Three surface antigens dominate the mucosal antibody response to gastrointestinal L3-stage strongylid nematodes in field immune sheep

Although gastrointestinal nematode parasites are a major human and veterinary health problem, little is known about how the host is sometimes able to mount an effective immune rejection response. In previous work, we identified a carbohydrate larval surface antigen (CarLA) as the target of mucosal antibodies that can elicit rejection of Trichostrongylus colubriformis L3s in sheep. Here we characterise the natural mucosal antibody responses to L3s from three major strongylid gastrointestinal parasites of sheep, Trichostrongylus colubriformis, Haemonchus contortus and Teladorsagia circumcincta. The mucosal antibody repertoire of naturally field-immune sheep was displayed on bacteriophage as single-chain antibodies (scFvs) and phage were selected for the ability to bind to the surface of living L3s of the three nematode species. All nematode-binding scFvs were found to recognize one of three different antigen classes that are each found in the three strongylid species. These three antigen classes appear to represent all of the major antigens recognized on Western blots by pooled mucosal antibodies from field-immune sheep. One of the antigen classes is a heterogeneous, high molecular weight molecule that is protease-sensitive. The scFvs recognizing this surface antigen also recognize a similar antigen in all strongylids tested. A second antigen class is a protease-insensitive, low molecular weight antigen found only in sheaths and scFvs recognizing this antigen cross-react with a similar molecule found in all strongylids tested. The third surface antigen class is CarLA and all of the anti-CarLA scFvs obtained from the field-immune sheep repertoire were specific to L3s of only one species and often recognized only a subset of the worms. Thus three different L3-stage surface antigens, two that lack a protein component, dominate the natural mucosal antibody response to L3-stage gastrointestinal strongylid nematodes in sheep.     

Secretion of anti-parasite substances and leukotrienes from ovine gastrointestinal tissues and isolated mucosal mast cells

The presence of larval migration inhibitory (LMI) compounds in the gastrointestinal mucus of nematode resistant sheep has been shown previously to be associated with increased numbers of gastrointestinal mucosal mast cells (MMC) and globule leukocytes (GL). This experiment was designed to determine if LMI compounds were secreted by MMC/GL in response to nematode antigenic challenge and if so, could secretion account for levels observed in mucus. Rommey sheep were immunized by repeated cycles of infection with Trichostrongylus colubriformis or Haemonchus contortus larvae and anthelmintic treatment. After slaughter, gastrointestinal tissue was taken for examination of histology and mucus anti-parasite activity. Segments of small intestine were ligatured to form sacs which were incubated with exsheathed nematode larvae or larval excretory/secretory antigens. Tissue slices from small intestine or abomasum were also incubated with nematode larvae or antigens. After homologous challenge, levels of leukotrienes secreted into small intestinal tissue sacs were significantly higher than levels in heterologously challenged sacs or unimmunized sheep intestinal sacs challenged with larvae of any nematode species (279.4±33.7, 141.0±27.8 and 39.5±15.2 ng h⁻¹ respectively). Tissue slices gave a similar pattern of leukotriene secretion. LMI activity was also significantly elevated in intestinal sacs from immunized sheep challenged homologously with nematode larvae or antigen (64±10 and 68±14% respectively cf. heterologous challenge 32±10% and unimmunized sheep sacs 15±6%). Histological examination of abomasal and small intestinal sections showed that immunized sheep had significantly greater numbers of MMC/GL than unimmunized sheep. MMC/GL isolated and purified from immunized sheep secreted leukotrienes and compounds having LMI activity when cultured with homologous nematode larvae or antigens. Secretion of leukotrienes and molecules having LMI activity from MMC/GL could account for the levels of these substances observed in small intestinal mucus.     

The role of immunologically specific and non-specific components of resistance in cross-protection to intestinal nematodes

Vaccination of 6–8-month-old Merino sheep with irradiated Trichostrongylus colubriformis larvae gave a high level of protection (81 %) against single-species challenge with normal infective larvae of the same species. The level of protection (34%) was substantially reduced against challenge with a closely related species (T. vitrinus) and no significant protection occurred against single-species challenge with a generically unrelated nematode (Nematodirus spathiger). These results suggest that antigen(s) which stimulate protective immunity are shared by the related Trichostrongylus species but not by N. spathiger. By contrast with the results obtained for single-species challenge, vaccination with irradiated T. colubriformis produced 98–100% protection against all 3 species in animals challenged simultaneously with infective larvae of the 3 species. Comparison of the levels of protection recorded following the 2 types of challenge indicate that although a specific antigenic trigger is required to provoke an appropriate response, the results obtained, particularly in the case of N. spathiger, suggest that the terminal effector mechanism is not immunologically specific. The implications of these conclusions are discussed in relation to theories of the mechanism of resistance to gastrointestinal nematodes and the potential efficacy of vaccination in the field.     

Haemonchus contortus and Trichostrongylus colubriformis did not adapt to long-term exposure to sheep that were genetically resistant or susceptible to nematode infections

We tested the hypothesis that Haemonchus contortus and Trichostrongylus colubriformis would adapt to long-term exposure to sheep that were either genetically resistant or susceptible to H. contortus. Sheep genotypes were from lines with 10 years prior selection for low (resistant, R) or high (susceptible, S) faecal worm egg count (WEC) following H. contortus infection. Long-term exposure of H. contortus and T.colubriformis to R or S genotypes was achieved using serial passage for up to 30 nematode generations. Thus, we generated four nematode strains; one strain of each species solely exposed to R sheep and one strain of each species solely exposed to S sheep. Considerable host genotype differences in mean WEC during serial passage confirmed adequate nematode selection pressure for both H. contortus (R 4900 eggs per gram (epg), S 19,900 epg) and T. colubriformis (R 5300 epg, S 13,500 epg). Adaptation of nematode strain to host genotype was tested using seven cross-classified tests for H. contortus, and two cross-classified and one outbred genotype test for T. colubriformis. In the cross-classified design, where each strain infects groups of R, S or randomly bred control sheep, parasite adaptation would be indicated by a significant host genotype by nematode strain interaction for traits indicating parasite reproductive success; specifically WEC and, for H. contortus strains, packed cell volume. We found no significant evidence of parasite adaptation to host genotype (P > 0.05) for either the H. contortus or T. colubriformis strains. Therefore, we argue that nematodes will not adapt quickly to sheep bred for nematode resistance, where selection is based on low WEC, although selecting sheep using a subset of immune functions may increase adaptation risk. Our results support the hypothesis that nematode resistance is determined by many genes each with relatively small effect. In conclusion, selection of sheep for nematode resistance using WEC should be sustainable in the medium to long-term.     

Subtractive phage display technology identifies zebrafish marcksb that is required for gastrulation

In the present study, we used a phage display technique to screen differentially expressed proteins from zebrafish post-gastrula embryos. With a subtractive screening approach, 6 types of single-chain Fv fragments (scFvs) were screened out from an scFv antibody phage display library by biopanning against zebrafish embryonic homogenate. Four scFv fragments (scFv1, scFv3, scFv4 and scFv6) showed significantly stronger binding to the tailbud embryos than to the 30%-epiboly embryos. A T7 phage display cDNA library was constructed from zebrafish tailbud embryos and used to identify the antigens potentially recognized by scFv1, which showed the highest frequency and strongest binding against the tailbud embryos. We acquired 4 candidate epitopes using scFv1 and the corresponding genes showed significantly higher expression levels at tailbud stage than at 30%-epiboly. The most potent epitope of scFv1 was the clone scFv1-2, which showed strong homology to zebrafish myristoylated alanine-rich C-kinase substrate b (Marcksb). Western blot analysis confirmed the high expression of marcksb in the post-gastrula embryos, and the endogenous expression of Marcksb was interfered by injection of scFv1. Zebrafish marcksb showed dynamic expression patterns during embryonic development. Knockdown of marcksb strongly affected gastrulation movements. Moreover, we revealed that zebrafish marcksb is required for cell membrane protrusion and F-actin alignment. Thus, our study uncovered 4 types of scFvs binding to zebrafish post-gastrula embryos, and the epitope of scFv1 was found to be required for normal gastrulation of zebrafish. To our knowledge, this was the first attempt to combine phage display technique with the embryonic and developmental study of vertebrates, and we were able to identify zebrafish marcksb that was required for gastrulation.     

Lactobacillus helveticus MIMLh5-Specific Antibodies for Detection of S-Layer Protein in Grana Padano Protected-Designation-of-Origin Cheese

Single-chain variable-fragment antibodies (scFvs) have considerable potential in immunological detection and localization of bacterial surface structures. In this study, synthetic phage-displayed antibody libraries were used to select scFvs against immunologically active S-layer protein of Lactobacillus helveticus MIMLh5. After three rounds of panning, five relevant phage clones were obtained, of which four were specific for the S-layer protein of L. helveticus MIMLh5 and one was also capable of binding to the S-layer protein of L. helveticus ATCC 15009. All five anti-S-layer scFvs were expressed in Escherichia coli XL1-Blue, and their specificity profiles were characterized by Western blotting. The anti-S-layer scFv PolyH4, with the highest specificity for the S-layer protein of L. helveticus MIMLh5, was used to detect the S-layer protein in Grana Padano protected-designation-of-origin (PDO) cheese extracts by Western blotting. These results showed promising applications of this monoclonal antibody for the detection of immunomodulatory S-layer protein in dairy (and dairy-based) foods.     

Acetylcholinesterase secretion by parasitic nematodes. II. Trichostrongylus spp

Unusually high levels of acetylcholinesterase (AChE) were found in the nematode parasites Trichostrongylus axei, T. colubriformis and T, retortaeformis. In T. colubriformis the enzyme was located in the oesophageal and excretory glands of the parasitic stages. The highest level/unit wt was found in the fourth-stage larvae, which per worm had a comparable level to that in adult worms because the excretory gland was fully developed in the fourth-stage larvae. In acrylamide gel electrophoresis, T. axei and T. colubriformis AChE and esterases were similar but differed from those present in T. retortaeformis. Globulins prepared from the sera of sheep and guinea-pigs infected with T. colubriformis complexed with T. colubriformis and T. axei AChE, but not with esterases nor with AChE from T. retortaeformis, Nippostrongylus brasiliensis, Oesophagostomum radiatum or O. venulosum. Complexing of AChE to globulins did not inhibit the enzymic function of this enzyme.     

Vaccination against the nematode Trichostrongylus colubriformis. III. Some observations on factors influencing immunity to infection in vaccinated guinea-pigs

Rothwell T. L. W. 1978. Vaccination against the nematode Trichostrongylus colubriformis. III. Some observations on factors influencing immunity to infection in vaccinated guineapigs. International Journal for Parasitology8: 33–37. Guinea-pigs were protected against infection with T. colubriformis when soluble material extracted from fourth-stage larvae was administered by the subcutaneous, intradermal, intraperitoneal and intraduodenal but not oral routes. The level of immunity following vaccination by the various effective routes was similar. Mature animals were found to respond significantly better to vaccination than immature animals. Significant immunity was present 10 days after vaccination but higher levels were found after 20 and 40 days. A single dose of vaccine was as effective as three divided doses. Finally, it was found that the adjuvant aluminium hydroxide gel, but not B. pertussis vaccine or levamisole improved the level of immunity to infection which followed vaccination.     

Schistosoma mansoni host-exposed surface antigens characterized by sera and recombinant antibodies from schistosomiasis-resistant rats

Antibodies from Schistosoma mansoni-infected rats, unlike mice, show a higher titer for schistosome apical tegumental antigens compared with non-apical membrane antigens. These antibodies bind to the surface of living lung-stage worms and to formaldehyde-fixed adult worms. We produced a single-chain antibody Fv domain (scFv) phage library displaying the antibody repertoire of rats highly immune to schistosome infection and we selected for scFvs that recognize the host-exposed surface of worms. Five unique rat scFvs (Teg1, Teg4, Teg5, Teg20 and Teg37) were obtained which recognize schistosome surface epitopes. Each of the scFvs recognizes the surface of living schistosomula and lung-stage schistosomules and/or the surface of formaldehyde-fixed adult worms. None of these scFvs reproducibly stained living adult worms. This suggests that a change occurs during the transition from lung schistosomules to 4-week adults such that at least some surface antigens, although remaining on the surface in living adult worms, can no longer be immunologically stained. Teg1 and Teg4 scFvs both recognize specific bands on Western blots. No bands were observed for the other three scFvs, suggesting that these scFvs may recognize non-protein or conformationally-dependent epitopes. Teg1 was unambiguously identified as recognizing the S. mansoni tetraspanin antigen, SmTSP-2, within the large extracellular domain. Teg4 recognizes a 35 kDa band tentatively identified as Sm29 by proteomic analysis. These scFvs can now be used to characterize schistosome epitopes at the host-parasite interface, to target worms in vivo, and to study the mechanisms by which these worms naturally evade immune damage to the tegument within permissive hosts.     

Development of phage-based single chain Fv antibody reagents for detection of Yersinia pestis

Background

Most Yersinia pestis strains are known to express a capsule-like antigen, fraction 1 (F1)^. F1 is encoded by the caf1 gene located on the large 100-kb pFra plasmid, which is found in Y. pestis but not in closely related species such as Yersinia enterocolytica and Yersinia pseudotuberculosis. In order to find antibodies specifically binding to Y. pestis we screened a large single chain Fv antibody fragment (scFv) phage display library using purified F1 antigen as a selection target. Different forms of the selected antibodies were used to establish assays for recombinant F1 antigen and Y. pestis detection.

Methods

Phage antibody panning was performed against F1 in an automated fashion using the Kingfisher magnetic bead system. Selected scFvs were screened for F1-binding specificity by one-step alkaline phosphatase enzyme linked immunosorbant assay (ELISA), and assayed for binding to recombinant antigen and/or Y. pestis by flow cytometry and whole-cell ELISA.

Results

Seven of the eight selected scFvs were shown to specifically bind both recombinant F1 and a panel of F1-positive Yersinia cells. The majority of the soluble scFvs were found to be difficult to purify, unstable and prone to cross-reactivity with F1-negative Yersinia strains, whereas phage displayed scFvs were found to be easy to purify/label and remarkably stable. Furthermore direct fluorescent labeling of phage displaying scFv allowed for an easy one-step flow cytometry assay. Slight cross-reactivity was observed when fixed cells were used in ELISA.

Conclusions

Our high throughput methods of selection and screening allowed for time and cost effective discovery of seven scFvs specifically binding Y. pestis F1 antigen. We describe implementation of different methods for phage-based immunoassay. Based on the success of these methods and the proven stability of phage, we indicate that the use of phage-displayed, rather than phage-free proteins, might generally overcome the shortcomings of scFv antibodies.     

The effect of vaccinating infection during pregnancy and dietary protein supply on the peri-parturient immune response of sheep to infection with Teladorsagia circumcincta and Trichostrongylus colubriformis larvae

It is well established that dietary protein supply can influence the peri-parturient breakdown of immunity to nematode parasites but there is no information on the importance of exposure to nematode larvae during pregnancy for this response. We investigated this by exposing housed pregnant sheep, scanned as carrying two lambs, to a vaccinating infection with a trickle mixed infection of Teladorsagia circumcincta and Trichostrongylus colubriformis larvae (L3) or to no infection during weeks − 9 to − 4 relative to parturition. At the beginning of week − 3 all sheep were treated with anthelmintic to remove any vaccinating worm burden and from week − 2 to week +6 received a trickle challenge infection with the same nematodes. Within each vaccinating treatment there were two nutritional treatments (no. = 20 per subgroup) designed to provide 1.5 or 1.0 and 1.3 or 0.8 of metabolisable protein (MP) requirement during pregnancy and lactation, respectively. Five ewes were necropsied during weeks +1 and +3 to measure worm burdens and mucosal inflammatory cells and the remainder maintained until week +6. Serum levels of total, IgA and IgE antibodies against L3 antigen of each nematode were measured.Scanning errors and lamb losses resulted in some ewes carrying and/or rearing only one lamb. Numbers of lambs reared was therefore introduced as a treatment effect. Vaccinating infection delayed the peri-parturient rise in faecal egg count (FEC) by an average of 2 weeks but its effect on FEC during the first 6 weeks of lactation was smaller and less persistent than that of dietary MP supply and single- v. twin-suckling.Populations of both nematodes were lower in association with high MP supply, vaccination and single suckling. These changes were associated with increases in numbers of mucosal mast cells (MMC) as a result of both increased MP supply and vaccination. Evidence for a more rapid return of host ability to limit populations of the abdominal nematode T. circumcincta than of the intestinal nematode T. colubriformis was associated with fewer eosinophils and more globule leucocytes (GL) in abomasal than in intestinal tissue.None of the serum antibody isotypes was affected by dietary protein supply. Total and IgA antibodies were maintained by a current larval (vaccinating) intake. IgA titres, however, increased progressively during pregnancy, especially in twin-bearing ewes. IgE titres appeared to be sensitive primarily to the reproductive cycle itself, peaking around parturition.This work supports the conclusion that availability of MP supply influences the recruitment and activity of cells of the immune armoury of the gastro-intestinal tract to nematode parasites. The precise outcome may differ with site and/or nematode species.     

Gastric dysfunction in sheep infected with Trichostrongylus colubriformis, a nematode inhabiting the small intestine

Barker I. K. and Titchen D. A. 1982. Gastric dysfunction in sheep infected with Trichostrongylus colubriformis, a nematode inhabiting the small intestine. International Journal for Parasitology12: 345–356. Six of 12 lambs infected with Trichostrongylus colubriformis had reduced abomasal acidification (pH 4.0–8.1) in comparison with uninfected pair-fed and replete controls (pH <3.5), though less than 0.8% of the worm burden was in the abomasum. Loss of prominence of parietal cells and encroachment of mucous cells deep into fundic glands was seen by light microscopy. Under the electron microscope, parietal cells had little canalicular or tubulovesicular development, had large vacuoles, many polyribosomes and few mitochondria in comparison with those in controls. In a further 8 sheep prepared with abomasal fistulae and separated fundic pouches and inoculated orally with T. colubriformis, the volume of fundic pouch secretion declined as feed intake dropped and in 7 out of 8 animals H⁺ concentration in fundic pouch secretion also fell. Infection generally reduced volume and acidity of pouch secretion more than did a pre-inoculation fast. In 5 sheep, abomasal content exceeded pH 4. Inoculation of T. colubriformis by enterotomy and Ostertagia circumcincta per os, in a lamb with a separated fundic pouch, caused depression of volume and acidity of pouch secretion characteristic of T. colubriformis infection, rather than the hypersecretion typical of abomasal infection with Ostertagia. Factors inhibitory to parietal cell differentiation and gastric acid secretion may be released from the small intestine of some sheep in response to changes in the gut induced by the presence of T. colubriformis. Abomasal dysfunction is a manifestation of severe intestinal trichostrongylosis.     

A Novel Dual Expression Platform for High Throughput Functional Screening of Phage Libraries in Product like Format

High throughput screenings of single chain Fv (scFv) antibody phage display libraries are currently done as soluble scFvs produced in E.coli. Due to endotoxin contaminations from bacterial cells these preparations cannot be reliably used in mammalian cell based assays. The monovalent nature and lack of Fc in soluble scFvs prevent functional assays that are dependent on target cross linking and/or Fc functions. A convenient approach is to convert scFvs into scFv.Fc fusion proteins and express them in mammalian cell lines for screening. This approach is low throughput and is only taken after primary screening of monovalent scFvs that are expressed in bacteria. There is no platform at present that combines the benefits of both bacterial and mammalian expression system for screening phage library output. We have, therefore, developed a novel dual expression vector, called pSplice, which can be used to express scFv.Fc fusion proteins both in E.coli and mammalian cell lines. The hallmark of the vector is an engineered intron which houses the bacterial promoter and signal peptide for expression and secretion of scFv.Fc in E.coli. When the vector is transfected into a mammalian cell line, the intron is efficiently spliced out resulting in a functional operon for expression and secretion of the scFv.Fc fusion protein into the culture medium. By applying basic knowledge of mammalian introns and splisosome, we designed this vector to enable screening of phage libraries in a product like format. Like IgG, the scFv.Fc fusion protein is bi-valent for the antigen and possesses Fc effector functions. Expression in E.coli maintains the speed of the bacterial expression platform and is used to triage clones based on binding and other assays that are not sensitive to endotoxin. Triaged clones are then expressed in a mammalian cell line without the need for any additional cloning steps. Conditioned media from the mammalian cell line containing the fusion proteins are then used for different types of cell based assays. Thus this system retains the speed of the current screening system for phage libraries and adds additional functionality to it.     

Observations on the coiled posture of trichostrongyle infective larvae using a freeze-substitution method and scanning electron microscopy

Wharton D. A. 1982. Observations on the coiled posture of trichostrongyle infective larvae using a freeze-substitution method and scanning electron microscopy. International Journal for Parasitology12: 335–343. A method for the preparation of the infective larvae of Trichostrongylus colubriformis for scanning electron microscopy is described. This involves freeze-substitution with methanol followed by critical-point drying. The method resulted in good preservation and the retention of the coiled posture. The morphology of ensheathed and exsheathed larvae is described and the mechanisms of coil formation discussed. Coiling was also observed in the infective larvae of nine other trichostrongyle species.     

Screening and identification of human ZnT8-specific single-chain variable fragment (scFv) from type 1 diabetes phage display library

Zinc transporter 8(ZnT8) is a major autoantigen and a predictive marker in type 1 diabetes(T1D). To investigate ZnT8-specific antibodies, a phage display library from T1 D was constructed and single-chain antibodies against ZnT 8 were screened and identified. Human T1 D single-chain variable fragment(sc Fv) phage display library consists of approximately 1í10~8 clones. After four rounds of bio-panning, seven unique clones were positive by phage ELISA. Among them, C27 and C22, which demonstrated the highest affinity to ZnT8, were expressed in Escherichia coli Top10F' and then purified by affinity chromatography. C27 and C22 specifically bound ZnT8 N/C fusion protein and ZnT8 C terminal dimer with one Arg325 Trp mutation. The specificity to human islet cells of these sc Fvs were further confirmed by immunohistochemistry. In conclusion, we have successfully constructed a T1 D phage display antibody library and identified two ZnT8-specific sc Fv clones, C27 and C22. These ZnT8-specific sc Fvs are potential agents in immunodiagnostic and immunotherapy of T1 D.     

Procaryotic Expression of Single-Chain Variable-Fragment (scFv) Antibodies: Secretion in L-Form Cells of Proteus mirabilis Leads to Active Product and Overcomes the Limitations of Periplasmic Expression in Escherichia coli

Recently it has been demonstrated that L-form cells of Proteus mirabilis (L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in Escherichia coli JM109 and P. mirabilis L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in E. coli was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with P. mirabilis L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with E. coli JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the P. mirabilis L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The P. mirabilis L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic cells.     

Studies of the responses of basophil and eosinophil leucocytes and mast cells to the nematode Trichostrongylus colubriformis: Comparison of cell populations in parasite resistant and susceptible guinea-pigs

Handlinger J. H. and Rothwell T. L. W. 1981. Studies of the responses of basophil and eosinophil leucocytes and mast cells to the nematode Trichostrongylus colubriformis: comparison of cell populations in parasite resistant and susceptible guinea-pigs. Internationaljournal for Parasitology11: 67–70. Basophil and eosinophil leucocytes and mast cells in T. colubriformis resistant and susceptible guinea-pigs were compared. There were significantly more circulating and small intestinal eosinophils in the resistant guinea-pigs. Intestinal eosinophils increased in both groups following infection with T. colubriformis but after 10 days the count in susceptible animals had only reached the pre-infection count in the resistant group. Pre-infection intestinal mast cell counts in the two groups were similar. Mast cell counts in susceptible guinea-pigs did not change during the period of observation but almost doubled within seven days of infection in the resistant animals.     

The initiation of coiling behaviour prior to desiccation in the infective larvae of Trichostrongylus colubriformis

Wharton D.A.1981. The initiation of coiling behaviour prior to desiccation in the infective larvae of Trichostrongylus colubriformis. International Journal for Parasitology11: 353–357. Infective larvae of Trichostrongylus colubriformis coil during the evaporation of water films. Decreasing the depth of the liquid film does not initiate coiling but enclosure in capillary tubes of similar diameter to the track width of actively swimming or crawling larvae results in a marked stimulation of coiling behaviour. It is suggested that larvae coil in response to the progressive restriction of lateral movement in an evaporating water film. The behavioural flexibility of the infective larvae of T. colubriformis maximizes both their survival and their chances of infection.     

Micrometeorological factors involved in development and survival of free-living stages of the sheep nematodesHaemonchus contortus andTrichostrongylus colubriformis. A review

Temperature and soil moisture are the most important factors affecting the development and survival ofHaemonchus contortus andTrichostrongylus columbriformis eggs and larvae on pasture. More than half of the eggs develop into infective larvae in the laboratory, but a very low percentage (0.03% forH. contortus) does so on pasture. There is a marked difference betweenH. contortus andT. colubriformis in survival of infective larvae.H. contortus larvae survived in the winter at Urbana poorly, whereasT. colubriformis did well. The former survived better than the latter in the spring and worse in the summer, while both survived equally well in the fall. Technics for larval recovery from pasture are not very efficient. Meteorologic conditions at ground level where the larvae exist are quite different from those in a standard weather shelter 1.6 m above the ground. Bioclimatographs in which mean monthly maximum temperatures are plotted against total monthly precipitation are fairly good in predicting the type of nematode liable to be important in a given region, but they are too simplistic to be relied on for more than approximations.