Luis Felipe Rodarte

CYSTICERCOSIS: IDENTIFICATION AND CLONING OF PROTECTIVE RECOMBINANT ANTIGENS

We describe the cloning and the evaluation of the protective capacity of 5 recombinant antigens expressed during the cysticercus stage of both Taenia crassiceps and Taenia solium. A cDNA library was constructed in bacteriophage lambda ZAP using mRNA isolated from larvae of T. crassiceps of the ORF strain. The recombinant phage library was screened with polyclonal antibodies against 56- and 74-kDa protective antigen fractions. This screening identified 13 recombinant clones, 5 of which were also strongly recognized by pooled sera from pigs experimentally infected with T. solium. The native antigens are proteins of 56 (clones KETcl, 4, 7) and 74 and 78 kDa (clones KETc11, 12) of T. crassiceps cysticerci. Vaccination experiments using these 5 recombinant clones against murine cysticercosis point to the relevance of KETcl, 4, 7, and 12 in host protection, whereas immunization with the clone KETc11 does not modify the parasite load in females and facilitates the parasitosis in males. We report here the DNA and the deduced amino acid sequence (100 amino acids) of the first protective antigen (KETc7) of potential interest for T. solium pig cysticercosis prevention.

Castration and pregnancy of rural pigs significantly increase the prevalence of naturally acquired Taenia solium cysticercosis

Cuentepec is a rural village of central Mexico, where 1300 pigs were bred at the time of the study in conditions that favor Taenia solium transmission. The tongues of 1087 (84%) of these pigs were visually examined and 33% were found to be cysticercotic. Castration of male pigs increased prevalence from 23 to 50% (P < 0.001) and pregnancy in sows also increased their prevalence from 28 to 59% (P < 0.001). Thus, endocrinological conditions characterized by low levels of androgens or high levels of female hormones probably influence the susceptibility of pigs to T. solium cysticercosis as observed in mice infected with Taenia crassiceps. Delaying castration of male pigs and confinement of sows during pregnancy might significantly decrease the prevalence of pig-cysticercosis and help curb transmission without much cost or difficulty.

Immunization of pigs against Taenia solium cysticercosis: factors related to effective protection

Fifty-six (56) pigs were immunized against Taenia solium cysticercosis with antigens from Taenia crassiceps metacestodes, in a variety of protocols, and then challenged orally with Taenia solium proglottids or eggs. Results of immunization (expressed as individual parasite loads) ranged from significant reduction of parasite loads (host protection) to clear increase (parasite facilitation) in apparent relation to the immunogen dose, adjuvant employed and genetic background of the pigs. In all trials, however, immunized pigs harboured more damaged cysticerci than controls, indicating that immunization does induce some restrictions to parasite these are eventually overwhelmed by other parasite-promoting factors. Western blots in immunized-protected pigs indicated antigens of 242, 234, 118, 77, 55 and 45 kDa as possibly being involved in immunological protection.

Experimental Taenia solium cysticercosis in pigs: characteristics of the infection and antibody response.

Pigs were infected with taeniid eggs to study the susceptibility to infection and reinfection of the animals of mixed breeds and of different ages, the viability and death of the metacestodes in the host tissue, and the antibody response which accompanies these events. Sixteen pigs were infected with Taenia solium eggs for this purpose. At necropsy metacestodes were counted in 2 kg of shoulder muscles and classified as vesicular or caseous, and all the metacestodes in brains were counted and classified. The results show that pigs inoculated at 49 and 60 days of age became infected to different degrees and reacted differently to the presence of parasites. In the brain the metacestodes remain viable for longer periods than in muscles. Enzyme-linked immunosorbent assay (ELISA) showed a significant rise in antibodies after infection, which started to decrease 92 days post-infection (p.i.). Pigs with viable cysts remained seropositive up to the end of the experiment (281 days p.i.). Antibody levels rose further after reinfection or after treatment. The results of Western blot were comparable to those of ELISA. Antigens of 13, 14 and 18 kDa were most frequently recognized in early infections and then started to decrease 92 days p.i., while the antigens of 42, 50 and 24 kDa were recognized during later stages of infection (200 days p.i.). The results suggest that older animals are more resistant to the infection [corrected].

Diagnosis of porcine cysticercosis: a comparative study of serological tests for detection of circulating antibody and viable parasites

Epidemiological studies of porcine cysticercosis require identification of pigs harbouring viable Taenia solium cysticerci and estimates of the degree of exposure to the parasite in the pig population destined for human consumption. Identification of infected pigs with viable larvae is achieved through detection of their secretory products. However, detectable levels of circulating antibody may also be present in the absence of viable larvae. In this study, both types of tests have been evaluated in groups of pigs experimentally infected with T. solium. Detection of viable cysticerci was achieved using a monoclonal antibody-based (HP10) antigen capture assay. HP10 epitope-bearing antigens have now been demonstrated in T. solium and T. crassiceps cyst fluid and excretion/secretions. Serum antibodies were measured in ELISA assays using two parasite preparations as antigens; T. solium cyst fluid and T. crassiceps cyst fluid antigens bearing the HP10 epitope. Low-background values were obtained with sera from non-infected animals in all the assays used. In heavily infected pigs, both antigens and antibodies were detected at least 29 days and up to 200 days post-infection (pi), while in lightly infected pigs antigen and antibodies were first observed between 61-97 days pi. Thus, the levels of the serum antigen and antibody varied with the intensity of the infection.

Further evaluation of the synthetic peptide vaccine S3Pvac against Taenia solium cysticercosis in pigs in an endemic town of Mexico

Taenia solium cysticercosis is a parasitic disease frequently affecting human health and the pig industry in many developing countries. A synthetic peptide vaccine (designated S3Pvac) against porcine cysticercosis has been developed previously as an aid to interrupt transmission and has been shown to be effective. The results of the present study support the effectiveness of the vaccine under endemic field conditions. However, given the time-frame of the vaccination trial, no changes in the local levels of transmission were detectable before and after vaccination using sentinel pigs. Thus, this investigation shows the limited usefulness of single vaccination as the sole means of interrupting Taenia solium transmission in an endemic region.

Taenia solium cysticercosis: immunity in pigs induced by primary infection

The present study demonstrates that pigs experimentally infected with Taenia solium eggs develop resistance to reinfection that lasts at least five months. Thirteen 2-month-old piglets were infected with eggs of Taenia solium. After 5 months, two pigs were euthanized and five were challenged with eggs from a second tapeworm. Nine months after the first infection, six pigs were challenged with a third tapeworm. All 11 challenged pigs were euthanized 2 months after reinfection. In order to confirm the infectivity of the eggs, several piglets were inoculated with each taenia. Two of the five pigs reinfected after 5 months did not develop metacestodes, two showed few caseous non-infective forms and in the fifth pig, 14% of the metacestodes were vesicular and 86% colloidal and caseous. In the six animals challenged 9 months after the first infection, three were heavily infected with vesicular metacestodes and the other three showed only colloid and caseous forms in muscles. All parasites found in brains were vesicular. We conclude that immunity due to primary infection lasts at least 5 months. At 2 months of infection antigens of 24 and 39-42 kDa were the most frequently recognised. In those pigs with only a few caseous cysts in muscles and/or vesicular ones in brains no antibodies were detected.

The Effect of Environmental Manipulation on Behavior, Salivary Cortisol, and Growth of Piglets Weaned at 14 Days of Age

Environmental enrichment can be a useful tool to reduce belly nosing behaviors in early weaned piglets. The aim of this study was to assess the effect of environmental enrichment on behavior, salivary cortisol, and productivity of piglets weaned at 14 days of age. The study assigned 112 piglets (line Camborough 22 of PIC™) into 2 treatments, control and enriched, and observed them for 192 hr in 3 periods: 14 to 28 days of age (Phase 1), 28 to 42 days of age (Phase 2), and 42 to 54 days of age (Phase 3). The study obtained saliva samples in each phase from 56 piglets selected randomly from each group for cortisol determination. Comparisons between both treatments and phases included the following: proportion of time belly nosing, latency of approaching a person, average levels of salivary cortisol, and daily weight gain. Belly nosing was higher and latency of approaching a person lower in the control group than in the enriched one (p < .05 and p < .01). Belly nosing was lower in Phase 3 (p < .05); latency of approaching a person was higher in Phase 1 with respect to Phase 2, and this was higher with respect to Phase 3 (p < .01). There were no differences in salivary cortisol levels between treatments or phases. Weight gain was higher in the enriched group (p < .001). Environmental enrichment in piglets weaned at 14 days of age resulted in a reduced proportion of time nosing, reduced latency of response to humans, and better growth than piglets in barren environments.

Influenza vaccine: development of a novel intranasal and subcutaneous recombinant adjuvant.

The synthetic peptide GK-1, derived from Taenia crassiceps, enhances the protection induced by human influenza vaccine in both young and aged mice. Herein, the adjuvant properties of GK-1 fused to the pVIII protein of a heat-inactivated phagemid vector (FGK1) when co-administered with the influenza vaccine were assessed, to evaluate its feasibility as a low-cost adjuvant. In mice, FGK1 significantly increased the expected IgG and IgA anti-influenza antibody levels both in sera and in bronchoalveolar fluids when intranasally or subcutaneously co-administered with influenza vaccine. Single-dose pig co-immunization with FGK1 and influenza vaccine induced serum levels of IgG anti-influenza antibodies similar to those elicited by a two-dose immunization with the influenza vaccine alone. Preclinical evaluation of FGK1 with the influenza vaccine is currently in progress, in order to recommend its use for veterinary purposes.