Albert Marinculić

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.

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.

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 excretory-secretory antigens for the serodiagnosis of swine trichinellosis

Groups of hog sera from endemic and non-endemic areas for swine trichinellosis in Yugoslavia were tested by ELISA using excretory-secretory (ES) antigens collected from T. spiralis muscle larvae maintained in vitro for 24, 48 or 72 h. The 24-h ES had the highest level of specificity for T. spiralis infection. Antigen preparations recovered after 48 or 72 h yielded an increasing rate of false-positive reactions. Additional antigens occurred in the 48- and 72-h ES preparations as determined by gel electrophoresis and monoclonal antibody binding. The occurrence of false-negative reactions was directly correlated with T. spiralis worm burdens. Hogs with muscle larvae densities greater than 10 larvae per gram were all positive by ELISA. Among 17 hogs with less than 10 larvae per gram, only one hog was negative by ELISA with 24-h ES antigen; the false-negative rate was higher with 48- and 72-h ES. These results show that ES antigen produced during the first 24 h of in vitro cultivation is highly specific for the immunodiagnosis of swine trichinellosis.

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.

Validation of a Western Blot for the detection of anti-Trichinella spp. antibodies in domestic pigs

Trichinellosis is a zoonotic disease in humans caused by Trichinella spp. According to international regulations and guidelines, serological surveillance can be used to demonstrate the absence of Trichinella spp. in a defined domestic pig population. Most enzyme-linked immunosorbent assay (ELISA) tests presently available do not yield 100% specificity, and therefore, a complementary test is needed to confirm the diagnosis of any initial ELISA seropositivity. The goal of the present study was to evaluate the sensitivity and specificity of a Western Blot assay based on somatic Trichinella spiralis muscle stage (L1) antigen using Bayesian modeling techniques. A total of 295 meat juice and serum samples from pigs negative for Trichinella larvae by artificial digestion, including 74 potentially cross-reactive sera of pigs with other nematode infections, and 93 meat juice samples from pigs infected with Trichinella larvae were included in the study. The diagnostic sensitivity and specificity of the Western Blot were ranged from 95.8% to 96.0% and from 99.5% to 99.6%, respectively. A sensitivity analysis showed that the model outcomes were hardly influenced by changes in the prior distributions, providing a high confidence in the outcomes of the models. This validation study demonstrated that the Western Blot is a suitable method to confirm samples that reacted positively in an initial ELISA.

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, Fishers 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.

A Case of Visceral Leishmaniosis in a Gray Wolf (Canis lupus) from Croatia

The southern habitats of Croatias gray wolf (Canis lupus) population are found in central and southern parts of Dalmatia. This region is recognized as an endemic region for canine visceral leishmaniosis, caused by Leishmania infantum. In November 2003, a 4-yr-old male gray wolf was found dead in the northwestern border of this endemic region. Pathologic and parasitologic analysis, confirmed by polymerase chain reaction, indicated that lesions associated with infection by Leishmania infantum are, in this case, typical for visceral leshmaniosis commonly described in dogs. Review of the literature suggests that this is the first reported case of gray wolf death due to lesions caused by L. infantum.

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.

γδTCR+ intestinal intraepithelial lymphocytes (i-IEL) in reaction against intestinal nematode Trichinella spiralis

To assess the gamma delta TCR T cells in the control of the timing of the mucosal response to enteric parasitic infections, we used C57BL mice, orally infected with 200 viable T. spiralis larvae. The small intestine, spleens and Peyers patches (PP) were excised on 1, 4, 7, 14, 21 and 29 postinfection days (p.i.) for immunophenotyping and histological studies. Uninfected mice served as control. Characterization of isolated lymphocytes of C57BL control mice, confirmed that T cell immunophenotype differs in spleen, PP and i-IEL. Practically all i-IEL were CD3+ cells (83%). In addition, most of the i-IEL expressed Ly-2 (65%). Among the i-IEL, the level of gamma delta TCR+ cells was significantly higher (29%) than that found in spleen (3%) and PP (3%). The expression was high on CD3+ and Ly-2+ (26 and 21%, respectively) and low on L3T4+ i-IEL (< 1%). During T. spiralis infection alpha beta TCR+ CD3+, gamma delta TCR+ CD3+ and gamma delta TCR+ Ly-2+ i-IEL increased on day 4 and 7. However, infected mice displayed a reduction in i-IEL number from 14 to 29 p.i. day. At the same time the proportion of gamma delta TCR on spleen Ly-2+ and on PP CD3+ and Ly-2+ cells increased on 14 and 21 p.i. day. Adult worms were expelled from the gut by day 14. Thus, the kinetics of gamma delta TCR+ i-IEL, but not spleen and PP gamma delta TCR, corresponded to the kinetics of worm expulsion in C57BL mice. Most murine i-IEL of the gamma delta T cell lineage tend to be cytolytic when activated. We speculated that gamma delta T cells of i-IEL during the early stages of infection recognize and eliminate damaged epithelial cells generated by parasite antigens, simultaneously accelerating the worm expulsion.