Working group for trichinellosis--a way of systematic prevention, control and eradication of trichinellosis in the Republic of Croatia.

At the end of the last century, human trichinellosis was an important public health problem in the eastern parts of Croatia. Moreover, the majority of clinically infected people were registered in Vukovar-Srijem County (up to 60% of all human cases registered in Croatia). Also, 95% of all Trichinella positive swine carcasses originated from Vukovar-Srijem County. Beside the health threat, trichinellosis implied not only notable economic expenses but also threatened to endanger traditional way of life and eating habits. In order to reduce all negative consequences of the disease, a multidisciplinary Working group for trichinellosis was founded. The group consisted of scientists and experts from different fields of work, who helped and significantly contributed to minimizing the threats of trichinellosis as well as to maintaining and preserving the method of traditional processing and consumption of swine meat. The members, the methods and the results of the Working group activities will be discussed in this paper.

[Ocular dirofilariasis: a case report]. / Okularna dirofilarioza--prikaz bolesnice.

A case of ocular dirofilariasis in a female patient is presented. The zoonosis caused by parasites of the genus Dirofilaria is relatively rare in humans, with a higher incidence in south and central Europe, Asia and Africa. In Europe, dirofilariasis is mostly caused by the species Dirofilaria repens. In the past 50 years, the number of individuals involved has been on an increase, with about 780 cases reported in the literature to date. Dirofilaria is a parasite found in the dog, cat, racoon and bear. The parasite replicates in the animal's body and enters circulation in the form of microfilariae. These microfilariae reach the insect's digestive tract and are transmitted to another animal or human with subsequent mosquito bites. When transmitted to humans, the parasite is found in the skin and subcutaneous tissue, mucous membranes, and less frequently visceral organs. Concerning ocular involvement, infections of the eye and adnexa oculi and tumorous noninfectious growth of eyelid or orbit have been described to date. The symptoms of the disease vary and include local pain, proptosis, diplopia, palpebral and conjunctival edema, redness, feeling of foreign body, and impaired vision. The diagnosis is generally made by histologic identification of the parasite micro- and macroscopic characteristics, Dirofilaria DNA analysis by the method of polymerase chain reaction, and serology (ELISA) demonstrating the presence of Dirofilaria antibodies in serum. Treatment includes surgical excision of the parasite as an appropriate and efficient therapeutic procedure. A 76-old-female patient presented to outpatient ophthalmology clinic for occasional sensation of pain, rubbing and redness in her right eye. Initial therapy was introduced, resulting in short-lasting improvement. In two weeks, the patient was re-examined for recurrence of discomforts. Slit lamp examination performed temporally revealed a whitish motile, live parasite under the injected and chemotic bulbar conjunctiva. Upon surgical extirpation of the parasite, the diagnosis of dirofilariasis was verified by microbiologic identification.

Evaluation of a new commercial enzyme-linked immunosorbent assay for the detection of porcine antibodies against Trichinella spp.

Trichinellosis is a zoonotic disease that is caused by the nematode Trichinella spp. Both European Union regulations and guidelines from the World Organization for Animal Health foresee the possibility of conducting serological surveillance for Trichinella spp. A newly developed commercial enzyme-linked immunosorbent assay (ELISA) was evaluated against 2 existing diagnostic techniques: an in-house ELISA and an in-house Western blot. A total of 875 Trichinella larva-negative samples of pigs and 93 Trichinella larva-positive samples of both naturally and experimentally infected pigs were included in the study. Bayesian modeling techniques were used to correct for the absence of a perfect reference test. The sensitivity and specificity of the commercial ELISA was 97.1-97.8% and 99.5-99.8%, respectively. Sensitivity analysis demonstrated high stability in the models. In a serological surveillance system, ELISA-positive samples should be tested by a confirmatory test. The Western blot is a suitable test for this purpose. With the use of the results of the models, the sensitivity and specificity of a test protocol in both ELISA and Western blot were 95.9% and 99.9%, respectively. The high sensitivity and specificity were achieved with a lower limit of detection than that of the routine artificial digestion test, suggesting that serological surveillance is a valuable alternative in surveillance for Trichinella spp. in pig production.

Sequence analysis of ribosomal and mitochondrial genes of the giant liver fluke Fascioloides magna (Trematoda: Fasciolidae): intraspecific variation and differentiation from Fasciola hepatica.

Complete sequences of ribosomal and mitochondrial genes of the giant liver fluke Fascioloides magna are presented. In particular, small subunit (18S) and internal transcribed spacers (ITS1 and ITS2) of the ribosomal gene (rDNA), as well as cytochrome c oxidase subunit I (cox1) and nicotinamide dehydrogenase subunit I (nad1) of the mitochondrial DNA (mtDNA), were analyzed. The 18S and ITS sequences were compared with previously published sequences of the liver fluke Fasciola hepatica. Fixed interspecific genetic differences were determined that allow molecular differentiation of F. magna and F. hepatica using either the PCR-RFLP method or PCR amplification of species-specific DNA regions. Additionally, intraspecific sequence polymorphism of the complete cox1 and nad1 mitochondrial genes in geographically distinct F. magna populations was determined. Based on the sequence divergences, short (< 500 bp) variable regions suitable for broader biogeographical studies of giant liver fluke were designed.

Evaluation of factors affecting the efficacy of treatment against Fascioloides magna in wild red deer population.

Deer fascioloidosis is a serious and potentially fatal disease caused by the non-native trematode Fascioloides magna. Infections of red deer with F. magna in Croatia have been reported for the first time in 2000 in the Baranja region. Subsequently, the disease spread throughout the Eastern parts of the country, involving all 3 deer species (red, roe, and fallow) and mouflons. Within the disease control programme (DCP), livers from all shot deer were thoroughly analysed and all detected trematodes and gross lesions were counted and categorized. Prevalence of positive animals, in this study for Spacva region, in the period ranging from 2007 to 2012 was 36.42% (46.39% when fawns are not considered). Epidemiological analysis was applied to evaluate risk factors and disease patterns at the population level with the aim to understand factors with negative influence on therapeutic effect. Each demographic variable was tested at the seasonal, individual and location level. Model for pathological lesions suggested that the likelihood of lesions was dependent on age (p = 0.003). We did not find any locality or sex related significant differences. Finally, environmental characteristics and migratory patterns were analysed using Geographic Information System (GIS) and showed that Spacva region represents an epidemiological unit for red deer fascioloidosis.

Alternative approaches for the control of gastrointestinal nematodes in sheep farming: a review.

Gastrointestinal nematodes (GIN) are a serious health problem and represent the most significant constraint in sheep grazing operations. Problems tend to be worse in organic sheep farming systems, as a consequence of a less restricted access of animals to outdoor environment with a higher exposure to infective larvae. In domestic animals, GIN are effectively controlled by an aggressive prophylactic administration of commercially available anthelmintics. As a consequence to a common overdose and misuse of readily available antiparasitic treatments, there is an inevitable development of populations of GIN resistant to all major classes of anthelmintics. Also, the control of GIN that is based entirely on the anthelmintic use, threatens sustainability of the sheep farming worldwide. The combination of the optimized use of anthelmintic drugs and alternative approaches seem to be a reasonable choice in sustainable parasitic control programs that offer a substantial reduction of anthelmintic treatments and conservation of anthelmintic efficacy. In that aspect, a "targeted selective treatment (TST)" directed towards animals clinically diagnosed with GIN, seems to be an effective approach to leave some parasite populations unexposed to anthelmintics (refugia) and to reduce development of anthelmintic resistance. Also, many current research efforts aim to find and validate sustainable non-chemotherapeutic approaches to GIN control, including changes in grazing management, optimized nutrition, dietary supplementation, consumption of plants with anthelmintic properties, biological control by nematophagous fungi, copper oxide wire particles (COWP), and homeopathic treatments. This manuscript outlines (outlines) and discusses relevant alternative approaches for GIN control in modern sheep farming systems.

Molecular characterisation of Babesia canis canis and Babesia canis vogeli from naturally infected European dogs.

The morphologically small Babesia species isolated from naturally infected dogs in Europe, Japan, and US are described as Babesia gibsoni despite the fact that molecular techniques show that they should be assigned to two or three separate taxons. The morphologically large Babesia isolated from dogs in Europe, Africa, and US were generally classified as B. canis until it was proposed to distinguish three related, albeit genetically distinct subspecies of this genus, namely B. canis canis, B. canis rossi, and B. canis vogeli. The insight into the molecular taxonomy of canine piroplasms is, however, limited because only partial small subunit ribosomal RNA (ssrRNA) sequence data exist for two species from the B. canis group. In this work, we molecularly characterised natural Babesia infections in 11 dogs from Croatia, France, Italy, and Poland. These infections were diagnosed as caused by B. canis canis and B. canis vogeli based on the analysis of the complete sequence of the ssrRNA genes. Phylogenetic analysis confirmed that the large Babesia species of dogs belong the to the Babesia sensu stricto clade, which includes species characterised by transovarial transmission in the tick vectors and by exclusive development inside the mammalian host erythrocytes. The new data facilitate the reliable molecular diagnosis of the subspecies of B. canis.

The epidemiological investigation of Trichinella infection in brown rats (Rattus norvegicus) and domestic pigs in Croatia suggests that rats are not a reservoir at the farm level.

Whether the brown rat (Rattus norvegicus) is a reservoir of Trichinella spp. infection or merely an accidental host, which may be vector of Trichinella spp., continues to be debated. We estimated the prevalence of Trichinella sp. infection in brown rat populations and in domestic pigs in 2 villages in Croatia, where Trichinella sp. infection in pigs has been endemic in the past 10 yr. Trichinella spiralis larvae, identified by a multiplex polymerase chain reaction analyses, were the only species detected in both rats and pigs. In 2001 and 2002, 2,287 rats were collected on 60 farms with different levels of sanitation and with, or without, T. spiralis-infected pigs. The prevalence of infection in rats ranged from 0.2 to 10.7%. Infected rats were detected only on farms with T. spiralis-positive pigs and low sanitation or formerly with low sanitation (P = 0.007, Fisher's exact test), yet no infected rat was detected on farms with T. spiralis-negative pigs. The finding that no infected rat was found on farms with T. spiralis-negative pigs suggests that, in the investigated area, the brown rat is not a reservoir but only a victim of improper pig slaughtering.

Molecular epizootiology of canine hepatozoonosis in Croatia.

An epizootiological survey was conducted to investigate the prevalence of hepatozoonosis in a population of 924 apparently asymptomatic dogs from different regions of Croatia. DNA was isolated from canine blood and screening PCR on the 666 bp fragment of 18S rRNA revealed that 108 (11.8%) of dogs were infected. Positive samples were confirmed by partial sequencing of the 18S rRNA gene. The consensus sequences, derived from various sequence data sets, were compared with sequences of 18S ssrRNA of Hepatozoon spp. available in GenBank. The alignments revealed 106 Hepatozoon canis and two Hepatozoon sp. sequences. Among H. canis isolates, we found a certain amount of heterogeneity, while both Hepatozoon sp. isolates were identical to the Spanish isolate (Accession No. AY600625) from Clethrionomys glareolus. On the basis of eight commonly mutated nucleotide positions in the partial 18S rRNA gene sequence, we divided the H. canis isolates into five groups. The results obtained indicate a higher prevalence and significance of hepatozoonosis in Croatia than previously believed and demonstrate that the organisms belonging to H. canis that infect European dogs are genetically very heterogeneous.

Trichinella pseudospiralis in pig from Croatia.

Trichinella spiralis and Trichinella britovi are species that are frequently found in domestic pigs and various sylvatic animals in Croatia. During routine trichinoscopy, non-encapsulated larvae were detected in the muscle tissue of a domestic pig. Artificial digestion revealed a larvae burden of 602 muscle larvae per gram of tissue. Tissue section analysis confirmed the presence of non-encapsulated larvae. Multiplex PCR identified the larvae as T. pseudospiralis. This observation is consistent with the reports of a local veterinary inspector who described the presence of non-encapsulated Trichinella in four individual cases over the last 2 years. This is the first report of T. pseudospiralis in Croatia and one of very few cases of T. pseudospiralis infection described in domestic pigs. The detection of non-encapsulated larvae stresses the need for implementation of artificial digestion instead of trichinoscopy for the detection and identification of Trichinella infections.

Trichinellosis in wolves from Croatia.

The aim of the present study was to investigate the prevalence of Trichinella infection in wolves (Canis lupus) in a 17,468 km(2) area in Croatia. Muscle samples were collected from 67 wolves between 1996 and 2007 and analyzed by artificial digestion. Muscle larvae were detected in 21 wolves (31%) and genotyped by multiplex PCR. Trichinella britovi was the predominant species confirmed in 90% (19 wolves) while Trichinella spiralis was detected in 9% (2 wolves). The presence of the so called "domestic"Trichinella species was a surprise since, to date, only T. britovi had been reported in wild animals in this region. The larval burdens in infected animals ranged from 0.3 to 45.9 larvae per gram. The prevalence of infected animals varied by geographic region; infected animals were found in the region of Gorski Kotar (20%) which has very similar environment to the region of Lika, where almost all wolves were found infected. Interestingly, this is the first report of infected wolves in Dalmatia.

Diversity of Babesia and Theileria species in symptomatic and asymptomatic dogs in Croatia.

Babesiosis, the disease caused by tick-borne hematozoan parasites of the genus Babesia, is particularly common in dogs, and is caused by several "large" species of Babesia, as well as by an increasing number of "small" species of Babesia, some of which appear to be more closely related to members of the genus Theileria. In this work, blood samples were collected from 848 randomly selected, asymptomatic dogs and from 81 symptomatic dogs, microscopically positive for Babesia, and characterised by PCR and sequence analysis of a fragment of the ssrRNA gene. A prevalence of 3.42% (29 of 848) was found in asymptomatic dogs and sequence analysis revealed the presence of Babesia canis canis in 20 dogs (69%), Babesia gibsoni in six dogs (21%), Babesia canis vogeli in two dogs (7%) and Theileria annae in one dog (3%). In the group of symptomatic dogs, which were all positive by PCR, B. canis canis was the predominant species (78 dogs, or 96%), followed by single infections with B. canis vogeli, Babesia caballi and Theileria equi. Our study has confirmed that dogs are infected with a wide range of both large and small piroplasm species and subspecies, including B. caballi and T. equi, two parasites usually found in horses. The detection of the pathogenic species B. canis canis and B. gibsoni in asymptomatic dogs indicates that the relationship between parasite species/subspecies and clinical signs of infection in dogs deserves further investigation. Finally, the identities of the tick vectors transmitting T. annae and B. caballi remain to be elucidated.

Trichinella diagnostics and control: mandatory and best practices for ensuring food safety.

Because of its role in human disease, there are increasing global requirements for reliable diagnostic and control methods for Trichinella in food animals to ensure meat safety and to facilitate trade. Consequently, there is a need for standardization of methods, programs, and best practices used in the control of Trichinella and trichinellosis. This review article describes the biology and epidemiology of Trichinella, and describes recommended test methods as well as modified and optimized procedures that are used in meat inspection programs. The use of ELISA for monitoring animals for infection in various porcine and equine pre- and post-slaughter programs, including farm or herd certification programs is also discussed. A brief review of the effectiveness of meat processing methods, such as freezing, cooking and preserving is provided. The importance of proper quality assurance and its application in all aspects of a Trichinella diagnostic system is emphasized. It includes the use of international quality standards, test validation and standardization, critical control points, laboratory accreditation, certification of analysts and proficiency testing. Also described, are the roles and locations of international and regional reference laboratories for trichinellosis where expert advice and support on research and diagnostics are available.

Antibodies against Leishmania cross-react with Crithidia luciliae: indirect immunofluorescence and Dot-ELISA study in dogs.

Leismaniasis is zoonotic parasitic disease distributed in many areas of Mediterranean basin. Because of its pathogenicity and difficulty to grow in vitro, we used Leishmania infantum as primary and Crithidia luciliae as secondary source of antigen. We compared 100 canine sera by indirect immunofluorescence and Dot-ELISA. Both atigens showed same results.