Detection and dynamics of tumor necrosis factor alpha in the diagnosis and treatment of canine heartworm disease.

The aim of this study was to determine the concentration of TNF-alpha (TNF-α) in dogs naturally infected with Dirofilaria immitis (D. immitis) and to assess whether there are any changes in TNF-α concentration and their dependence during therapy for heartworm disease (HWD). For this study, 14 client-owned dogs with HWD were selected. Clinical and parasitological examinations (modified Knott test for circulating microfilariae and SNAP Test IDEXX for circulating D. immitis antigen) had been used for diagnosing D. immitis and HWD. All dogs were treated with an alternative therapy for HWD (oral doxycycline 10 mg/kg b.w., once daily for 6 weeks, then alternately 4 weeks without and 2 weeks with the medication, and oral ivermectin 6-14 µg/kg b.w., every 2 weeks). The dogs blood sera at the moment of HWD diagnosis, during and at the end of therapy were frozen for further quantifying of TNF-α (Canine TNF-alpha ELISA kit, Thermo scientific). At the moment of HWD diagnosis TNF-α was detected in 9 dogs (7.21±12.44 pg/ml). Concentration of TNF-α was not significantly change during the therapy, neither related to the level of D. immitis antigen nor to antigen level changes. The alternative therapy for HWD has no influence on TNF-α concentration dynamics.

First molecular detection of shiga toxin-producing Escherichia coli in dogs from serbia: a potential threat to human health?

Shiga toxin­producing Escherichia coli (STEC) are considered one of the most significant E. coli pathotypes transmitted by food, causing life­threatening conditions in children and elderly people. The aim of this study was to investigate the presence and determine the prevalence of STEC in dogs in Serbia by conventional PCR method, targeting three major virulence genes (stx1, stx2, and eae). The overall percentage of positive samples was 12.87% (13/101), with the stx2 gene, the more potent of the two toxins, found in all the positive samples. The finding of eae gene in combination with stx genes (8/13) within the same genetic pool implies the potential presence of enterohemorrhagic E. coli or the potential emergence of these strains, considering an efficient mechanism of horizontal transfer of three major virulence genes. Our results also highlight dogs' lifestyle as a risk factor for STEC colonisation. These E. coli strains, according to our results, are more likely to be found in dogs living outdoors than those kept in house. Due to significant prevalence of STEC in dogs determined in this research and due to close contact between dogs and humans, dogs could be considered a source of human infections.

Subcutaneous Taenia crassiceps Cysticercosis in a Ring-Tailed Lemur (Lemur catta) in a Serbian Zoo.

BACKGROUND: Different rodent species serve as natural intermediate hosts for carnivore tapeworm Taenia crassiceps. However, this cestode occasionally infects various dead-end hosts including humans and other primates and may cause serious pathological implications with potentially fatal outcome. In this paper, we present subcutaneous cysticercosis caused by T. crassiceps, found in a previously healthy 17-years-old male ring-tailed lemur (Lemur catta) in a Serbian Zoo. CASE PRESENTATION: The animal was presented to a veterinarian with a history of periarticular subcutaneous swelling in medial right knee region. After fine needle aspiration revealed cycticerci-like structures, a surgery was performed for complete extraction of the incapsulated multicystic mass containing numerous cysticerci. Collected material was sent for parasitological, histological and molecular analysis. One month after surgery, the lemur died due to respiratory failure unrelated to cysticercosis. Based on morphological features of large and small hooks and characteristic proliferation of cysticerci, a metacestode of T. crassiceps was identified, which was confirmed after sequencing of obtained amplicons and comparing them to the GenBank database. CONCLUSIONS: This is one of the few reported cases of T. crassiceps cysticercosis in a ring-tailed lemur, and the first one in Serbia. This endangered species seem to be more sensitive for T. crassiceps than other non-human primates, which represents serious conservation challenge for captive animals. Due to zoonotic nature of the parasite, challenging diagnosis, severity of the disease, difficult treatment and possible fatalities, high biosecurity measures are of particular importance, especially in endemic regions.

Subtyping Blastocystis in pigs and humans revealed unusual avian-specific subtype ST6 in humans in Serbia.

Blastocystis is a common protist colonizing the gastrointestinal tract of humans and various animals. The first subtyping of Blastocystis isolates in pigs and humans in Serbia revealed unusual avian-specific subtype ST6 in humans. In total, 48 pig faecal specimens collected on seven pig farms and 50 human faecal specimens positive to Blastocystis by microscopic examination were selected for the study. Eleven randomly selected PCR-positive pig samples and 10 samples from human patients (with gastrointestinal complaints) were subjected to SSU rDNA sequencing. Three subtypes were identified (ST3, ST5 and ST6) by phylogenetic analysis. ST5 was found in all pig samples; while in human samples, we detected ST3 and ST6. The latter subtype is relatively uncommon in Europe and highly adapted to avian hosts; therefore, the possibility of sporadic zoonotic transmission to human patients should not be ignored.

Comparison of Conventional and Molecular Diagnostic Techniques for Detection of Blastocystis sp. in Pig Faeces.

BACKGROUND: Blastocystis is a common protist colonizing the gastrointestinal tract of humans and various animals. Pigs have been suggested to be a reservoir for human Blastocystis infections because of high prevalence of the parasite in these animals and the presence of zoonotic subtypes. Nevertheless, epidemiological data is often misinterpreted due to the lack of standard diagnostic procedures. This study aimed to compare the sensitivity of different diagnostic techniques in detection of Blastocystis sp. in pigs. METHODS: Overall, 48 individual faecal samples were collected from pigs reared in an intensive farming system (Autonomous Province of Vojvodina, Serbia) and were tested by microscopic examination of direct wet mount, in vitro cultivation in modified Jones' medium and conventional PCR for rRNA gene. RESULTS: Xenic in vitro cultivation in Jones' medium showed higher sensitivity than direct wet mount when we compared it with PCR. Namely, the estimated sensitivity of direct wet mount was 46.15%, while the sensitivity of in vitro cultivation was 84.62%. CONCLUSION: Low sensitivity of conventional parasitological compared to molecular methods is proven. Thus, reports on prevalence that rely solely on microscopy of faecal samples (unprocessed or concentrated) are probably underestimating the true prevalence of the parasite.

Prevalence of quinolone resistance and mutations in the topoisomerase genes in Salmonella enterica serotype Enteritidis isolates from Serbia.

The prevalence of quinolone resistance was studied in Salmonella enterica serotype Enteritidis isolates collected during 2005-2010 in Southern Backa County, Serbia. A total of 878 clinical isolates were examined, among which 19 (2.2%) nalidixic acid (NAL)-resistant S. Enteritidis were detected by selection on agar plates containing 64 mg/L NAL. Antimicrobial susceptibility of the isolates was tested by the agar dilution method. According to Clinical and Laboratory Standards Institute (CLSI) breakpoints, ciprofloxacin (CIP) resistance was not present in the strains. Multiple drug resistance was rare, and resistance to NAL was most often present as a single resistance property. All but one NAL-resistant S. Enteritidis showed reduced susceptibility to CIP [i.e. minimum inhibitory concentration (MIC)≥0.125 mg/L]. This isolate of human origin had a CIP MIC of 0.064 mg/L and DNA sequencing revealed that it contained an Asp87Gly gyrA mutation. Most of the remaining isolates had MICs for NAL and CIP of 256 mg/L and 0.256 mg/L, respectively. Mutations in the Asp87 codon resulted in substitutions to Asn in most of the isolates, but Asp87Gly and Ser83Phe exchanges were also detected. No mutations were present in the gyrB, parC or parE genes. Although CIP resistance was absent, reduced susceptibility characterised by mutations in gyrA was apparent among S. Enteritidis isolates from Serbia.

[Sensitivity of bacteria to antimicrobial drugs and interpretation of results].

INTRODUCTION: The discovery of antimicrobial drugs was a turning point in the permanent conflict between the mankind and microorganisms. However, due to the wide use and misuse of antibiotics in therapy and prophylaxis of infections the mankind is threatened by an alarming rise in the resistance of bacteria to drugs. Will this phenomenon turn us back to the pre-antibiotic era? DISCUSSION: The increasing resistance of bacteria has become a global public health problem: bacteria are showing a remarkable capacity to develop different mechanisms and avoid drug effect. Mechanisms of resistance are numerous and various: production of beta-lactamases (Ambler class A): TEM-I, TEM-2 and SHV-1 and mutants of classical enzymes with extended spectrum (ESBL) (e.g. in Klebsiella spp.) which results in the resistance to the 3rd generation cephalosporines and new metallo-beta-lactamases among Pseudomonas and Acinetobacter (resistance to carbapenems). The alteration of the target enzymes (PBP) leads to the Staphylococci resistance to methicillin and the responsible gene is mecA gene). The alteration of DNA gyrase due to the mutations of gyrA, gyrB, parC genes (accumulation of multiple mutations) results in the development of resistance to fluoroquinolones); and the active efflux system - "pumping out" of the drug from the bacterial cell leads to the resistance of a wide spectrum of different antibiotics. In order to choose the most efficient drug for therapy, it is necessary to investigate susceptibility of bacteria to antimicrobial agents. For that purpose, a disc-diffusion method according to CLSl standard procedure is performed. For invasive strains it is often necessary to determine minimal inhibitory concentrations (MIC) of antimicrobials. The methods that are in use are agar-dilution methods, E-test and automated MIC determination by VITEK 2 system. CONCLUSION: By molecular-biological methods it is possible to identify the mechanisms of resistance and detect the specific genes behind it (mecA gene). The targeted therapy prevents compromising of antibiotics valuable in treatment of severe infections (carbapenems).

Significance of screening tests in diagnosis of infectious mononucleosis.

The investigation included 91 patients in who an acute or previous EBV infection was established by ELISA test. All patients were also subjected to the Paul-Bunnell-Davidsohn test, while 20 patients were tested by the rapid screening test Clearview IM. The diagnosis of acute infective mononucleosis was in 61 patients (67%) confirmed by the Elisa test, and in 12 patients (19.67%) by the Paul-Bunnell-Davidsohn test, while the rapid screening test Clearview IM demonstrated too low a detection of heterophile antibodies. The rapid screening test was not reliable. In 25% cases, the test was invalid, at early infection stages the rapid test failed to diagnose any case of the EBV virus infection. Paul-Bunell-Davidsohn was often negative, especially with young children. Therefore, priority should be given to virology tests based on the detection of specific antibodies to EBV antigen.