. et Sm. The effects of different concentrations of 6-benzylaminopurine (BAP) and naphtalenacetic acid (NAA), and different explant types (single-node cuttings, terminal buds and shoot cuttings) on shoot multiplication were examined on Murashige and Skoog (MS) and half-strength MS media. The best results were obtained for shoot cuttings on the MS medium supplemented with 0.5 mg/L BAP and 0.1 mg/L NAA, achieving a maximum rate of regeneration (100%) and a total of 15.4 newly-developed shoots per explant. The highest rooting rate (96.7%) was obtained on MS medium containing 1 mg/L NAA, while the acclimatization of the microplants obtained to ex vitro conditions was successful (88.9%).

Effect of explant type on the rooting and acclimatization of Dianthus serotinus Waldst. & Kit.

The effect of the concentration of MS salts and explant type on D. serotinus n rooting and acclimatization was investigated in order to optimize a protocol n for the micropropagation of this species. The obtained results showed that n explant type as well as the concentration of MS salts had a significant n effect on rooting, and the highest rooting rate (85-86,7%) was achieved when n culturing single-node cuttings and terminal buds on a half-strength MS medium n supplemented with 0,5 mgL-1 NAA. Nevertheless, mean number of roots per n explant was higher on the MS media (15,3-18,6) than on the half-strength MS n media (11,8-13,4). The best acclimatization rate was obtained in a 4:1 n mixture of peat and sand (83,3-86,7%). The explant type from which in vitro n plantlets developed had no effect on the acclimatization rate. [Projekat n Ministarstva nauke Republike Srbije, br. 43007]

Effects of the time of cuttings collection and IBA concentration on the rooting of softwood cuttings from elite trees of Cornelian cherry (Cornus mas L.) in Belgrade area.

In this study, the effect of concentration of indole-3-butyric acid (IBA) n (powder dip), cutting type as well as the time of taking cuttings on the n rooting of softwood cuttings of cornelian cherry was examined. Four types of n cuttings were used: basal cuttings, terminal cuttings, basal cuttings with n 2-year-old wood and terminal cuttings with 2-year-old wood. The obtained n results showed that IBA concentration, cutting type and time of collecting n have significant effect on rooting. The best results were obtained using 1% n IBA and cuttings collected in the second term (in mid-July) had a higher n rooting percentage. Terminal cuttings treated with 1% IBA (powder dip) should n be used for optimum results. In that case the rooting percentage was very n high (over 90%) in both terms of cuttings collection. [Projekat Ministarstva n nauke Republike Srbije, br. 43007: Istraživanje klimatskih promena na životnu n sredinu: pracenje uticaja, adaptacija i ublažavanje]