Immunohistochemical distribution of antigens in liver of infected and immunized pigs with Ascaris suum

In the present work, we carry out an immunopathological study of the swine ascariosis, under different conditions (control, infection and immunization). Twenty-one Iberian pigs were used and divided in seven groups. Groups 1 and 2 were the uninfected and challenged controls, respectively. Groups 3 and 4 were weakly infected with increasing doses of Ascaris suum eggs and treated with pyrantel (Group 4). Groups 5-7 were immunized with 14, 42 and 97 kDa proteins from the parasite, respectively. Groups 2-7 were challenged with 10,000 infective eggs 7 days before the sacrifice. The focal parasitic granulomata with eosinophils and lymphocytes were the main histopathological lesions in the liver of reinfected pigs, while more marked cellular infiltrate and abundant connective tissue were seen in the livers of immunized animals. There were important deposits of antigens in the livers of immunized and infected pigs. Antigens were mainly located in the connective tissue, with positive staining detection of the somatic larvae antigen, the body wall from the adult worms and the 14-, 42- and 97-kDa proteins. However, cholangiols, biliary ducts and macrophages presented an immunohistochemical positive stain against excretory-secretory and somatic antigens from the larvae and the body fluid antigen from the adult parasite. The detection of A. suum antigens in the liver of infected pigs improves the diagnosis of swine ascariosis. It may be possible to apply these procedures for diagnosis of human ascariosis in liver biopsies since A. suum from swine have been previously used as a substitute for the study of the human parasite Ascaris lumbricoides.     

Production and distribution of immunoglobulin-bearing cells in the intestine of young pigs infected with Ascaris suum

Pigs of 10 days-1 month old were orally infected with eggs of Ascaris suum at different rates and inoculation schedules. Histological sections from various parts of the small intestines were prepared to observe the production and localization of immunoglobulin-bearing cells. Fluorescent antibody and immunoperoxidase staining methods were used to determine the number of IgM-, IgA- and IgG-producing plasma cells in the intestinal lamina propria. Significant increases in immunoglobulin-bearing cells were observed in those pigs which received single inoculations of A. suum eggs. Pigs infected every 2,4,8 and 10 days with 10,000-20,000 embryonated eggs showed numerical increases in IgM-bearing cells. Increases in IgA-bearing cells were noted in pigs which received the higher number of eggs every 8-10 days. Higher concentrations of IgA- and IgM-bearing cells were observed in the jejunal mucosa of infected pigs as compared to those in the duodenum and ileum.     

Tumor necrosis factor-like cytotoxicity and anorexia in Ascaris suum infected pigs

Tumor Necrosis Factor-Like Cytotoxicity (TNF-LC) was examined in sera from 12 pigs experimentally infected with Ascaris suum. The difference of TNF-LC levels between eight infected and four uninfected controls was not significant. When an endotoxin challenge was intravenously administered 1 month after the first dose of A. suum, the levels of TNF-LC in the sera of infected pigs were one-third that of the controls 125 min post-challenge (PC). In a more detailed study on four infected and two uninfected control pigs, TNF-LC was monitored every 10-15 min until 125 min after endotoxin challenge. The TNF-LC levels in these pigs increased at 40 min PC, reached maximum in another 10-25 min and then decreased. This pattern was seen in all except one infected pig. The infected pigs showed milder shock symptoms and their serum TNF-LC levels returned to pre-challenge levels 30 min earlier than controls.     

Evidence of interaction between Ascaris suum and Metastrongylus apri in experimentally infected pigs

A study has been carried out with the aim to determine possible interactions between Ascaris suum and Metastrongylus apri under experimentally infected pigs. Twenty-eight Iberian pigs were allocated into four groups. Group 1 was inoculated with 5000 infective A. suum eggs; group 2 received concurrently 5000 infective A. suum eggs and 5000 infective M. apri larvae; group 3 received 5000 infective M. apri larvae; group 4 served as uninfected controls. In each group, pigs were necropsied on day 7 (n = 4) and day 28 (n = 3) post-infection (p.i.). Pigs with single M. apri infections showed earlier and more severe respiratory symptoms compared to pigs with mixed infection, while no clinical signs were observed in pigs single infected with A. suum. Mean burdens of immature A. suum and immature and adult M. apri were reduced in pigs with concomitant infection both on day 7 and 28 p.i., respectively. In contrast, the number of white spots was significantly increased on day 7 in pigs with mixed infection. In addition, pigs of group 1 showed the highest eosinophil levels in blood compared to pigs in groups 2 (intermediate levels) and 3 (moderate levels). The results suggest an antagonistic interaction between A. suum and M. apri in concomitantly infected pigs.     

Persistent activity of doramectin and ivermectin against Ascaris suum in experimentally infected pigs.

A study was conducted to investigate the persistent nematocidal activity of two avermectins against experimentally-induced infections of Ascaris suum in swine. Seventy-two nematode-free cross-bred pigs of similar bodyweight were randomly allotted to nine treatment groups of eight pigs each. Eight of the groups were treated with injectable solutions containing 300 microg of doramectin/kg (IM) or 300 microg of ivermectin/kg (SC) either 0 (same day), 7, 14, or 21 days prior to an oral challenge of 50000 embryonated A. suum eggs. The ninth group (control) was challenged in parallel without any avermectin treatment. At 41 or 42 days after challenge, pigs were euthanatized and adult and larval stages of A. suum were collected from the gastrointestinal tract of each pig and counted. Both avermectins significantly (P < 0.0002) reduced nematode counts when given on the day of challenge (0 days prior), and the efficacy was 100% and 97.5% for doramectin and ivermectin, respectively. Doramectin given 7 days prior to challenge significantly (P < 0.0001) reduced nematode counts, and the efficacy was 98.4%. For all other avermectin-treatment groups, nematode counts were not significantly reduced compared to those in control pigs. These data indicated that anthelmintic activity of ivermectin against A. suum persisted for less than 7 days and the activity of doramectin persisted for more than 7, but less than 14 days.     

Efficacy of piperazine dihydrochloride against Ascaris suum and Oesophagostomum species in naturally infected pigs

A controlled trial was performed to evaluate the efficacy of piperazine dihydrochloride in a new granular formulation (Ascarex D) against naturally occurring infections with Ascaris suum, Oesophagostomum dentatum and O quadrispinulatum. Treatment effects were estimated on the basis of parasites recoverable from the intestinal contents. Given orally at 200 mg per kg body weight the compound showed an efficacy of 99 to 100 per cent against A suum and the nodular worms. Egg excretion of the respective species was reduced by 98 per cent and 100 per cent six days after treatment. No adverse reactions were observed after the treatment.     

Mature Ascaris suum in naturally infected calves

In two small farms in Sweden young calves were found to be naturally infected with Ascaris suum. One of the calves expelled mature worms with the faeces, one had a great number of worms in the ductus choledochus and two others had worms in the intestine. Most of the worms were mature and at the egg-producing stage. The morphology of the eggs and the adult worms indicated that these parasites were A. suum and that Toxocara (syn. Neoascaris) vitulorum could be excluded.     

Concurrent Ascaris suum and Oesophagostomum dentatum infections in pigs.

The aim of this study was to examine interactions between Ascaris suum and Oesophagostomum dentatum infections in pigs with regard to population dynamics of the worms such as recovery, location and length; and host reactions such as weight gain, pathological changes in the liver and immune response. Seventy-two helminth-na?ve pigs were allocated into four groups. Group A was inoculated twice weekly with 10000 O. dentatum larvae for 8 weeks and subsequently challenge-infected with 1000 A. suum eggs, while Group B was infected with only 1000 A. suum eggs; Group C was inoculated twice weekly with 500 A. suum eggs for 8 weeks and subsequently challenge-infected with 5000 O. dentatum larvae, whereas Group D was given only 5000 O. dentatum larvae. All trickle infections continued until slaughter. Twelve pigs from Group A and B were slaughtered 10 days post challenge infection (p.c.i.) and the remaining 12 pigs from the each of the four groups were slaughtered 28 days p.c.i.. No clinical signs of parasitism were observed. The total worm burdens and the distributions of the challenge infection species were not influenced by previous primary trickle-infections with the heterologous species. Until day 10 p.c.i. the ELISA response between A. suum antigen and sera from the O. dentatum trickle infected pigs (Group A) pigs were significantly higher compared to the uninfected Group B. This was correlated with a significantly higher number of white spots on the liver surface both on Day 10 and 28 p.c.i. in Group A compared to Group B. The mean length of the adult O. dentatum worms was significantly reduced in the A. suum trickle infected group compared to the control group. These results indicate low level of interaction between the two parasite species investigated.     

Serum levels of gastrin, insulin and glucagon as possible factors of anorexia in pigs infected once with Ascaris suum

In order to determine possible mediators for development of anorexia in pigs infected with Ascaris suum, serum levels of gastrin, insulin and glucagon were measured. After a single high oral dose of 100,000-200,000 embryonated eggs the serum levels of gastrin and insulin in the infected pigs did not significantly differ from those in controls. Serum glucagon levels in the infected groups, however, were lower than those in controls and the difference was more evident 24 days postinoculation and later.     

A novel technique for identification of Ascaris suum cohorts in pigs

The objective of the present study was to develop a fast, cheap and reliable technique for identifying different cohorts of the swine parasite, Ascaris suum. A polymerase chain reaction linked restriction fragment length polymorphism (PCR-RFLP) technique on mt-DNA was used to identify unique haplotypes of four gravid A. suum females on agarose gels after eggs were recovered from each of the worms. Each of four pigs was inoculated with 2000 embryonated eggs originating from one of the four identified Ascaris haplotypes, respectively. Ascaris larvae were isolated from the small intestine at day 14 post-infection using an agar technique. Single larvae from each pig were transferred to 96-well PCR plates and a simple DNA extraction using a worm lysis buffer was carried out and followed by the PCR-RFLP analysis. More than 100 larvae from each of the four pigs were analysed and all were found to have the same haplotype as the parental female. We conclude that unique haplotypes of female A. suum and offspring can be identified by means of PCR-RFLP on mt-DNA and suggest that this method can be used in future research on Ascaris population biology using cohorts with distinct mt-DNA profile.     

Blocking of nonspecific IgG in indirect radioimmunoassay for detecting Ascaris suum antigens

The sensitivity of an indirect radioimmunoassay (IRIA) used for detecting larval body wall (LBW) antigens of Ascaris suum was enhanced by using various blocking agents which prevented nonspecific binding of immunoglobulin G (IgG) or free ¹²⁵I without preventing binding of specific antibodies to the antigen. The use of blocking agents reduced counts for both positive and negative sera, resulting in an increase in calculated binding ratios (BR) and deltas and, thus, in the sensitivity of the assay. The relative effectiveness of blocking agents, in decreasing order, were turkey serum (TS), rabbit gamma globulin (RGG), rabbit whole serum (RS), bovine serum albumin (BSA), rabbit IgG (RIgG), bovine alpha globulin (BAG), bovine gamma globulin (BGG) and Tween-20.     

Effect of fenbendazole and pyrantel tartrate on the induction of protective immunity in pigs naturally or experimentally infected with Ascaris suum

An experiment was conducted with 96 weanling pigs (avg initial wt 18.5 kg) divided into six treatment with two replicates of eight pigs each. Pigs in Treatments 1, 2 and 3 were penned in outside pens with dirt lots that previously were contaminated with A. suum ova to induce a natural ascaris infection. Pigs in Treatments 4, 5 and 6 were penned in an open-front building with solid concrete floors and were experimentally infected with 2,000 embryonated A. suum. ova on d 1, 15 and 29 of the experiment. Pigs in Treatments 1 and 4 were medicated with fenbendazole (FBZ, 3 mg/[kg BW.d]) for three consecutive days during three consecutive time periods. Pigs in Treatments 2 and 5 were medicated with pyrantel tartrate (PT, 106 mg/kg feed) for 28 d. Pigs in Treatments 3 and 6 served as infected, unmedicated controls. All pigs were challenged with 100 A. suum eggs 7 d after termination of the final FBZ treatment. All pigs were killed 66 d after challenge and worms were recovered. Fenbendazole treatment resulted in greater (P less than .07) average daily gain than PT treatment in pigs penned outside. Among inside pigs, FBZ treatment resulted in better (P less than .02) feed utilization than in controls. The FBZ and PT treatments reduced (P less than .03) the total number of A. suum, the length and weight of female ascarids and the length of male ascarids compared with controls. A natural continual infection with A. suum was less effective than experimental infection in inducing protective immunity in pigs.     

A comparison of the efficacy of two ivermectin formulations against larval and adult Ascaris suum and Oesophagostomum dentatum in experimentally infected pigs

A study was conducted to evaluate and compare the efficacy of two injectable formulations of ivermectin (IVM-1 and IVM-2) at a dose rate of 0.3 mg/kg bodyweight versus placebo in the treatment and control of larval and adult stages of Ascaris suum and Oesophagostomum spp. in experimentally infected pigs. Seventy helminth free pigs were allocated on a liveweight basis to 7 groups each comprising 10 pigs (A-G). Group A served as an untreated control group. Groups B and C were used to investigate the efficacy of both formulations against adult stages of A. suum and Oesophagostomum spp., Groups D and E for efficacy against larval stages of A. suum and Groups F and G for efficacy against larval stages of Oesophagostomum spp. Pigs of groups A, B, C, D and E were infected on Day-0 with 1000 infective A. suum eggs each. Infective larvae of Oesophagostomum spp. (10,000/pig) were given on Day-0 to pigs of Groups F and G and on Day-21 to pigs of Groups A, B and C. Treatment was given to pigs of Group A (saline as placebo) on Day-7 and -28, IVM-1 to pigs of Group F on Day-7, pigs of Group D on Day-14 and pigs of Group B on Day-49. IVM-2 was given to pigs of Group G on Day-7, Group E on Day-28 and Group C on Day-49. Pigs of Groups F and G were sacrificed on Day-28, pigs of Groups A, D and E on Day-49 and pigs of Groups B and C on Day-56. Post mortem worm counts showed the following efficacies: (IVM-1) against larval A. suum 100%, against adult A. suum 94.4%, against larval Oesophagostomum spp. 52.0% and against adult Oesophagostomum spp. 83.0%. (IVM-2) against larval A. suum 100%, against adult A. suum 90.3%, against larval Oesophagostomum spp. 94.0% and against adult Oesophagostomum spp. 94.7%.     

Influence of an experimental infection of Ascaris suum on performance of pigs

Thirty-two pigs (average 26.6 kg live weight) were individually housed and fed to study the effect of an infection of Ascaris suum (either 0, 600, 6,000 or 60,000 A. suum eggs/pig) on performance of growing-finishing pigs. Increasing the level of A. suum infection produced linear (P less than .07) and quadratic (P less than .09) effects on final weight, weight gain and average daily gain. Feed to gain ratio and number of A. suum worms recovered from the intestines of pigs at slaughter increased linearly (P less than .01) with increasing doses of A. suum eggs. Pigs receiving 60,000 A. suum eggs were 13% less (P less than .01) efficient than the noninfected controls. In each of two trials, eight crossbred barrows (15.7 kg in trial 1 and 16.1 kg body weight in trial 2) were examined for the effects of two levels of A. suum infection (0 and 20,000 eggs/pig) on digestibility coefficients for dry matter, crude protein and gross energy. The infection did not affect (P greater than .05) digestibility coefficients during the first two collection periods (d 6 through 10 and 19 through 23). However, digestion coefficients for dry matter, crude protein and gross energy obtained from the total collection period on d 33 through 37 postinfection were greater (P less than .01) for control pigs than for pigs given 20,000 A. suum eggs each. Also, N retention was greater (P less than .05) for control pigs than for infected pigs.     

Ecological influences on transmission rates of Ascaris suum to pigs on pastures

A study was conducted to determine the distribution and transmission rate of Ascaris suum eggs and Oesophagostomum dentatum larvae in a pasture/pig house facility, which during the preceding summer was contaminated with helminth eggs by infected pigs. In May, four groups of 10 helminth na?ve tracer pigs were exposed to fenced sections of the facility for 7 days and necropsied for parasite recovery 9-10 days later (trial 1). The highest rate of A. suum transmission (201 eggs per day) occurred in the pig house (A). On the pasture, egg transmission decreased with the distance from the house: 8 eggs per day in the feeding/dunging area (B); 1 egg per day on the nearest pasture (C); <1 egg per day on the distant pasture (D). Only a few O. dentatum infections were detected, indicating a poor ability of the infective larvae to overwinter. Soil analyses revealed that the highest percentage (5.8%) of embryonated A. suum eggs were in the house (A). Subsequently, the facility was recontaminated with A. suum eggs by infected pigs. A replicate trial 2 was conducted in the following May. A major finding was the complete reversal of egg distribution between the 2 years (trials 1 and 2). In contrast to previous results, the highest rates of transmission (569 and 480 eggs per day) occurred in pasture sections C and D, and the lowest transmission rates (192 and 64 eggs per day) were associated with the feeding/dunging sections and the house (B and A). Soil analyses again supported the tracer pig results, as the pasture sections had the highest concentrations of embryonated eggs. Detailed soil analysis also revealed a non-random, aggregated egg distribution pattern. The different results of the two trials may be due to the seasonal timing of egg deposition and tracer pig exposure. Many eggs deposited during the summer prior to trial 1 may have died rapidly due to high temperatures and dessication, especially when they were not protected by the house, while deposition in the autumn may have favored egg survival through lower temperatures, more moisture, and greater sequestration of eggs in the soil by rain and earthworms. The latter eggs may, however, not have become embryonated until turnout the next year. The results demonstrate that yearly rotations may not be sufficient in the control of parasites with long-lived eggs, such as A. suum, and that a pasture rotation scheme must include all areas, including housing.