Halina Wędrychowicz

Prevalence of Babesia canis, Borrelia afzelii, and Anaplasma phagocytophilum infection in hard ticks removed from dogs in Warsaw (central Poland).

The purposes of this study were to specify the occurrence and prevalence of Babesia canis, Borrelia burgdorferi sensu lato, and Anaplasma phagocytophilum in ticks removed from dogs in Warsaw, and to determine the Borrelia species occurring in Ixodes ricinus ticks. Among 590 collected ticks, 209 were identified as I. ricinus, and 381 as Dermacentor reticulatus. DNA of B. canis was detected in 11% of D. reticulatus ticks. We found that 6.2% of I. ricinus ticks harbored B. burgdorferi s.l. specific DNA and 2.9% harbored A. phagocytophilum DNA. In these samples sequencing of the detected Borrelia amplicon confirmed infection with Borrelia afzelii genospecies. New sequences were submitted to the GenBank database (accession no. EU152128, EU152127, EU152126). This work is the first detection of B. afzelii and A. phagocytophilum in ticks from Warsaw, and the first survey for the prevalence of B. canis, B. afzelii, and A. phagocytophilum in ticks in central Poland.

c-DNA vaccination against parasitic infections: advantages and disadvantages

Recently developed technology for DNA vaccination appears to offer the good prospect for the development of a multivalent vaccines that will effectively activate both the humoral and cell mediated mechanisms of the immune system. Currently, DNA vaccination against such important parasitic diseases like malaria, leishmaniosis, toxoplasmosis, cryptosporidiosis, schistosomosis, fasciolosis offers several new opportunities. However, the outcome of vaccination depends very much on vaccine formulations, dose and route of vaccine delivery, and the species and even strain of the vaccinated host. To overcome these problems much research is still needed, specifically focused on cloning and testing of new c-DNA sequences in the following: genome projects: different ways of delivery: design of vectors containing appropriate immunostimulatory sequences and very detailed studies on safety.

Detection of the DNA of Borrelia afzelii, Anaplasma phagocytophilum and Babesia canis in blood samples from dogs in Warsaw

Each month, from March 2003 to February 2004, 34 blood samples from dogs were randomly selected from the blood samples delivered to two veterinary laboratories in Warsaw and tested for the DNA of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Babesia canis and Hepatozoon canis. Borrelia DNA was detected in seven of the 408 dogs, A phagocytophilum DNA was found in two, and B canis DNA was found in 48 (11.8 per cent). The DNA of H canis was not found in any of the blood samples. Sequencing of the seven Borrelia amplicons showed that only the genospecies Borrelia afzelii was present, the first time it has been detected in dogs in Poland.

The performance of a PCR assay for field studies on the prevalence of Fasciola hepatica infection in Galba truncatula intermediate host snails.

The aim of this work was to develop a PCR assay for the detection of F. hepatica in Galba truncatula snails and to evaluate its performance in field studies. Primers were designed to amplify a 124bp non-coding tandem repeat found in the Fasciola genome. The result was a banding pattern corresponding to multiples of the initial target sequence. The sensitivity of the PCR was determined on experimentally infected snails. The test was sensitive enough to detect fluke DNA in snails experimentally infected with 1 miracidium, within 12h after exposure. The specificity was determined with Dicrocoelium dendriticum, Paramphistomum cervi and snail DNA. No cross-reactions occurred with DNA of the trematodes or snail DNA. G. truncatula specimens were collected from 4 localities in Eastern Poland, with a total of 192 snails from 12 habitats. The overall prevalence of F. hepatica infection was 26.6% (51/192), ranging from 21.4% to 84.6% in the individual sites. The designed assay was shown to be a valuable epidemiological tool for the purpose of snail infection monitoring. The results on F. hepatica prevalence in snail hosts are the first data from Poland since the 1950s and the only such data based on molecular methods.

Somatic antigens of adult Dicrocoelium dendriticum recognised by bile antibodies of naturally infected cattle

Bile samples, from slaughtered cattle harbouring between 120 and 280 adult lancet flukes, were used to investigate the range of somatic proteins inducing local antibody responses in naturally infected animals. Lancet fluke infections induced local (bile) antibody responses against Tris-buffered saline (TBS) soluble, sodium dodecyl sulphate (SDS) soluble and 2-mercaptoethanol (2-Me) soluble somatic proteins of adult Dicrocoelium dendriticum. IgA antibody isotypes predominated in the response against buffer-soluble somatic antigens, whereas SDS-soluble and 2-Me-soluble proteins induced similar level of both IgA and IgG1 antibodies. Analysis of the antigens recognised by particular isotype-specific bile antibodies suggests that different antigens preferentially induce isotype restricted antibody responses. The bile antibody response was highly species specific, only one antigen from somatic protein extracts of Fasciola hepatica being precipitated by bile samples showing the highest reactivity against D. dendriticum.

Blocking Fasciola hepatica's energy metabolism - a pilot study of vaccine potential of a novel gene - phosphoglycerate kinase.

Fasciola hepatica infections cause huge economic losses to livestock production and are a serious problem in human and veterinary medicine. The main difficulty in the control of infections is progressing drug resistance. Moreover, pharmacological therapy is expensive and harmful for the environment. The best way of prophylaxis against infections seems to be vaccination. A new generation of vaccines could be the best possible way of controlling fasciolosis. This paper is focused on first vaccination trials based on a new vaccine candidate antigen, F. hepatica phosphoglycerate kinase (FhPGK) performed on a rat model. We obtained protection levels ranging from 0% to 69% depending on the way of delivery and form of vaccine.

Molecular cloning and characterisation of in vitro immune response against astacin-like metalloprotease Ace-MTP-2 from Ancylostoma ceylanicum.

Ancylostoma ceylanicum belongs to the group of parasites commonly known as hookworms, blood-sucking nematodes which infect around 576 million people and hundreds of millions of animals. The interactions between these parasites and host immune systems are complicated and yet to be determined. Hookworm infections are usually long lasting and recurrent, due in part to their ability to synthesize macromolecules capable of modulating the host immune response. The interaction of parasite proteins with host immune systems has been proven, but so far there is no data describing the influence of astacin-like metalloproteases (expressed among different parasitic nematodes) on the human immune system. The cDNA encoding A. ceylanicum metalloprotease 2 (Ace-mtp-2) was cloned using RACE-PCR. Computational analysis was used to examine the immunogenicity and recombinant Ace-MTP-2 was used to investigate its influence on human THP-1 monocytes and macrophages. The Ace-mtp-2 gene encodes an astascin-like metalloprotease, with a theoretical molecular mass of 26.7 kDa. The protease has a putative signal peptide, 11 potential phosphorylation sites, and two disulfide bridges revealed by computational analysis. Maximal expression of Ace-mtp-2 by A. ceylanicum occurs in the adult stage of the parasite, and Western blot indicates the secretory nature of the protease. This suggests the protease is working at the host-parasite interface and would likely be exposed to the hosts immune response. Recombinant protein were expressed in Escherichia coli and Pichia pastoris. Recombinant Ace-MTP-2 amplified the in vitro release of TNFα and induced release of IFNγ by lipopolysaccharide activated THP-1 macrophages. The presence of Ace-MTP-2 in secretory products of the adult parasite and the induction of IFNγ release may suggest an important role for Ace-MTP-2 in host-parasite interactions since IFNγ is suggested to be responsible for the protective immune response against adult hookworms.

Characterization and differential expression of cathepsin L3 alleles from Fasciola hepatica.

Fasciola hepatica infections cause significant global problems in veterinary and human medicine, including causing huge losses in cattle and sheep production. F. hepatica host infection is a multistage process and flukes express papain-like cysteine proteases, termed cathepsins, which play pivotal roles in virulence through host entry, tissue migration and immune evasion. Expression of these proteases is developmentally regulated. Recent studies indicate that excystment of infective larvae is dependent on cysteine proteases and together FhCL3 and FhCB account for over 80% of total protease activity detectable in newly excysted juvenile (NEJ) fluke. This paper focuses on members of the cathepsin L gene family, specifically those belonging to the CL3 clade. The cDNA of two novel cathepsin L3 proteases--FhCL3-1 and FhCL3-2 were cloned. The mRNA transcript expression levels for these enzymes were significantly different at various time points in life development stages obtained in vitro, from dormant metacercariae to NEJ 24h after excystment. Maximum expression levels were observed in NEJ immediately after excystment. In all stages examined by Real Time PCR, FhCL3-2 was expressed at a higher level compared to FhCL3-1 which was expressed only at very low levels. Western blot and immunohistochemical analysis also indicated higher expression of the FhCL3-2 allele and its secretory nature. The ability of antibody responses from rats and sheep challenged with F. hepatica to recognize recombinant FhCL3-1 and FhCL3-2 was shown to differ. Differences were also confirmed through the use of anti-rFhCL3-1 and anti-rFhCL3-2 sera in Western blot analysis of juvenile excretory/secretory (ES) material separated by 2D electrophoresis. These results indicate analysis of relative expression of parasite virulence factors from different populations is required, as this will likely impact the effectiveness of vaccines based on these antigens.

Evaluation of the immune response of male and female rats vaccinated with cDNA encoding a cysteine proteinase of Fasciola hepatica (FhPcW1)

Not only do males and females of many species vary in their responses to certain parasitic infections, but also to treatments such as vaccines. However, there are very few studies investigating differences among sexes following vaccination and infection. Here we demonstrate that female Sprague-Dawley rats vaccinated with cDNA encoding a recently discovered cysteine proteinase of Fasciola hepatica (FhPcW1) develop considerably lower liver fluke burdens after F. hepatica infection than their male counterparts. This is accompanied by differences in the course of their immune responses which involve different eosinophil and monocyte responses throughout the study as well as humoral responses. It is evident that host gender influences the outcome of parasitic infections after vaccination and research on both sexes should be considered when developing new treatments against parasites.

Fasciola hepatica - the pilot study of in vitro assessing immune response against native and recombinant antigens of the fluke.

Fasciola hepatica is a liver fluke that infects 2.4 million of people and causes great economical loss in animal production. To date a 100% effective vaccine has not been developed and the disease is controlled by drug therapy. Great efforts are put into development of effective vaccine against parasite what is difficult since Fasciola spp. (like other helmints) during evolutionary process has developed sophisticated and efficient methods to evade immune response. During preliminary experiments it is convenient to use cell lines which are relatively cheap and allow for reproducible comparison of results between laboratories. We stimulated BOMA (bovine monocyte/macrophage cell line) and BOMAC (bovine macrophage cell line) with native or recombinant antigens of Fasciola hepatica and assessed IFN-γ, IL-4 and TNF-α level upon stimulation. We observed diminished secretion of proinflammatory TNF-α in LPS activated BOMA cells stimulated with Excretory/Secretory products of adult fluke (Fh-ES). We also observed greater changes in gene expression in LPS activated BOMA cells than in non activated BOMA cells upon stimulation using Fh-ES. The results show possibility of using cell lines for in vitro research of bovine immune response against liver fluke, although this model still requires validation and further characterization.