Assessment of selected biochemical parameters and humoral immune response of Nile crocodiles (Crocodylus niloticus) experimentally infected with Trichinella zimbabwensis.

Fifteen crocodiles were randomly divided into three groups of five animals. They represented high-infection, medium-infection and low-infection groups of 642 larvae/kg, 414 larvae/kg and 134 larvae/kg bodyweight, respectively. The parameters assessed were blood glucose, creatine phosphokinase (CPK), lactate dehydrogenase (LDH), aspartate transaminase (AST) and alanine transaminase (ALT). The humoral immune response to Trichinella zimbabwensis infection was evaluated in all three groups by an indirect ELISA method. The results showed deviations from normal parameters of blood glucose, CPK, LDH, AST and ALT when compared with reported levels in uninfected reptiles. Contrary to studies involving mammals, hypoglycaemia was not observed in the infected groups in this study. Peak values of blood glucose were reached on post-infection (PI) Day 49, Day 42 and Day 35 in the high-infection, medium-infection and low-infection groups, respectively. Peak values of LDH and AST were observed on PI Day 56, Day 49 and Day 42 in the high-infection, medium-infection and low-infection groups, respectively. Peak values of CPK were observed on Day 35 PI in all three groups. Peak ALT values were reached on Day 56 in the high-infection group and on Day 28 PI in both the medium-infection and low-infection groups. No correlations between the biochemical parameters and infection intensity were observed. Peak antibody titres were reached on Day 49 PI in the medium-infection group, and on Day 42 PI in both the high-infection and low-infection groups. Infection intensity could not be correlated with the magnitude of the humoral immune response or time to sero-conversion. Results from this study were in agreement with results reported in mammals infected with other Trichinella species and showed that antibody titres could not be detected indefinitely.

Cystatin capture-dot-enzyme-linked immunosorbent assay for immunodiagnosis and assessment of cure of experimental trichinellosis in mice.

Cystatin capture dot enzyme-linked immunosorbent assay (CC-dot ELISA) was evaluated as a new version of ELISA for diagnosis of trichinellosis by the detection of anti-Trichinella spiralis cysteine proteinase (CyP) IgG, using nitrocellulose membrane sensitized with cystatin as a capture reagent for CyP from T. spiralis muscle larvae excretory secretory products (ESP) without purification, compared to the detection of anti-T. spiralis IgG by conventional (conv.) ELISA, using whole ESP. Experimentally infected mice with light (100 larvae/mouse) and heavy (300 larvae/mouse) infections by T. spiralis larvae at 7, 14, 21 and 56 days post infection, and after flubendazole treatment were examined. As early as one week post infection CC-dot ELISA gave high positive rates of 86.6% and 100% in light and heavy infections, respectively, in contrast to negative results by conv. ELISA. CC-dot ELISA showed in light and heavy trichinellosis a higher efficiency in comparison to conv. ELISA (97.7% versus 67.7% and 98.8% versus 80%, respectively) and a higher overall sensitivity (96.6% versus 55% and 98.3% versus 73.3%, respectively). No cross reactions with sera of other parasitic infected or non infected control mice were recorded by CC-dot ELISA giving 100% specificity compared to 93.3% by conv. ELISA. After treatment, CC-dot ELISA gave positive results only in uncured mice with remaining muscle larvae, while conv. ELISA was positive in mice with and without remaining muscle larvae. CC-dot ELISA used lower quantities of antigen, was performed at room temperature, and read by naked eye in less than 2.5 hours.

Assessment of efficacy and safety of various adjuvant formulations with a total soluble extract of Trichinella spiralis.

Trichinellosis, a re-emerging zoonosis in several countries and pig, is the main species responsible for its transmission to human. Vaccination of swine could be an alternative to prevent the risk of human contamination. In order to develop an efficient and safe inactivate vaccine, the choice of the adjuvant is an important issue. The aim of this study was to develop and select potent and safe adjuvants by screening them in an experimental model with a crude soluble antigen from L1 muscular larvae (ML) of Trichinella spiralis (Ts). The efficacy was checked by the quantification of specific antibody levels. Specific and non-specific IgE antibody levels were also assessed. Safety was checked by the assessment of the local reaction at the injection site. Various Montanide ISA adjuvant formulations including water in oil, oil in water and multiphasic emulsions, but also nanoparticles or microbeads were tested. The results clearly showed differences between the antibody responses induced by the adjuvants and demonstrated the necessity to use an adjuvant to obtain a specific IgG (IgG1 or IgG2a) response directed against the total soluble extract of Ts. All the formulations enhanced the humoral immune response. The origin of the oil contained in the emulsions played an important role on the efficacy. Indeed emulsions based on mineral oils were more efficient than those based on metabolisable oils. However it was linked with stronger local reactions. Multiphasic and oil in water emulsions but also nanoparticles failed to induce IgG2a antibody levels. Microbeads and water in oil formulations based on mineral oils were more efficient. This experimentation allowed then the selection of several adjuvants which efficacy will be further investigated by a challenge test and an analysis of the cellular populations involved in the mechanism of the immune response.

Assessment of host resistance to Trichinella spiralis in mice following preinfection exposure to 2,3,7,8-TCDD.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been reported to decrease host resistance to a variety of infectious agents when exposure occurs prior to infection. Resistance to viral infection has been observed at doses as low as 0.1 microgram TCDD/kg body wt, well below the thymolytic dose in mice. In the present study, female B6C3F1 mice were exposed to a single intraperitoneal injection of 0, 0.1, 1.0, 10.0, or 30.0 micrograms TCDD/kg 7 days prior to infection to determine the effects of TCDD exposure on resistance to the nematode parasite Trichinella spiralis. Exposure to 10 or 30 micrograms TCDD/kg delayed adult parasite elimination from the small intestine. Significantly more larvae were released by female parasites and greater numbers of encysted larvae were recovered from the muscle of mice exposed to TCDD. Proliferative responses of splenocytes and mesenteric lymph node cells stimulated with T. spiralis antigen were significantly suppressed at exposure levels of TCDD > or = 1.0 microgram/kg 7 days after infection and in splenocytes only at 14 days after infection, demonstrating the greater sensitivity of proliferative responses to TCDD exposure than actual host resistance to Ts infection. Suppressed proliferation was observed at doses which produced TCDD concentrations > or = 0.2 pmol/g of lymphoid tissue on Day 7 of infection. In addition, it was determined that infected mice had higher TCDD levels than noninfected mice given the same dose. These results suggest an interaction between TCDD exposure and infection, i.e., that exposure to TCDD altered the host response to infection, while infection delayed elimination of TCDD from the host.