Primary Heligmosomoides polygyrus bakeri infection induces myeloid-derived suppressor cells that suppress CD4+ Th2 responses and promote chronic infection.

Primary infection with the gastrointestinal nematode Heligmosomoides polygyrus bakeri is chronic in C57BL/6 (B6) mice whereas challenge infection is rapidly eliminated. F4/80-CD11b+Gr+ cells, presumed to be neutrophils, were reported to accumulate around encysting larvae in intestinal tissue during primary infection, but their exact identity and role remain unclear. We observed significant increases in F4/80-CD11bhiGr1hi cells in mesenteric lymph nodes (MLNs) and spleen after primary but not challenge infection; a high proportion of these cells expressed Ly6G and Ly6C. These cells, which phenotypically resemble myeloid-derived suppressor cells (MDSC), increased in lamina propria (LP) early during primary infection. Increased MDSC were associated with low numbers of alternatively activated macrophages (AAMØ) in LP and CD4+GATA3+ T cells and AAMØ in MLN and spleen. Purified CD11c-CD11b+Gr1+ cells from H. polygyrus bakeri-infected mice suppressed OVA-specific CD4+ T-cell proliferation via a nitric oxide-dependent mechanism and parasite-specific IL-4 secretion in vitro. Adoptive transfer of CD11c-CD11b+Gr1+ cells from mice with primary infection resulted in significantly higher adult worm burdens and increased egg production in naïve B6 recipients infected with H. polygyrus bakeri. Altogether, these findings indicate that primary H. polygyrus bakeri infection induces a novel subset of MDSC that suppress CD4+ Th2 responses and promote chronic infection.

Haemonchus contortus: Applications in Drug Discovery.

Haemonchus contortus is an important pathogen of small ruminants and is therefore a crucially important target for anthelmintic chemotherapy. Its large size and fecundity have been exploited for the development of in vitro screens for anthelmintic discovery that employ larval and adult stages in several formats. The ability of the parasite to develop to the young adult stage in Mongolian jirds (Meriones unguiculatus) provides a useful small animal model that can be used to screen compounds prior to their evaluation in infected sheep. This chapter summarizes the use of H. contortus for anthelmintic discovery, offers a perspective on current strategies in this area and suggests research challenges that could lead to improvements in the anthelmintic discovery process.

Identification of Dirofilaria immitis proteins recognized by antibodies from infected dogs.

The identification of excreted-secreted (ES) proteins of filarial nematodes as potential diagnostic reagents is an important requirement for the development of methods to determine level of infection in the host, especially for human filariae. Dirofilaria immitis, the canine heartworm, is a widespread and important veterinary pathogen and is a useful model for filarial parasites of humans. An analysis of proteins released from adult D. immitis (the secretome) in culture is available. We sought to identify D. immitis ES proteins found in vivo to validate the in vitro secretome and to investigate them as potential diagnostic reagents. Cultures of D. immitis adults obtained from infected dogs were maintained for 72 hr with daily changes of media. Proteins were concentrated from spent media by standard methods and were passed through Protein-A columns containing purified IgG antibodies from heartworm-infected dogs. Following extensive washing, heartworm proteins recognized by the antibodies were eluted from these columns and submitted for analysis by tandem mass spectrometry (MS/MS). As a comparison, somatic proteins from adult D. immitis female parasites and microfilaria were also processed and analyzed by the same protocol. Six, 9, and 12 proteins were identified by MS/MS in the ES, adult female, and microfilaria samples, respectively. The identification of the most abundant parasite proteins present in the serum of infected hosts offers a rational approach to the development of new diagnostic assays that may be applicable across the Filarioidea.

The association of adult Onchocerca volvulus with lymphatic vessels.

Immunocytochemical examination of onchocercal nodule tissues containing adult Onchocerca volvulus using immuno-markers for blood and lymphatic vessels (vWF, D2-40, podoplanin, Prox-1, and Lyve1) shows a distinct pattern of distribution of these vessels within nodules. Blood vessels were commonly seen associated with organized lymphoid cellular aggregates in the both the outer and inner areas of the nodules. In contrast, the majority of the lymphatic vessel positivity was seen in the central zone in close apposition to the adult parasites, and the remainder usually associated with microfilariae in the outer areas of the nodule. These findings suggest an intimate relationship between adult O. volvulus and lymphatic vessels, including the likely proliferation of lymphatic endothelial cells (lymphangectasia) akin to that seen with other filariae. These findings indicate that adult O. volvulus may migrate via the lymphatic system, and that clinical manifestations of this disease that involve tissue edema may be the result of the location of these worms in the lymphatic system.

Foreword: towards markers for anthelmintic resistance in helminths of importance in animal and human health.

Anthelmintic resistance is a serious problem in veterinary medicine and appears to be developing in some helminths of importance to human health. Anthelmintic drugs remain the principal means of control of helminth infections in animals and humans and the continued dependence on these pharmaceuticals will continue to impose selection pressure for resistance development. Our ability to detect anthelmintic resistance before control breaks down and to monitor the spread of anthelmintic resistance is quite limited. We are currently dependent on biological methods which are not sufficiently sensitive to detect low levels of drug resistance and are particularly difficult to perform on helminth parasites of humans. There is a serious need for new molecular markers for detecting and monitoring for anthelmintic resistance. The problem of anthelmintic resistance is already very serious in nematode parasites of livestock. In addition, there should be great concern about possible anthelmintic resistance development and the lack of tools and efforts for monitoring it as part of the major worldwide programmes to control helminth parasites in people. An international Consortium has been formed to develop Anthelmintic Resistance Single nucleotide polymorphism markers (CARS). Discussions within the Consortium have addressed the need for such markers, the current state of knowledge about them, possible mechanisms of anthelmintic resistance and approaches and constraints to the development of markers. Summaries of the state of the art in these areas are presented in a series of papers in this Special Issue of Parasitology.

Interrelationships among physicochemical properties, absorption and anthelmintic activities of 2-desoxoparaherquamide and selected analogs.

The interrelationships between physicochemical properties, absorption and potency of 2-desoxoparaherquamide and five analogs, representing a new anthelmintic class, were evaluated in in vitro and in vivo assays. At pH 7.5, rates of drug absorption by the gastrointestinal nematode Haemonchus contortus and jird small intestine, parameterized by the permeability coefficient, P(e), ranged from 1.2-2.4 x 10(-4) cm/min (nematode) to 2.5-5.5 x 10(-3) cm/min (jird). In the jird intestine, absorption was pH-dependent, with P(e) at pH 7.5 being twice that at pH 4.5, reflecting the negative influence of protonation on transport of these weakly basic molecules. Each compound rapidly paralyzed H. contortus during in vitro exposure to therapeutically relevant concentrations (1-10 microm). The kinetics of drug action on motility in vivo mirrored their in vitro effects; motility concentrations were reduced in nematodes collected from jird stomach 3 h following oral drug dosing, by which time > or =50% clearance of the parasites had occurred. The nematode/medium partition coefficient K ranged from 10.1 to 16.1, consistent with the lipophilic nature of the compounds. The time required to reduce motility in vitro by 50% (t50*) and P(e) were used to determine C(n)*, the concentration of drug in the nematode at t50*, as an indicator of intrinsic potency. In the jird, the apparent potencies of the compounds were insensitive to route of administration (i.e. oral = i.v. = i.p. = i.m.) for H. contortus and two other gastrointestinal nematodes, Ostertagia ostertagi and Trichostrongylus colubriformis; topical administration, however, required three to 10-fold higher doses for equivalent efficacy.

Benzimidazole-resistant beta-tubulin alleles in a population of parasitic nematodes (Cooperia oncophora) of cattle.

Three anthelmintic classes with distinct mechanisms of action are commercially available. Selection of nematode populations resistant to all these drugs has occurred, particularly in trichostrongyloid parasites of sheep. Anthelmintic resistance in cattle parasites has only recently been recognized and appears to be less pronounced, even though very similar species infect both hosts. To understand the bases for differences in the rate of resistance development in sheep versus cattle parasites, it is important to first demonstrate that the same kinds of resistance alleles exist in both. The benzimidazoles (BZ), which have been used for more than 40 years, were chosen as an example. BZ-sensitive (BZ(S)) and BZ-resistant (BZ(R)) nematodes that parasitize sheep have been distinguished at the molecular level by a single nucleotide change in the codon for amino acid 200 of a beta-tubulin gene, a switch from TTC (phenylalanine) to TAC (tyrosine). PCR primers were designed to completely conserved regions of trichostrongyloid beta-tubulin genes and were used to amplify DNA fragments from Haemonchus contortus (cDNA from a BZ(S) and a BZ(R) library) as positive controls. The technique was then extended to the cattle parasites, Cooperia oncophora and Ostertagia ostertagi (from genomic DNA). Sequence analysis proved the presence of amplified BZ(S) alleles in all three species and BZ(R) alleles in the BZ(R) population of H. contortus. Based on these data, nested PCR primers using the diagnostic T or A as the most 3' nucleotide were designed for each species. Conditions for selective PCR were determined. To demonstrate feasibility, genomic DNA was recovered from individual H. contortus L(3) larvae from both BZ(S) and BZ(R) populations. Genomic DNA was also isolated from >70 individual adult male C. oncophora collected from a cattle farm in New Zealand with reported BZ resistance. Allele-specific PCR discriminated among heterozygotes and homozygotes in both species. This method could find utility in studying the molecular epidemiology of BZ resistance in cattle parasites and for defining the variables that limit the development and spread of anthelmintic resistance in this host.

The ovijector of Ascaris suum: multiple response types revealed by Caenorhabditis elegans FMRFamide-related peptides.

Caenorhabditis elegans possesses 22 FMRFamide-like peptide (flp) genes predicted to encode 60 different FMRFamide-related peptides with a range of C-terminal signatures. Peptides from five flp genes (1, 6, 8, 9 and 14) are known to modulate the ovijector of Ascaris suum in vitro. This study examines the physiological effects of peptides from the remaining 17 flp genes such that the variety of FMRFamide-related peptide-induced ovijector response types can be delineated. Five categories of response were identified according to the pattern of changes in contractile behaviour and baseline tension. Peptides encoded on 16 flp genes (1, 2, 3, 4, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 20) had qualitatively similar inhibitory (response type 1) actions, with the lowest activity thresholds (1 nM) recorded for peptides with FIRFamide or FLRFamide C-terminal signatures. Peptides encoded on four flp genes (2, 18, 19 and 21), and on the A. suum afp-1 gene, had excitatory actions on the ovijector (response type 2), with PGVLRFamides having the lowest activity threshold (1 nM). An flp-2 peptide (LRGEPIRFamide) induced a transient contraction of the ovijector (activity threshold, 10nM) that was designated response type 3. Response type 4 comprised a transient contraction followed by an extended period of inactivity and was observed with peptides encoded on flp-5 (AGAKFIRFamide, APKPKFIRFamide), flp-8 (KNEFIRFamide) and flp-22 (SPSAKWMRFamide). SPSAKWMRFamide was the most potent peptide tested with an activity threshold of 0.1 nM. A single peptide (AMRNALVRFamide; activity threshold 0.1 microM), encoded on flp-11, induced response type 5, a shortening of the ovijector coupled with an increase in contraction frequency. Although most flp genes encode structurally related peptides that trigger one of the five ovijector response types, flp-2 and flp-11 co-encode FMRFamide-related peptides that induce distinct responses. Within the ovijector of A. suum FaRPs play a complex role involving at least five receptor subtypes or signalling pathways.

Effects of KHEYLRFamide and KNEFIRFamide on cyclic adenosine monophosphate levels in Ascaris suum somatic muscle.

KHEYLRF-NH(2) (AF2) is a FMRFamide-related peptide (FaRP) present in parasitic and free-living nematodes. At concentrations as low as 10 pM, AF2 induces a biphasic tension response, consisting of a transient relaxation followed by profound excitation, in neuromuscular strips prepared from Ascaris suum. In the present study, the effects of AF2 on cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP) and inositol-1,4,5-triphosphate (IP(3)) levels were measured following muscle tension recordings from 2 cm neuromuscular strips prepared from adult A. suum. AF2 induced a concentration- and time-dependent increase in cAMP, beginning at 1 nM; cAMP levels increased by 84-fold following 1 h exposure to 1 microM AF2. cGMP and IP(3) levels were unaffected by AF2 at concentrations

Caenorhabditis elegans: how good a model for veterinary parasites?

The organism about which most is known on a molecular level is a nematode, the free-living organism Caenorhabditis elegans. This organism has served as a reasonable model for the discovery of anthelmintic drugs and for research on the mechanism of action of anthelmintics. Useful information on mechanisms of anthelmintic resistance has also been obtained from studies on C. elegans. Unfortunately, there has not been a large-scale extension of genetic techniques developed in C. elegans to research on parasitic species of veterinary (or human) parasites. Much can be learned about the essentials of nematode biology by studying C. elegans, but discovering the basic biology of nematode parasitism can only be gained through comparative studies on multiple parasitic species.

Isolation and preliminary biological assessment of AADGAPLIRFamide and SVPGVLRFamide from Caenorhabditis elegans.

To date, 9 FMRFamide-related peptides (FaRPs) have been structurally characterised from Caenorhabditis elegans. Radioimmunometrical screening of an ethanolic extract of C. elegans revealed the presence of two additional FaRPs that were purified by reverse-phase HPLC and subjected to Edman degradation analysis and gas-phase sequencing. Unequivocal primary structures for the two FaRPs were determined as Ala-Ala-Asp-Gly-Ala-Pro-Leu-Ile-Arg-Phe-NH(2) and Ser-Val-Pro-Gly-Val-Leu-Arg-Phe-NH(2). Using MALDI-TOF mass spectrometry, the molecular masses of the peptides were found to be 1032 Da (MH) and 875 Da (MH)(+), respectively. Two copies of AADGAPLIRFamide are predicted to be encoded on the precursor gene termed flp-13, while one copy of SVPGVLRFamide is located on flp-18. Synthetic replicates of the peptides were tested on Ascaris suum somatic muscle to assess bioactivity. ADDGAPLIRFamide had inhibitory effects on A. suum muscle strips, which occurred over a range of concentrations from a threshold for activity of 10 nM to 10 microM. SVPGVLRFamide was excitatory on A. suum somatic musculature from a threshold concentration for activity of 1 nM to 10 microM. The inhibitory and excitatory effects of AADGAPLIRFamide and SVPGVLRFamide, respectively, were the same for dorsal and ventral muscle strips as well as innervated and denervated preparations, suggesting that these physiological effects are not nerve cord dependent. Addition of ADDGAPLIRFamide (10 microM) to muscle strips preincubated in high-K(+) and -Ca(2+)-free medium resulted in a normal inhibitory response. Peptide addition to muscle strips preincubated in Cl(-)-free medium showed no inhibitory response, suggesting that the inhibitory response of the peptide may be chloride mediated. A normal excitatory response was noted following the addition of 10 microM SVPGVLRFamide to muscle strips preincubated in high-K(+), Ca(2+)- and Cl(-)-free media.

Individual expression of recombinant alpha- and beta-tubulin from Haemonchus contortus: polymerization and drug effects.

Three tubulin isotypes from the parasitic nematode Haemonchus contortus were individually expressed in Escherichia coli, purified, and induced to polymerize into microtubules in the absence of microtubule-associated proteins. The effect of different conditions on the rate of polymerization of pure tubulin was assessed. This is the first time that recombinant alpha-tubulin has been shown to be capable of polymerization into microtubule-like structures when incubated with recombinant beta-tubulin. In addition, the present study has shown that: (1) microtubule-associated proteins are not required for tubulin polymerization; and (2) pure beta-tubulin isotype, beta12-16, alone was capable of forming microtubule-like structures in the absence of alpha-tubulin. Polymerization of the recombinant invertebrate tubulin, as measured by a spectrophotometric assay, was found to be enhanced by a concentration of tubulin >0.25 mg/mL; temperature > or =20 degrees C; 2 mM GTP; glycerol; EGTA; and Mg(2+). Polymerization was inhibited by GTP (>2 mM) and albendazole. Calcium ions and a pH range of 6 to 8.5 had no measurable effect on polymerization. Individual isotypes of tubulin polymerized to approximately the same extent as an alpha-/beta-tubulin mixture. Samples of tubulin assembled under the above conditions for 60 min were also examined under a transmission electron microscope. Although the spectrophotometric assay indicated polymerization, it did not predict the structure of the polymer. In many cases tubulin sheets, folded sheets, and rings were observed in addition to, or instead of, microtubule-like structures.

Classical neurotransmitters in the ovijector of Ascaris suum: localization and modulation of muscle activity.

Ascaris suum possesses a well-developed nervous system which is regulated by a number of classical neurotransmitters including acetylcholine (ACh), gamma-aminobutyric acid (GABA), glutamate and serotonin. The vagina vera, the distal part of the ovijector, displays intrinsic, rhythmic activity which has been shown to be modulated by FMRFamide-related peptides (FaRPs) in vitro. Confocal scanning laser microscopy coupled with immunocytochemistry, and histochemical studies, revealed that the nerve plexus of the ovijector contains GABAergic and glutamatergic innervation. Although no distinctive cholinergic or serotoninergic innervation was apparent, cholinesterase activity was localized to discrete areas of the musculature of the vagina vera. The effects of classical transmitters on the activity of the vagina vera in vitro were examined. ACh was excitatory, stimulating a brief but powerful contraction of the vagina vera with a threshold for activity of 1 microM. Both GABA and glutamate were inhibitory, causing a cessation of contractile activity at high concentrations (> 10 microM). Although less potent than glutamate, GABA had more profound effects and induced longer-lasting paralysis of the tissue. The threshold concentrations for activity were 5 microM for glutamate and 10 microM for GABA. Serotonin had no consistent effect on the vagina vera. This study demonstrates that classical transmitters modulate the activity of the ovijector of A. suum.

Nematode neuropeptide modulation of the vagina vera of Ascaris suum: in vitro effects of PF1, PF2, PF4, AF3 and AF4.

Ascaris suum possesses a large number of FMRFamide-related peptides (FaRPs) of which KNEFIRFamide (AF1), KHEYLRFamide (AF2) and KSAYMRFamide (AF8/PF3) have been shown to modulate the intrinsic, rhythmic activity of the vagina vera of A. suum in vitro. In the present study, the effects of the nematode FaRPs, SDPNFLRFamide (PF1), SADPNFLREamide (PF2) and KPNFIRFamide (PF4) (from Panagrellus redivivus) and AVPGVLRFamide (AF3) and GDVPGVLRFamide (AF4) (from A. suum) on the in vitro activity of the vagina vera were examined. The effects of each of the peptides were qualitatively and quantitatively distinct. All 3 FaRPs from P. redivivus were inhibitory, causing a cessation of contractions. PF2 was 3 times more potent than PF1, with a threshold of 1 nM. Although PF4 was the least potent (threshold, 10 nM), its effects at > or = 10 nM were quantitatively the greatest. Both AF3 and AF4 (1 microM) induced complex, multiphasic responses consisting of an initial contraction and spastic paralysis followed by a return of contractile activity of increased amplitude. AF3 was 3 times more potent than AF4. The effects of these peptides had some similarities to those observed on A. suum somatic body wall muscle in vitro, with PF1, PF2 and PF4 being inhibitory and AF3 and AF4 being excitatory.

Transport of model peptides across Ascaris suum cuticle.

Several FMRFamide-related peptides (FaRPs) found in nematodes exert potent excitatory or inhibitory effects on the somatic musculature of Ascaris suum and other nematode species when injected into the pseudocoelom or applied directly to isolated neuromuscular preparations. These peptides, however, generally fail to induce detectable effects on the neuromusculature when applied externally to intact nematodes. The apparent lack of activity for these peptides when administered externally in whole-organism assays is likely a function of both absorption and metabolism. To delineate the factors that govern transport of peptides across the cuticle/hypodermis complex of nematodes, we measured the rates of absorption of a series of structurally related model peptides using isolated cuticle/hypodermis segments from A. suum and two-chamber diffusion cells. [14C]-Labeled peptides were prepared from D-phenylalanine, with the amide nitrogens sequentially methylated to give AcfNH2, Acf3NH2, Acf(NMef)2NH2, and Ac(NMef)3NHMe. These model peptides were designed to allow systematic analysis of the influence of peptide size, hydrogen bonding and lipophilicity on transport. Results of these studies show that, within this series, permeability across the cuticle increases with addition of each methyl group. The permeability coefficient of Ac(NMef)3NHMe, with four methyl groups, was 10-fold greater than that of the smaller peptide, AcfNH2, even though both peptides contain five hydrogen bonds. When compared with vertebrate membranes, transport of the model peptides across A. suum cuticle was about 10-fold slower. A biophysical model for transcuticular transport of peptides predicted that nematode FaRPs, which are larger, less methylated and less lipophilic than the model peptides tested, would not be absorbed across the cuticle of nematodes. This prediction was confirmed for the excitatory FaRP, AF2 (KHEYLRFamide), which did not diffuse across the cuticle/hypodermis complex, but diffused rapidly across lipid-extracted cuticle preparations.

Frontiers in anthelmintic pharmacology.

Research in anthelmintic pharmacology faces a grim future. The parent field of veterinary parasitology has seemingly been devalued by governments, universities and the animal industry in general. Primarily due to the success of the macrocyclic lactone anthelmintics in cattle, problems caused by helminth infections are widely perceived to be unimportant. The market for anthelmintics in other host species that are plagued by resistance, such as sheep and horses, is thought to be too small to sustain a discovery program in the animal health pharmaceutical industry. These attitudes are both alarming and foolish. The recent history of resistance to antibiotics provides more than adequate warning that complacency about the continued efficacy of any class of drugs for the chemotherapy of an infectious disease is folly. Parasitology remains a dominant feature of veterinary medicine and of the animal health industry. Investment into research on the basic and clinical pharmacology of anthelmintics is essential to ensure chemotherapeutic control of these organisms into the 21st century. In this article, we propose a set of questions that should receive priority for research funding in order to bring this field into the modern era. While the specific questions are open for revision, we believe that organized support of a prioritized list of research objectives could stimulate a renaissance in research in veterinary helminthology. To accept the status quo is to surrender.

Bombesin-like neuropeptides in nematodes.

This paper reports evidence of members of the bombesin-like peptide family in a number of nematodes, including Caenorhabditis, Panagrellus, Dirofilaria, Onchocerca, Brugia, Haemonchus, Ostertagia, Toxocara, and Ascaris. One of these (Ostertagia) secretes the bombesin-like material into its environment. Specific binding of gastrin-releasing peptide to the hypodermis consistent with the presence of receptors was demonstrated. These data suggest that this class of peptides may play an important role in nematode hypodermal physiology.

Synthesis and biological activity of anthelmintic thiadiazoles using an AF-2 receptor binding assay.

Following our discovery of the strong binding of thiadiazole 1 to the AF-2 neuropeptide receptor of gastrointestinal nematodes (e.g., Ascaris suum), we prepared two series of analogs. Only the series containing the thiadiazole ring had potencies comparable to that of compound 1. Analog 50 exhibited an apparent potency in the AF-2 binding assay 300 times that of compound 1.

Structural characterisation and pharmacology of KHEYLRFamide (AF2) and KSAYMRFamide (PF3/AF8) from Haemonchus contortus.

The FMRFamide-related peptides (FaRPs), KHEYLRFamide (AF2) and KSAYMRFamide (PF3) were structurally characterised from the parasitic nematode of sheep, Haemonchus contortus (MH isolate). Both peptides were sequenced in a single gas-phase sequencing run and their structure confirmed by mass spectrometry which identified peptides of 920 Da (C-terminally amidated AF2) and 902/918 Da (C-terminally amidated non-oxidised/oxidised PF3, respectively). AF2 had inhibitory effects on H. contortus muscle and inhibited acetylcholine (ACh, 10 microM)-induced contractions, with a threshold for activity of 1 microM. PF3 induced concentration-dependent contractions of H. contortus (activity threshold, 10 nM) and enhanced ACh contractions. Compared with the MH isolate, an isolate of H. contortus which has reduced sensitivity to cholinergic drugs (Lawes isolate) was less sensitive to the effects of PF3. The concentration-response curves for the cholinergic compounds ACh and levamisole (LEV), and PF3, but not a control, KPNFIRFamide (PF4), showed a statistically similar shift. This study implicates PF3 in the modulation of cholinergic function in H. contortus.