Evaluation of pyrantel-tartrate abbreviated Ascaris suum infections for the development of resistance in young pigs against migrating larvae

Crossbred young pigs were used to test whether abbreviated infections with eggs of Ascaris suum can stimulate the acquisition of resistance to challenge. Weanling pigs from an Ascaris-free colony were kept free of A. suum until they were divided into groups at the age of 7-8 weeks. The experimental animals received pyrantel tartrate during the period when they were being exposed to increasing numbers of infective A. suum eggs and challenged 10 days after the last infective dose. Liver milk-spot counts and larval recoveries from the lungs indicated that the strongest resistance was acquired by the animals that received the drug continuously for 6 weeks while being exposed to six weekly infective egg doses. The data do not suggest any drug-related suppression of the resistance response to A. suum infection.     

Ascaris suum: protective immunity in pigs immunized with products from eggs and larvae

Parasite products were collected at three distinct phases of development of Ascaris suum, and their immunogenicity was determined after injection into rabbits and pigs. Products were derived from (1) the hatching fluid of infective eggs; (2) the conditioned medium of 2nd-stage larvae that developed to 3rd stage in vitro in defined medium; and (3) the conditioned medium of 3rd-stage larvae that developed to 4th stage in vitro in defined medium. Protein profiles from these three preparations, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were less complex than that of extracts from homogenized A. suum larvae. Hyperimmune rabbit antiserum raised against either egg products, 2nd- to 3rd-stage larval excretory-secretory products, or 3rd- to 4th-stage larval excretory-secretory products showed strong homologous reactions after immunoelectrophoresis, but relatively weak cross-reactions with the other preparations. A combined enteral immunization of pigs with egg products and parenteral immunization with the 2nd- to 3rd-stage larval excretory-secretory products, and 3rd- to 4th-stage larval excretory-secretory products induced antibody to each preparation and significant protective immunity to a challenge exposure with 10,000 A. suum eggs. However, a marked pathological response to larvae migrating in the liver after challenge exposure was also induced.     

Detection of a quantitative trait locus associated with resistance to Ascaris suum infection in pigs

Helminths almost invariably have an over-dispersed distribution in the host population. Human and animal studies have provided evidence suggesting that a large part of this variation is due to host genetic factors. Recently, the heritability for roundworm (Ascaris suum) infection levels in pigs was estimated to be 0.45. We used single nucleotide polymorphism markers to perform a whole-genome scan on 195 pigs experimentally infected with A. suum. A putative quantitative trait locus for worm burden on chromosome 4 covering 2.5 Mbp was identified by measured genotype analysis, although none of the SNPs reached genome-wide significance. To validate the putative quantitative trait locus, we genotyped two of the SNPs within the region in unrelated, informative animals exposed to experimental or natural infections and from which we had worm counts and/or faecal egg counts; the validation studies showed that one of the SNPs (TXNIP) was associated with total worm burden (P < 0.001) and adult worm burden(P < 0.0001), whereas the other SNP (ARNT) was associated with adult worm burden (P < 0.025) in these populations. We were thus able to confirm the existence of the quantitative trait locus on chromosome 4.This is to our knowledge the first report of a quantitative trait locus associated with helminth burden in pigs.     

Subcellular localization of glyoxylate cycle enzymes in Ascaris suum larvae

Evidence is presented on the particulate nature of glyoxylate cycle enzymes in metazoa with the use of 15-day old larvae of the nematode Ascaris suum. Homogenization procedures were developed to disrupt the resistant nematode cuticle. Malate synthase and isocitrate lyase, key enzymes of the glyoxylate cycle, consistently sedimented with mitochondrial enzymes in differential pellets while catalase, a major peroxisomal enzyme, was always soluble. Isopycnic sucrose gradient centrifugation of the differential pellet yielded two protein peaks: one at 1.18 g/cm3 (characteristic for mitochondria), and another at 1.23 g/cm3 (common for glyoxysomes and peroxisomes). Electron microscopy of these fractions revealed that the lighter peak consisted primarily of mitochondria, while the heavier band contained proteinaceous bodies termed "dense granules" morphologically resembling microbodies. SIgnificantly, both malate synthase and isocitrate lyase cosedimented with the mitochondrial marker enzymes in the lighter peak (1.18 g/cm3) and not with the dense granules. Further purification of mitochondria, accomplished by separating dense granules with a step gradient before isopycnic centrifugation, substantiated the evidence that microbodies (glyoxysomes) do not occur in these nematode larvae. Rough-surfaced membranes were alternatively considered as the subcellular site, but the evidence tends to favor localization of the glyoxylate bypass enzymes in the mitochondria.     

An ultrastructural study of the invasion of Ascaris suum larvae by neutrophils

Neutrophils are capable of penetrating the cuticle of the second-stage larvae of the nematode Ascaris suum. The integrity of the internal tissues of the larvae is then destroyed by cytoplasmic extensions of the neutrophils.     

Ascaris suum: development of intestinal immunity to infective second-stage larvae in swine

The development of protective immunity to Ascaris suum was examined in pigs naturally exposed to eggs on a contaminated dirt lot. Pigs became almost totally immune to second-stage larvae migrating from the intestines because few larvae from a challenge inoculum could be found in the lungs, and liver white-spot lesions (an immunopathologic response to migrating larvae) were absent. Blood from these pigs contained lymphocytes that responded blastogenically to larval antigens in vitro, while the serum contained antibody to larval antigens. Immunity was related to parasite exposure and not to the age of the host, and was not affected by the removal of adult A. suum from the intestines. Naturally exposed pigs responded to a variety of A. suum antigens with an immediate-type skin reactivity, and their intestinal mucosa contained relatively large numbers of mast cells and eosinophils. Other pigs were maintained on a dirt lot not contaminated with A. suum eggs and the effects of common environmental conditions on development of resistance to A. suum were studied. Resistance also developed in these pigs because 72% fewer larvae were detected in their lungs following a challenge exposure than in control pigs confined indoors on concrete floors and challenged similarly. This response was not expressed at the intestinal level, however, because their livers had numerous, intense white-spot lesions. To verify that the intestinal immunity that developed in pigs after natural exposure to A. suum was a direct result of homologous infection and not related to other stimuli encountered on a dirt lot, pigs maintained indoors on concrete floors, free from inadvertent helminthic infection, were inoculated orally with A. suum eggs daily for 16 weeks. Intestinal immunity was induced because larvae from a challenge inoculum were not detected in the lungs, and few white-spot lesions appeared on the livers of these pigs. Apparently, continual exposure of the intestinal mucosa to larvae eventually elicits the appropriate effector components necessary to prevent larval migration from the intestines.     

Strongyloides ratti: Acquired resistance in the rat to the preintestinal migrating larvae

Potential sites for expression of acquired resistance to Strongyloides ratti larvae in rats were investigated. In rats immunized by exposure to a single live infection and challenged 30 to 40 days later, 46 to 98% of the challenge larvae failed to reach the small intestine. Multiply immunized rats nearly completely eliminated migrating challenge larvae. This early killing of migrating larvae occurred during the first 48 hr after challenge infection. Resistance to migrating challenge larvae was also induced by repeated injections with heat-killed infective larvae. That the intestine may also serve as an effective site for worm expulsion was confirmed by intestinal transfers of worms from rats with primary infections into resistant rats.     

Serological responses to Ascaris suum adult worm antigens in Iberian finisher pigs

Adult Ascaris suum were dissected to obtain different worm components (body wall, body fluid, ovaries, uterus and oesophagus) which were used as antigens when testing 95 sera of naturally A. suum-infected Iberian pigs by enzyme-linked immunosorbent assay (ELISA) and Western blot (WB). Pigs with patent Ascaris infections had significantly lower ELISA optical density values than pigs without adult worms when using the body fluid and the body wall as antigens. A poor negative correlation was found between adult intestinal worm burden or eggs in faeces and specific antibody responses, measured by ELISA and WB using all antigens. By WB, the recognition of specific bands was variable, but three groups of bands with molecular weights of 97 kDa, 54-58 kDa and 42-44 kDa were generally recognized by sera from naturally infected pigs as well as from hyperimmunized pigs when using the five antigen extracts. The ELISA and WB techniques may be used for immunodiagnosis, using somatic adult worm antigens, to declare young pigs to be Ascaris-free but cannot be used for individual Ascaris-diagnosis in adult Iberian pigs.     

Development of Ascaris suum larvae from the third to fourth stage, in vitro

Larvae of Ascaris suum recovered from the lungs of experimentally infected guinea pigs or rabbits, seven to ten days after infection, underwent the third moult in vitro to become fourth-stage larvae. Ecdysis was first observed on the first and second day of culture and was completed by the eighth day. The culture medium consisted of tissue culture Medium 199 supplemented with either guinea pig or porcine serum and glucose in a gaseous atmosphere of Nitrogen:Oxygen:Carbon dioxide (90:5:5). By the twelfth day in culture, early development of both the male and female reproductive systems was observed.     

Synergistic effect of migrating Ascaris larvae and Escherichia coli in piglets

The effect of intestinal flora on the establishment, development and pathogenicity of Ascaris suum larvae in piglets (Large White breed) was investigated. The infected piglets with Ascaris and Escherichia coli showed signs of pneumonia, cough with respiratory difficulties initially even though these moderated with time. They lost appetite and showed signs of unthriftiness with loss of weight. The packed cell volume was normal but the differential leucocyte counts of the pigs infected with Ascaris larvae and bacteria had high neutrophils, unlike the very high lymphocyte count observed in piglets with ascarids only. The piglets had generalized serous atrophy of body fat. The pericardial and perirenal fats were gelatinous. There was a firm and nodular grey and red hepatization with abscess pockets in the intermediate and anterior one third of the diaphragmatic lobes of the lungs. The liver contained greyish-white and depressed focus immediately dorsal to the area of attachment to the gall bladder with multifocal areas. There was no significant gross lesion in the control animals. Cultural and microscopic examinations of some internal organs of the infected animals showed that bacteria were carried to the lungs by the migrating Ascaris larvae. The combined synergistic effect of Ascaris larvae and E. coli was also investigated and it was concluded that the two agents (A. suum larvae and E. coli) worked together synergistically.