Application of the Midi Parasep® SF technique for the detection of L1 larvae of Angiostrongylus cantonensis in faeces of infected rats.

We investigated parasitic zoonoses caused by protozoans and helminths in urban and peri-urban rat populations (Rattus norvegicus and R. rattus) in Spanish cities. Rats were trapped and then dissected to remove adult helminths, and the contents of the large intestine were retrieved for the study of parasitic forms. The Midi Parasep® solvent free (SF) technique was used to concentrate the parasites in the intestinal contents. Some of the rats studied (n = 8) were infected by the rat lungworm, Angiostongylus cantonensis, whose first stage larvae (L1) are shed in rat faeces. After the concentration technique, L1 larvae were found in the sediment of 6 of the 8 positive rats. The two negative sediment samples were due to the presence of either only adult females or, in addition to males, only young females in the lungs of the rats. In view of our results, Midi Parasep® SF turned out to be a simple, rapid, inexpensive, and sensitive method to detect nematode larvae, such as the L1 larvae of A. cantonensis (or A. costaricensis), in natural and experimentally infected rats.

Intraperitoneal administration of the anti-IL-23 antibody prevents the establishment of intestinal nematodes in mice.

Previous studies have established that an increased Th-9 response creates a hostile environment for nematode parasites. Given that IL-23, a cytokine required for maintenance of the IL-17-secreting phenotype, has inhibitory effects on IL-9 production, we hypothesized that reducing circulating IL-23 by treatment with anti-IL-23 antibodies would reduce the establishment and development of parasitic intestinal nematodes. In this study, we show that animals treated with anti-IL-23 monoclonal antibodies showed a drastic reduction in the number of mouse pinworms (Aspiculuris tetraptera) recovered from the intestine (p < 0.001) at 23 days post-infection compared to the untreated animals. The cytokine levels in Peyer's patches (PP) in treated and infected animals increase the expression of interleukins such as IL-25, IL-21, and IL-9, augmenting mucus production in the crypts, and boosting chemokines, such as OX40 and CCL20 in the mucosa. Our results suggest that the Th17/Th2 regulatory mechanism provoked by the administration of the anti-IL-23 antibody prevents the implantation of the intestinal nematode in mice. The diminished inflammatory IL-17 levels alter the Th9 environment perhaps as a consequence of IL-17 inhibiting IL-9 expression. These Th9 conditions may explain the successful treatment against Inflammatory Bowel Disease (IBD) both with antibodies against IL-23 or through parasitization with nematodes.

Self-adjuvanting C18 lipid vinil sulfone-PP2A vaccine: study of the induced immunomodulation against Trichuris muris infection.

Despite the importance of the adjuvant in the immunization process, very few adjuvants merge with the antigens in vaccines. A synthetic self-adjuvant oleic-vinyl sulfone (OVS) linked to the catalytic region of recombinant serine/threonine phosphatase 2A from the nematode Angiostrongylus costaricensis (rPP2A) was used for intranasal immunization in mice previously infected with Trichuris muris The animal intranasal immunization with rPP2A-OVS showed a reduction of 99.01% in the number of the nematode eggs and 97.90% in adult. The immunohistochemical analysis of the intestinal sections showed that in immunized animals with lipopeptide the mucus was significantly higher than in the other experimental groups. Also, these animals presented significantly different chemokine, CCL20 and CCL11, levels. However, although the number and size of Tuft cells did not vary between groups, the intensity of fluorescence per cell was significant in the group immunized with the rPP2A-OVS. The results of the present study suggest that mice immunized with the lipopeptide are capable of activating a combined Th17/Th9 response. This strategy of immunization may be of great applicability not only in immunotherapy and immunoprophylaxis to control diseases caused by nematodes but also in pathologies necessitating action at the level of the Th9 response in the intestinal mucosa.

Toxoplasma gondii detection and viability assays in ham legs and shoulders from experimentally infected pigs.

Epidemiological studies of toxoplasmosis show that infection in humans is mainly caused by the consumption of raw, undercooked or cured meat. Cured "Serrano" ham is a typical pork product from the Mediterranean area, highly valued for its flavour. The "Serrano" ham is prepared from pork meat and undergoes a process known as curing and a subsequent fermentation without thermal or smoking treatments. The viability of Toxoplasma gondii in hams and shoulders from experimentally infected pigs that have been subject to different curing processes has been studied in order to evaluate the best method to completely eliminate the viable protozoa. The different treatments include, i) freezing the legs and shoulders below -20 °C for 3 days before salting with marine salt, ii) salting the meat with marine salt and nitrites, iii) salting only with marine salt (traditional process) and iv) salting with marine salt and then freezing at -20 °C for 3 days after the curing period. The ham leg samples were cured for 7 months and the shoulder samples for 5 months. The presence of T. gondii in the different treatments was studied by a "magnetic-capture" method for the isolation of T. gondii DNA and a quantitative real-time PCR to estimate the T. gondii burden in the ham legs and shoulders. The infectivity capacity of T. gondii in positive samples was assayed by bioassays in mice and some physicochemical parameters, such as pH, water activity (aw) and salt content, were evaluated at the end of the curing time. In all the cases where the samples were frozen the T. gondii infectivity was eliminated. In samples in which the meat was salted in marine salt plus nitrites, the parasite viability remained for longer than in the traditional salting process. The methods described here could be useful for producers to guarantee the safety of their products.

Quantification and viability assays of Toxoplasma gondii in commercial "Serrano" ham samples using magnetic capture real-time qPCR and bioassay techniques.

"Serrano" ham is a typical pork product from the Mediterranean area, highly valued for its flavour. To make Serrano ham, pork undergoes a salting and a subsequent fermentation process known as curing. Certain pigs used for meat production are an important source of Toxoplasma gondii infection in humans. We have developed a method for quantifying and assaying the viability of the T. gondii present in commercial Serrano ham samples. A magnetic capture method for the isolation of T. gondii DNA and a qRT-PCR were used to estimate the T. gondii burden in 475 commercial samples of "Serrano" ham in two presentation formats: ham pieces and sliced ham. The infectivity capacity of T. gondii in positive samples was assayed in mice. The global prevalence of T. gondii was 8.84%, ranging from 32.35% in one of the companies to 0% prevalence in three other companies. The infectivity assays revealed that only 4.84% of the positive samples were infective. To the best of our knowledge this is the first report focussing on the prevalence of T. gondii in commercial "Serrano" ham. The method described here could be useful for producers to guarantee the safety of their products.