Effects of a series of infections of Ostertagia circumcincta on gastric secretion of sheep.

Sheep which had been either previously infected with 3. circumcincta or maintained worm-free, were surgically prepared with separated fundic pouches and abomasal cannulae and subsequently infected with 20,000 O. circumcincta larvae three times weekly. A reduction in food intake and increases in total acid output from the pouches and plasma pepsinogen levels were evident in both groups of sheep 4 days after repeated infections commenced; effects which increased in severity after 12 or more days. Except for a transient period of slight failure, previously infected sheep retained the capacity to acidify their abomasal contents whereas previously worm-free sheep lost this capacity. These changes were reversed between 2 and 7 days after treatment with thiabendazole (88 mg.kg-1). Secretory capacity of the fundic pouches was tested with histamine (40 mug.kg-1), the histamine antagonist (burimamide 8 mg.kg-1) and atropine (100 mug.kg-1). Ostertagiasis reduced or abolished the stimulatory effects of histamine. An increase in secretion volume and acid output was obtained after food was freshly provided, even though as little as 25 gm was consumed. Atropine and burimamide both caused a profound decrease in pouch secretion and acid output. These data are consistent with the hypothesis previously stated that in ostertagiasis the hypersecretion from fundic pouches is due to increased levels of circulating gastrin.     

The distribution and localization of the stage-specific GP31 antigen from infective Ostertagia circumcincta larvae.

The 31,000 mol. wt glycoprotein (GP31) antigen of infective third-stage (L3) Ostertagia circumcincta larvae was shown, by surface labelling experiments and immunofluorescent antibody staining of whole larvae and larval sections, to be distributed internally. When transverse sections of L3 O. circumcincta, taken from the anterior pharyngeal region, were further examined by electron microscopy, after immunogold staining with rabbit anti-GP31 antiserum, the GP31 antigen was found to be specifically located in 'secretory organelles' within the cells of the oesophageal glands. By in vitro culturing L3 O. circumcincta in medium supplemented with 35S-methionine and then analysing the excretory-secretory material released by the larvae, it was found that the GP31 molecule was one of the major components of the excretory-secretory complex. The purified GP31 molecule had no detectable proteolytic activity in protein degradation assays. On examination of Triton X-100 extracts of infective larvae from other nematode parasite species, a predominant antigen similar to GP31 was found in Trichostrongylus colubriformis and Haemonchus contortus, but in Toxocara canis a minor component corresponding in mol. wt to GP31 was also detected. Based on these results the possible role of GP31 as a candidate antigen for a broad spectrum molecular vaccine against gastrointestinal nematode parasites in sheep is discussed.     

Parasite antigens and host antibodies in Ostertagia circumcincta infection of the sheep

and 1972. Parasite antigens and host antibodies in Ostertagia circumcincta infection of the sheep. International Journal for Parasitology, 2: 449–457. An allergenic component was separated from Ostertagia circumcincta antigens and specific reaginic antibody was separated from the corresponding 7S antibodies in sheep sera. Further evidence was obtained that the immunoglobulin class defined as IgG1A contains the reaginic or homocytotropic antibodies in sheep. Both the IgG1A antibody and P.C.A. levels continued to increase after the expulsion of the parasites, whereas the levels of anti-Ostertagia IgG1 did not.     

Hypergastrinaemia, abomasal bacterial population densities and pH in sheep infected with Ostertagia circumcincta.

Serum gastrin and pepsinogen concentrations, food intake, abomasal pH and abomasal aerotolerant and anaerobic bacterial populations were measured in sheep infected with Ostertagia circumcincta to search for links between hypergastrinaemia, food intake and changes in the abomasal environment. Abomasal pH and serum gastrin and pepsinogen concentrations were elevated in each of five sheep infected via abomasal cannulae with 150000 exsheathed larval stage three, followed 11 days later by 100000 sheathed larvae given intraruminally. Unparasitised abomasa contained aerotolerant bacterial population densities of between 10(3) and 10(6) cells ml(-1) and these did not change significantly following parasitism. In contrast, anaerobic bacterial population densities increased markedly by about 10(4)-fold following parasitism. Anaerobic numbers changed rapidly when abomasal pH increased from 2.5 to 3.5. At pH 4 and above, anaerobic bacterial numbers approached levels expected in rumen contents but parameters other than pH did not relate to bacterial numbers. Brief periods when serum gastrin was lower than expected, coinciding with raised abomasal pH, were not explicable by increased bacterial numbers. Food intake, which decreased for a variable period from around Day 5 p.i., correlated poorly with serum gastrin concentration, suggesting hypergastrinaemia is not the sole cause of anorexia in parasitised animals. The survival of substantial numbers of rumen bacteria in the abomasum at only slightly raised pH may significantly lower the bacterial protein available to the sheep.     

Immunodiagnosis of angiostrongyliasis with monoclonal antibodies recognizing a circulating antigen of mol. wt 91,000 from Angiostrongylus cantonensis

In the analysis of excretory-secretory (ES) antigens from infective third-stage larvae (L3) of Angiostrongylus cantonensis, one major component of mol.wt 91,000 was not precipitated by pooled sera of patients with eosinophilic meningoencephalitis. Monoclonal antibodies (Mc Ab) secreted from two hybridoma cell lines, established against somatic antigens of L3, recognized this molecule but with different epitope specificities indicated by an additivity index (A.I.) of 83%. The 2 Mc Ab (TD2 and 3A5) belonged to IgG2a and IgM classes, respectively. Combinations of TD2 and 3A5 were used in a sensitive enzyme-linked fluorescent assay (ELFA) for the immunodiagnosis of human angiostrongyliasis. The double-antibody sandwich ELFA method was applied firstly to sera from experimentally infected rats using either TD2 or 3A5 to coat the assay plates. Two fluorescence unit (F.U.) peaks appeared in sera from infected rats collected 18 and 44 days after infection. Specimens from 35 patients were tested, all cerebrospinal fluids (CSF) and most sera (88%) showed positive reactions and the average F.U. of CSF was greater than that of serum.     

Investigations of 'transfer factor' activity in immunity to Ostertagia circumcincta and Trichostrongylus colubriformis infections in sheep.

Immunity was successfully transferred by ‘Transfer Factor’ prepared from leucocytes of two adult Scottish Blackface donor rams infected with O. circumcincta and T. colubriformis to 4-month-old susceptible Fin X Dorset lambs. The immunity was expressed by a significantly reduced faecal egg count and worm burden compared to challenged, untreated controls. The immunity was comparable to that produced in another group of lambs given an initial infection prior to challenge with both parasites.     

Variation in size of Ostertagia circumcincta,a nematode parasite of sheep,induced experimentally and during preparation and preservation

Summary This investigation was undertaken to determine the effect of formol-saline fixation and subsequent preparation on the length of Ostertagia circumcincta, a gastrointestinal parasite of sheep. Such induced changes in worm lengths were compared with the size distribution in two experimental populations in lambs, one of which was helminthologically susceptible and the other highly resistant. It was concluded that variation in worm length caused by fixation was not significant when compared to the variation encountered naturally in worm populations. ac]19851213     

Resistance of field isolates of Trichostrongylus colubriformis and Ostertagia circumcincta to ivermectin.

Twelve Romney lambs and 10 Angora goats were infected with 7000 infective third-stage larvae (89% Trichostrongylus, 11% Ostertagia) collected from goats suspected of harbouring ivermectin-resistant nematodes. On 28 days p.i., the lambs and goats were divided into treatment and control groups of six and five animals, respectively. The animals in the treatment groups were treated with ivermectin (0.2 mg/kg) and necropsied 35 days p.i. Faecal egg counts were estimated on days 28 and 35 p.i. and larval development assays (LDAs) were conducted on 22, 24, 26, 28, 30, 32, 34 and 35 days p.i. The ivermectin treatment reduced Trichostronglus colubriformis burdens by 39% and 13% and Ostertagia circumcincta by 33% and 0% in lambs and goats, respectively. When compared with a susceptible strain, the LDAs indicated a resistance factor before treatment in lambs for T. colubriformis of 2.6 and 1.5 with ivermectin and avermectin B2, respectively, which rose to 3.4 and 2.0 after treatment. The LD50 values of the two control groups were relatively constant throughout the experiment. Prior to ivermectin treatment the LD50 values of the treated groups were similar (P > 0.05) to the control groups but following ivermectin treatment their LD50 values increased steadily until the animals were killed on 35 days p.i. The LD50 values for ivermectin and avermectin B2 of sheep were always slightly higher and significantly different (P < 0.01) than those of goats indicating a host effect on this parameter. The greater reduction in worm counts in goats suggests a difference in the efficacy of ivermectin between lambs and goats. This is the first confirmed report of ivermectin resistance in a field strain of T. colubriformis.     

Population dynamics of Trichostrongylus colubriformis and Ostertagia circumcincta in single and concurrent infections.

Twenty-one-week-old worm-free pen-reared lambs were infected weekly with either 10,000 T. colubriformis larvae, 5000 O. circumcincta larvae, or with both species (15,000 larvae per week). Larval establishment and total worm burdens were estimated after 4, 7, 10 and 13 weeks of infection. Faecal egg counts and lamb bodyweights were measured weekly, and numbers of eosinophils in blood were estimated before infection and at weeks 5, 8 and 14. For both species of worms, the dynamics of infection (establishment, worm burdens, egg counts) were not affected by concurrent or pre-existing infection with the other species. Infection with T. colubriformis alone did not protect against O. circumcincta, but infection with O. circumcincta alone provided slight protection against the T. colubriformis larvae. Blood eosinophils increased between 5 and 8 weeks of infection and were similar for the three infections. This corresponded to the reduction in establishment for both species.     

Characterization of the low mol. wt zinc-binding ligand from rat small intestine by comparison to the organic zinc-binding ligands

1. 65Zn complexes of picolinate (PA), citrate (CA), L-histidine (L-his), arachidonic acid (AA) or low mol. wt zinc-binding ligand from rat intestine (LMW-ZBL) gave 65Zn eluting peak fraction numbers of 53, 53, 56, 59 and 59 respectively, in a Sephadex G-75 column chromatography. 2. The 65Zn eluting peak fraction numbers with CA, L-his, PA, prostaglandin (PG)E2, AA, no ligand, arachidonate (AT) or LMW-ZBL were 49, 50, 54, 55, 58, 64, 75 and 76 respectively in a Sephadex G-25 column chromatography. 3. In a Sephadex G-15 column chromatography, the 65Zn eluting peak fraction numbers with CA, PGE2, AA, L-his, LMW-ZBL or PA were 49, 50, 51, 52, 52 and 55 respectively. 4. The LMW-ZBL in rat small intestine appears to be an AA-like substance.     

Further studies with a strain of Ostertagia ostertagi resistant to morantel tartrate.

Two experiments with a morantel resistant strain of Ostertagia ostertagi were carried out. In the first experiment five groups of five calves were infected with 60,000 larvae of this resistant strain. Calves of one group remained untreated, calves of the other groups were treated with morantel tartrate, oxfendazole, levamisole or ivermectin in the recommended doses. It was demonstrated that there was side resistance to levamisole, but not to oxfendazole or ivermectin. Compared with the untreated controls the reduction percentages of the worm burdens were 45.3 (morantel tartrate), 99.7 (oxfendazole), 83.5 (levamisole) and 100 (ivermectin). In the second experiment a comparison was made between the effect of levamisole against a morantel susceptible strain of O. ostertagi and the resistant strain. Two groups of five calves were infected with the susceptible strain and two groups with the resistant one. One group of each pair remained untreated, the other was treated with levamisole. The reduction percentages of the worm burdens were 99.6 (susceptible strain) and 63.6 (resistant strain). This result confirms the efficacy of levamisole to susceptible O. ostertagi and the side resistance of the morantel resistant strain.     

Inhibition of in vitro fertilization by mouse anti-mouse sperm sera and preliminary antigen identification

Antisperm antibodies are implicated as one causative factor of infertility, but the target antigens have not been identified. Immune responses to sperm antigens are qualitatively variable even within a single mouse strain. We took advantage of this variability and immunized individual female mice to allogeneic sperm to reflect their natural exposure during mating. We determined the ability of the individual sera to inhibit in vitro fertilization and to bind to sperm antigens separated by electrophoresis. Compared to preimmune sera, four of five immune sera significantly inhibited in vitro fertilization. The serum from individual mice bound variable panels of sperm antigens. By comparing the panels, we identified two polypeptides with molecular weights of 40,000 and 44,000 that were bound by all sera. We propose that these molecules may be good candidates for further investigation of the immunoprophylaxis of pregnancy.     

Widespread distribution of a 210,000 mol wt microtubule-associated protein in cells and tissues of primates

Antisera prepared against a 210,000 mol wt microtubule-associated protein (210k MAP) isolated from the human cell line, HeLa, were used to survey a variety of cells and tissues for the presence of immunologically related proteins. The antisera were employed to test extracts of the cells and tissues, using a sensitive indirect immunofluorescence technique applied to polyacrylamide gels. Cross- reactive material of 210,000 mol wt was found in 10 kinds of cells and tissues derived from humans and four lines of cells from monkeys. Indirect immunofluorescent staining was also carried out on fixed cells and showed that the cross-reactive material was localized to interphase and mitotic microtubules as assayed in nine human and seven monkey cell lines. No protein that cross-reacted with 210k MAP antisera was detected in cells and tissues derived from two rodents, an ungulate, a marsupial, or a chicken. Therefore, the 210k MAP isolated from HeLa cells is present in a wide variety of cells and tissues of humans and other primates but is antigenically distinct from MAPs present in lower organisms.     

Secretion of a lactosaminoglycan-containing glycoprotein by peri-implantation sheep conceptuses

Sheep conceptuses from day 16 of pregnancy were cultured in the presence of [3H]glucosamine and [14C]leucine and a high-molecular-weight glycoprotein (HMWG) secreted into the culture medium was purified by a combination of anion-exchange and gel filtration chromatography. The HMWG was found to have a molecular weight between 800,000 and 900,000 and to be highly resistant to digestion with pronase. Characteristics of the carbohydrate portion of the purified glycoprotein were examined by selective chemical and enzymatic digestions and lectin binding studies. Mild alkaline reduction was ineffective in disassociating carbohydrate chains from the protein core. Furthermore, the protein was resistant to both O-glycanase and peptide:N-glycanase F. Harsh alkaline reduction caused the release of carbohydrates, however. After pronase digestion of these products, three molecular weight classes of carbohydrates were resolved by Sephadex G-25 chromatography. Two lines of evidence indicate that the HMWG contains lactosaminoglycan components. The intact molecule and two of the molecular weight classes of carbohydrates resolved by harsh alkaline reduction bind Datura stramonium lectin. Binding of HMWG to lectin could be partially inhibited by N-acetyllactosamine and completely inhibited by a mixture of N,N'-diacetylchitobiose and N,N',N"-triacetylchitotriose. Secondly, digestion with endo-beta-galactosidase causes the release of 16% of the [3H]glucosamine from the intact molecule. Therefore, the HMWG of the sheep conceptus is the first reported example of secretion of lactosaminoglycan-containing glycoprotein by peri-implantation embryos.     

Serial passage in resistant sheep drives the infectivity and fitness of Teladorsagia circumcincta in susceptible lambs: Experimental evidence

Gastrointestinal nematodes (GIN) of small ruminants have adapted their life history strategies to thrive in diverse and fluctuating environments. Environments which alter their expression of life traits may also drive changes in the infection or transmission dynamics, particularly if transferred to a foreign setting. This study aimed to explore how repeated exposure to a resistant sheep host environment would alter the life history traits and infection dynamics of Teladorsagia circumcincta when consequently infected in susceptible lambs. Following just three generations of passage in resistant sheep, T. circumcincta significantly increased their infectivity and fitness in susceptible lambs compared to a control population. This is the first evidence to indicate the resistant host environment can drive such rapid changes in the expression of GIN life traits, with potentially undesirable epidemiological outcomes.