Excretory/secretory proteins of adult Toxocara canis induce changes in the expression of proteins involved in the NOD1-RIP2-NF-κB pathway and modulate cytokine production in mouse macrophages.

Dog roundworm (Toxocara canis) is the major causative agent of toxocarosis, a parasitic disease of both veterinary and medical importance. Knowledge gaps in fundamental and applied aspects hinder the control of this important zoonotic disease. To have a better understanding of Toxocara infection and host immune responses, mouse macrophages were exposed to excretory/secretory (ES) proteins released by adult worms of T. canis in vitro. The messenger RNA transcription and protein expression of nucleotide-binding oligomerization domain-containing protein 1 (NOD1), receptor interacting protein 2 (RIP2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in macrophages were analysed using quantitative real-time PCR (qRT-PCR) and Western blot. The levels of tumour necrosis factor alpha (TNF-ɑ), interleukin-1 beta (IL-1ß) and IL-6 released by the stimulated macrophages were analysed using enzyme-linked immunosorbent assay. It was found that 20 µg/mL ES proteins of adult T. canis induced the expression of NOD1, RIP2 and NF-κB in mouse macrophages at both transcriptional and translational levels after 9 h of incubation in vitro. Incubation with 20 µg/mL ES proteins also modulated the production of pro-inflammatory cytokines TNF-ɑ, IL-1ß and IL-6 by the macrophages. Taken together, ES proteins of adult T. canis appeared to be able to affect the macrophage NOD1-RIP2-NF-κB signalling pathway, which might play a role in regulating the production of proinflammatory cytokines. Further investigation of these aspects should lead to a better understanding of immune recognition of and modulation by Toxocara canis in host animals.

Molecular characterization and transcriptional analysis of the female-enriched chondroitin proteoglycan 2 of Toxocara canis.

Toxocara canis is an important but neglected zoonotic parasite, and is the causative agent of human toxocariasis. Chondroitin proteoglycans are biological macromolecules, widely distributed in extracellular matrices, with a great diversity of functions in mammals. However, there is limited information regarding chondroitin proteoglycans in nematode parasites. In the present study, a female-enriched chondroitin proteoglycan 2 gene of T. canis (Tc-cpg-2) was cloned and characterized. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to measure the transcription levels of Tc-cpg-2 among tissues of male and female adult worms. A 485-amino-acid (aa) polypeptide was predicted from a continuous 1458-nuleotide open reading frame and designated as TcCPG2, which contains a 21-aa signal peptide. Conserved domain searching indicated three chitin-binding peritrophin-A (CBM_14) domains in the amino acid sequence of TcCPG2. Multiple alignment with the inferred amino acid sequences of Caenorhabditis elegans and Ascaris suum showed that CBM_14 domains were well conserved among these species. Phylogenetic analysis suggested that TcCPG2 was closely related to the sequence of chondroitin proteoglycan 2 of A. suum. Interestingly, a high level of Tc-cpg-2 was detected in female germline tissues, particularly in the oviduct, suggesting potential roles of this gene in reproduction (e.g. oogenesis and embryogenesis) of adult T. canis. The functional roles of Tc-cpg-2 in reproduction and development in this parasite and related parasitic nematodes warrant further functional studies.

Prevalence and risk factors associated with intestinal parasites in pigs in Chongqing, China.

From 2007 to 2009, the prevalence of intestinal parasites was investigated in intensive and extensive pig farms in Chongqing, China. A total of 2971 samples from both sexes and five age categories (breeding boars, breeding sows, fatteners, growers and weaners) were evaluated by standard methods for the presence of helminth ova and protozoan oocysts, cysts and/or trophozoites. Of the 2971 pigs sampled, 362(12.18%) were infected with Ascaris suum, 301(10.13%) with Trichuris suis, 301(10.13%) with Oesophagostomum spp., 491(16.53%) with Eimeria spp., 149(5.02%) with Isopora suis, 677(22.79%) with Balantidium coli and 196(6.60%) with Cryptosporidium spp. Growers had the highest infection rate while breeding boars had the lowest among the five age categories. B. coli was the most common protozoan in all pig age groups. Pigs infected with multiple parasites were common. Risk factors such as management methods, seasons, ages, etc. can influence the infection rate to a certain degree. This investigation provides relevant data about risk factors for pig farmers, thus allowing them to make more appropriate antiparasitic treatments according to farm conditions and local climate in Chongqing.

Combined PCR-oligonucleotide ligation assay for detection of dairy cattle-derived Cyclospora sp.

A rapid and sensitive assay for the detection of Cyclospora species in dairy cattle faecal specimens has been developed. The method utilizes a nested PCR to amplify a 168-bp DNA fragment of the 18S rRNA gene of cattle-derived Cyclospora sp. and an enzyme-linked immunosorbent assay (ELISA)-based oligonucleotide ligation assay (OLA) to detect the amplified product. In this study, the OLA technique was compared with conventional gel electrophoresis for the detection of amplified product. In evaluating the PCR-OLA for Cyclospora sp. and non-Cyclospora parasites, A(405) reading value for Cyclospora species was significantly higher than those for non-Cyclospora control. At known concentrations of purified amplicons from cattle-derived Cyclospora sp., the OLA was able to detect more than 0.5 ng of the amplified DNA. Of 168 clinical specimens collected from four dairy cattle farms, 6 were positive by both PCR-gel electrophoresis and the PCR-OLA procedure, and 2 were positive only by PCR-OLA, indicating the PCR-OLA procedure was more sensitive than the common way with gel electrophoresis. The results indicated that the PCR-OLA is simple, rapid and suitable in clinical detection of cattle-derived Cyclospora species.