Long Time No Hear, Magnificent Wohlfahrtia! Morphological and Molecular Evidence of Almost Forgotten Flesh Fly in Serbia and Western Balkans.

The "beautiful viviparous fly", Wohlfahrtia magnifica, may have a magnificent appearance due to its striking morphology; however, it is a potentially deadly agent of obligate traumatic myiasis in humans and animals, with a serious impact on welfare and economics. The fly is found across the Palearctic realm, including the Western Balkan region, with reports from former Yugoslavian countries from the first half of the 20th century. In this paper, a recent case of wohlfahrtiosis recorded in Northern Serbia is evidenced using morphological and molecular techniques. Larvae were collected from two adult sheep with severe hoof myiasis and two young sheep with genital and interdigital myiasis. Morphological identification was performed for adults bred from the infested vulva and third-stage larvae (L3) collected from the hoof wounds, supported with barcoding sequences of the COI gene obtained from larval pairs from the hoof wounds of older and the genitalia of younger sheep. W. magnifica was identified according to the appearance of male fly terminalia and the morphology of L3, which was confirmed after the comparison of representative sequences of the COI gene (deposited in GenBank™ under accession numbers MT027108-MT027114) to those available in GenBank™. This finding represents the first reported case of wohlfahrtiosis in the Western Balkans in 80 years, highlighting the need to re-inform relevant stakeholders to achieve adequate disease control.

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.

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.

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.

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 bio-assays using a multiplex multilocus nested polymerase chain reaction-restriction fragment length polymorphism (Mn-PCR-RFLP) method with seven markers (GRA6, alt. 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.

Disodium Cromolyn and Anti-podoplanin Antibodies Strongly Inhibit Growth of BHK 21/C13-derived Fibrosarcoma in a Chick Embryo Chorioallantoic Membrane Model.

AIM: To characterize baby hamster kidney fibroblast (BHK 21/C13) cells and test the effects of antibodies against podoplanin and disodium cromolyn on BHK 21/C13 cell line-derived tumors grown on chick embryo chorioallantoic membrane (CAM). MATERIAL AND METHODS: BHK 21/C13 cell-derived fibrosarcomas developed in hamsters were implanted on CAM and treated with anti-podoplanin antibodies and disodium cromolyn. BHK 21/C13 cell immunophenotype was assessed. RESULTS: Fibrosarcoma cells were positive for vimentin, CD117, smooth muscle actin, vascular endothelial growth factor epidermal growth factor receptor, homebox prospero gene 1 and negative for platelet-derived growth factor B, neuron-specific enolase, S100, CD34, Ewing sarcoma and podoplanin. CAM-grown fibrosarcomas were highly sensitive to disodium cromolyn and anti-podoplanin antibodies. CONCLUSION: Immunophenotyping BHK 21/C13 cells and their response to drugs represent the first step in revealing cell line utility and a reliable tool for experimental cancer research.

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.

Detection of Toxoplasma gondii in naturally infected domestic pigs in Northern Serbia.

Insufficiently cooked pork is considered as an important source of human infection with Toxoplasma gondii. The aim of our study was to investigate the presence of T. gondii in pigs intended for human consumption from Northern Serbia. Blood and diaphragm samples were collected from 182 naturally infected market-weight pigs, originating from both commercial farms and smallholdings. Sera were examined using modified agglutination test (MAT), and diaphragms from seropositive, as well as from some MAT-negative pigs, were bioassayed in mice. In addition, digests were examined for the presence of T. gondii DNA using a real-time polymerase chain reaction (qPCR) which was targeted at the 529 bp repetitive element of the T. gondii genome. The overall seroprevalence of T. gondii in pigs was 17% (31/182), with no difference between pigs from large commercial farms (17.8%) and those raised on smallholdings (16.3%). However, the seroprevalence in farm pigs was largely influenced by the findings on a single farm, where all examined animals tested positive. Parasites and/or parasite DNA were detected in the tissues of 15 of the 45 (25 seropositive and 20 seronegative) animals examined by either direct method. Tissue cysts were isolated in eight bioassays and an additional bioassay was positive by serology; all nine were confirmed positive by qPCR. All positive bioassays originated from seropositive pigs, but no correlation was observed between isolation rate and antibody titer. T. gondii DNA was detected in diaphragm tissues of eight pigs, of which three were seronegative. The results of our study provide further evidence for pork as a source of human T. gondii infection.

Moving the boundaries to the South-East: first record of autochthonous Angiostrongylus vasorum infection in a dog in Vojvodina province, northern Serbia.

BACKGROUND: Angiostrongylus vasorum is a cardiopulmonary canine nematode, potentially fatal to its host. In the last decade, there has been an increasing number of autochthonous cases in areas previously considered non-endemic. However, information about the parasite's occurrence and distribution among Central and Eastern (Southeastern) European countries are scarce. This paper reports the first recorded case of autochthonous A. vasorum infection in a hunting dog from Serbia. FINDINGS: In March 2013, a female hunting dog was presented to a veterinary clinic in Novi Sad, Serbia, for examination of a chronic skin problem. The dog had no history of respiratory or cardiovascular diseases. Faecal and urine samples were collected and examined for the presence of parasite ova/cysts. A modified Baermann test detected 8.8 larvae per gram of faeces. Based on their overall body length (mean 381.7 ± 15.9 µm; range from 342.5 to 404.3 µm; n = 12) and characteristic tail morphology, they were identified as the first-stage larvae of A. vasorum. CONCLUSIONS: The spread of A. vasorum to the southeast of Europe is further confirmed after finding autochthonous infected dog from Serbia. Therefore, veterinary professionals in Serbia should consider A. vasorum in differential diagnosis of dogs.

Neospora caninum antibodies in dairy cows and domestic dogs from Vojvodina, Serbia.

Neospora caninum, the causative agent of neosporosis, is a protozoan parasite responsible for high rate of abortion in cattle worldwide. In dogs, consequences of infection vary from severe neuromuscular disorders to asymptomatic infection and shedding of environmentally resistant oocysts. In this study, we determined the occurrence of N. caninum antibodies in dairy cattle and dogs in Vojvodina (Northern Province of Serbia) and possible risk factors. N. caninum antibodies were found in 15.4% (55/356, CI 95%:12.0-19.6) of cows and 17.2% (17/99, CI 95%: 10.8-26.2) of dogs. Cows from smallholdings showed significantly greater odds (OR = 5.28, CI 95%: 2.0-13.6, p = 0.0006) of being seropositive in comparison to the farm cows. Epidemiological importance of results is discussed.

High infection rate of zoonotic Eucoleus aerophilus infection in foxes from Serbia.

The respiratory capillariid nematode Eucoleus aerophilus (Creplin, 1839) infects wild and domestic carnivores and, occasionally, humans. Thus far, a dozen of human infections have been published in the literature but it cannot be ruled out that lung capillariosis is underdiagnosed in human medicine. Also, the apparent spreading of E. aerophilus in different geographic areas spurs new studies on the epidemiology of this nematode. After the recognition of the first human case of E. aerophilus infection in Serbia, there is a significant merit in enhancing knowledge on the distribution of the nematode. In the present work the infection rate of pulmonary capillariosis was investigated in 70 red foxes (Vulpes vulpes) from the northern part of Serbia by autopsy. The estimated infection rate with Eucoleus aerophilus was 84%. In contrast, by copromicroscopic examination only 38% of foxes were positive. In addition, 10 foxes were investigated for the closely related species in nasal cavity, Eucoleus boehmi, and nine were positive. Our study demonstrates one of the highest infection rates of pulmonary capillariosis in foxes over the world.

Adult human case of toxocariasis with pulmonary migratory infiltrate and eosinophilia.

INTRODUCTION: Toxocariasis is a zoonosis which is in Serbia characterized with a very high infection rate of dogs and excessive contamination of the soil with the eggs of Toxocara canis, the agent of the disease. Toxocara-induced infections have in recent years been established in a few hundreds of children, but toxocariasis has rather rarely been diagnosed in adults. CASE REPORT: We reported toxocariasis (visceral larva migrans) in an adult, manifested by migratory pulmonary infiltrates and positive serological test finding to Toxocara. CONCLUSION: Human toxocariasis is a rare disease in adults, therefore it should be considered in adult patients presented with eosinophilia and migratory pulmonary infiltrates.

Borrelia-like organism in heart capillaries of patient with Lyme-disease seen by electron microscopy.

A case of a patient who developed an acute myocarditis due to Lyme disease is reported. An increased serum antibody titer to Borrelia burgdorferi suggested a diagnosis and in addition of basic clinical methods, endomyocardial biopsy performed and analyzed by transmission electron microscopy. The lumen of myocardial capillaries was founded mostly filled with detritus and fibrin precipitate, between them several bacterial fragments were identified. The electron-microscopic characteristics of the microorganisms in this specimen, revealing irregularly coiled appearance and consistent thickness of 0.2 µm, correspond to the spiral-like structure of Lyme disease borrelia. The presence of fibrin deposits on the capillary endothelium and necrosis of myocardiocytes, suggests that the cardiopathy in our patient was represent borrelia-mediated damage of the heart microcirculation.

Pulmonary capillariasis miming bronchial carcinoma.

Pulmonary capillariasis is a zoonotic disease caused by the cosmopolitan nematode Capillaria aerophila, which circulates among wild carnivorous and omnivorous mammals. Only 11 cases have been documented to date. We describe a cryptic case of pulmonary capillariasis in a Serbian woman that resembled a bronchial carcinoma.

Dilemmas in diagnostics of disseminated echinococcosis.

A case of disseminated peritoneal echinococcosis localized in the small pelvis and ovary was diagnosed in a 12-year old girl. Secondary hydatid echinococcosis following the rupture of a primary liver cyst was presumed. A morphological differential diagnosis of the unilocular and multilocular or alveolar echinococcus in biopsy material was considered. Finding of a protoscolex or at least chitin hooklets suggests a hydatid echinococcus, whereas alveolar or multilocular echinococcus does not produce protoscoleces in humans, hence the absence of their hooklets in the material is considered an important morphological diagnostic criterion.

[Current differential diagnosis of hypereosinophilic syndrome].

INTRODUCTION: Hypereosinophilic syndrome (HES) is a group of idiopathic disorders associated with single or multiple organ system dysfunction. HES must be distinguished from reactive eosinophilia in parasitic infections, allergic diseases, and especially from hematological diseases of clonal origin. REACTIVE EOSINOPHILIA DUE TO INFECTIOUS AND PARASITIC DISEASES: Tissue helminth infections, especially toxocariasis, cause severe and long-standing hypereosinophilia. Despite specific therapy, eosinophilia may persist for over a year after diagnosis, and decreases slowly. Other helminth infections, such as trichinellosis, strongyloidosis, and rarely taeniasis and cysticercosis may also be diagnosed in patients with eosinophilia. HEMATOLOGIC AND OTHER NEOPLASTIC DISEASES: Numerous neoplastic diseases, like Hodgkin's and other malignant lymphomas, myeloproliferative diseases, systemic mastocytosis etc., may be associated with marked eosinophilia. We had two patients with clinical and histological features resembling chronic eosinophilic leukemia, and many others with T-cell lymphoma, planocellular or adenocarcinoma etc. where eosinophilia persisted DRUG-INDUCED EOSINOPHILIA: Drugs associated with eosinophilia include penicillins, tetracyclines, especially minocycline, clarithromycin, tetrazepam, mefloquine, and many, others. Toxins associated with L-tryptophan cause eosinophilia-myalgia syndrome and toxic oil syndrome, also belonging in this group. Treatment includes drug discontinuation and administration of corticosteroids. HYPEREOSINOPHILIA WITH ORGAN DYSFUNCTION: Many severe diseases, such as sarcoidosis, Churg-Strauss syndrome, pemphigus vulgaris, eosinophilic gastrointestinal diseases, inflammatory bowel disease and many others are associated with hypereosinophilia and target organ damage, e.g. involvement of the heart, lungs, skin, or nervous tissue. CONCLUSION: Eosinophilia can be classified as either familial or acquired. Hypereosinophilic syndrome is a subcategory of idiopathic eosinophilia. If the differential diagnosis of hypereosinophilia fails to resolve the etiology succesfully, the diagnosis of idiopathic HES remains.

[Fatal outcome after postexposure rabies vaccination in a patient with Parkinson's disease]. / Smrtni slucaj tokom postekspozicione vakcinacije protiv besnila obolelog od Parkinsonove bolesti.

INTRODUCTION: In Serbia and Montenegro postexposure rabies vaccination is performed using five doses of rabies vaccine with a potency of 2.5 I.U. It is given on 0, 3rd, 7th, 14th and 28th day, combined with human rabies immunoglobulin with the first dose. Modern rabies vaccines produced in cell cultures rarely cause neurologic complications, among which Guillain-Barre syndrome and parkinsonism. CASE REPORT: The authors report a case of a 78-year-old woman with a documented five-year history of Parkinson's disease, who was bitten by a rabid cat. Twelve hours later, when the rabies infection of the cat was confirmed by an immunofluorescence test, the patient received the first dose of rabies vaccine Verorab (Aventis), a cell culture vaccine, together with the human rabies immunoglobulin produced in Belgrade. After the third dose of rabies vaccine, the symptoms of Parkinson's disease progressed and vaccination was interrupted. However, one month later, the patient died with predominantly neurological symptoms. As the patient died at the time when incubation of rabies might have been expected, autopsy and rabies diagnostics were performed. AUTOPSY AND PATHOHISTOLOGIC FINDINGS: The autopsy and pathohistologic findings from the specimens treated with routine hematoxylin and eosin staining, together with immunofluorescence test, excluded rabies as a cause of death and revealed neurodegenerative changes typical for Parkinson's disease. Using two different fluorescent rabies antibodies, we performed a direct immunofluorescence antibody tests, but no rabies antigens were detected. However, in histologic slides of the brain stem, large intracytoplasmic inclusions were found in some neurons, identified as Lewy bodies characteristic for Parkinson's disease. CONCLUSION: Parkinson's disease, with its complications, was the cause of death of the patient bitten by a rabid cat. Furthermore, the coincidence of the progression of Parkinson's disease symptoms, at the time of postexposure rabies vaccination, points to the vaccine as a possible contributing factor to aggravation of the disease and lethal outcome.