Marker genes for activation of the RNA interference (RNAi) pathway in the free-living nematode Caenorhabditis elegans and RNAi development in the ovine nematode Teladorsagia circumcincta.

The nematode Teladorsagia circumcincta is a major cause of parasitic gastroenteritis in sheep in temperate regions. The development of resistance to the major anthelmintic classes used for its control is a threat to small ruminant farming sustainability. Vaccination is a potential alternative control method for this nematode. Gene datasets can be exploited to identify potential vaccine candidates and these validated further by methods such as RNA interference (RNAi) prior to vaccine trials. Previous reports indicate that RNAi in parasitic nematodes is inconsistent and, to date, there are no internal controls that indicate activation of the RNAi pathway in response to double-stranded RNA (dsRNA). The present aims were to determine whether or not the transcription levels of potential marker genes in the RNAi pathway could indicate activation of the pathway in Caenorhabditis elegans and to develop an RNAi platform in T. circumcincta. In C. elegans, transcript levels of three candidate marker genes, Ce-dcr-1 (Dicer), Ce-ego-1 (Enhancer of Glp-One family member) and Ce-rsd-3 (RNAi Spreading Defective), were analysed and results indicated that activation of the pathway had no effect on transcript levels of these genes. In T. circumcincta, two vaccine candidate genes from the Activation-associated Secreted Protein (ASP) family were targets for knockdown. RNAi experiments showed successful silencing of both targets, although inconsistencies in efficacy were observed. After testing a number of parameters that might affect variability, it was found that the length of the storage period of the larvae plays an important role in the consistency of the RNAi results.

Amelioration of the periparturient relaxation of immunity to parasites through a reduction in mammalian reproductive effort.

The degree of periparturient relaxation of immunity to gastrointestinal parasites has a nutritional basis, as overcoming protein scarcity through increased protein supply improves lactational performance, enhances local immune responses and reduces worm burdens. Herein lactating rats, re-infected with Nippostrongylus brasiliensis, are used to test the hypothesis that a similar and rapid improvement of immunity can be achieved through reducing nutrient demand at times of dietary protein scarcity. Reducing litter size from 12 to three pups during lactation resulted, as expected, in cessation of maternal body weight loss and increased pup body weight gain compared with dams which continued to nurse 12 pups. This increase in performance concurred with a rapid decrease in parasitism; within 3 days post nutrient reduction, a 87% reduction in the number of worm eggs found in the colon and 83% reduction in worm burdens was observed, which concurred with increased local immune responses, i.e. 70% more mast cells and 44% more eosinophils in the small intestinal mucosa, to levels similar to those in dams nursing three pups throughout. However, there were no concurrent changes in goblet cell hyperplasia, serum anti-N. brasiliensis-specific antibody levels or mRNA expression of IL-4, IL-10 or IL-13 in the mesenteric lymph nodes. To our knowledge the current study is the first to employ a litter reduction strategy to assess the rate of immune improvement upon overcoming nutrient scarcity in a non-ruminant host. These data support the hypothesis that periparturient relaxation of immunity to gastrointestinal nematodes can be reduced by restoring nutrient adequacy and, importantly, that this improvement can occur very rapidly.

Dissecting the impact of protein versus energy host nutrition on the expression of immunity to gastrointestinal parasites during lactation.

Many mammals exhibit a periparturient relaxation of previously established immune responses (PPRI) to gastrointestinal nematodes culminating in increased worm burdens. It has been suggested that the extent of PPRI may have a nutritional basis as it is considerably augmented when protein supply is scarce. Subsequent studies have shown that increased dietary protein intake can ameliorate this phenomenon. However, this effect is often confounded with increased food intake and thus increased energy levels. Herein, we aimed to dissect the effects of protein and energy nutrition on the immune status and resistance to re-infection with gastrointestinal nematodes in the periparturient host. The lactating, Nippostrongylus brasiliensis re-infected rat was utilised as an established model for mammalian PPRI. Experimental animals were assigned to restricted feeding regimens designed to achieve four pre-determined levels of crude protein (CP) at one of two levels of metabolisable energy (ME) and parasitological and immunological measurements taken at either day 6 or day 9 post re-infection. We clearly show that increased supply of dietary CP, but not increased dietary ME, significantly reduced worm burdens. The increased magnitude of worm expulsion with increased dietary CP supply strongly correlated with mucosal mast cell accumulation in the small intestine. In addition, increased CP and not ME supply increased mucosal eosinophil numbers. Furthermore, increased CP led to higher levels of total IgG at high ME only and there were interactive effects of CP and ME on serum levels of IgG1 and IgG2a. Perhaps surprisingly, CP nutrition did not affect expression of either Th1 (IFN-γ) or Th2 (IL-4, IL-13) cytokines in the mesenteric lymph nodes. These data emphasise the role of immunonutrition, and particularly dietary protein, in combating infectious disease such as gastrointestinal parasitism.

A calcium-activated apyrase from Teladorsagia circumcincta: an excretory/secretory antigen capable of modulating host immune responses?

A cDNA representing the gene Teladorsagia circumcincta apyrase-1 (Tci-apy-1) was isolated, by PCR, from a T. circumcincta fourth-stage larval (L4) cDNA library. The closest orthologue of this gene is a Ca(2+)-dependent apyrase from Ostertagia ostertagi, with 92% amino acid identity across all 339 residues. Tci-apy-1 is transcribed in a stage-specific manner, the transcript being predominant in L4, detectable in the adult cDNA, but absent from eggs and infective third-stage larvae (L3). The protein, Tci-APY-1, was detected by immunoblotting in extracts of L4 nematodes and was present in excretory/secretory products from the same developmental stage. A recombinant version of Tci-APY-1 was expressed in bacteria as an active enzyme that hydrolysed nucleoside triphosphate substrates with a preference of ATP over other nucleoside triphosphates. Recombinant Tci-APY-1 hydrolysed ATP and ADP but not AMP. Apyrase activity was divalent cation-dependent, with no hydrolysis in the presence of Mg(2+), but activation in the presence of Ca(2+). Recombinant Tci-APY-1 was bound by IgG present in serum and both IgG and IgA present in abomasal mucus from trickle-infected, immune sheep but not in material derived from lambs exposed to a single infection. The potential immunomodulatory roles of this Tci-APY-1 are discussed in relation to purinergic signalling.

Using lectins to identify hidden antigens in Fasciola hepatica.

Fasciola hepatica is the causative agent of fascioliasis, one of the most economically important helminth diseases of livestock worldwide. Traditionally, fascioliasis has been controlled by the strategic use of fasciolicidal drugs, but the emergence of resistant parasites has spurred an interest in developing vaccines as an alternative means of control. Most vaccine studies to date have evaluated conventional antigens, which are exposed to the host's immune system during the course of a natural infection. 'Hidden' antigens have proven to be effective vaccine candidates in other parasite species, most notably the blood-feeding nematode parasite, Haemonchus contortus, and tend to be expressed in the intestine or gut of the parasite. Fasciola hepatica is known to ingest large quantities of blood and may be vulnerable to this approach. Most, if not all, of the candidate antigens identified thus far have been membrane-bound glycoproteins which were solubilized by detergents. Here, we have attempted to employ lectins to select gut-associated glycoproteins from complex mixtures of somatic extracts of adult F. hepatica. We have conducted a comprehensive lectin-binding screen on adult histological sections with a panel of 16 fluorescently labelled lectins. Seven of the lectins bound to molecules within the gastrodermis but also bound to a range of other tissues. Within the gut tissues, jacalin and peanut lectins bound selectively to the gut lamellae and gastrodermal cells, respectively. These lectins were then used to isolate proteins from the integral membrane protein component of the adult fluke. Both lectins showed selectivity for relatively simple subsets of proteins compared to the original crude extracts.

A macrophage migration inhibitory factor-like tautomerase from Teladorsagia circumcincta (Nematoda: Strongylida).

A macrophage migration inhibitory factor (MIF)-like molecule, Tci-MIF-1, was isolated from Teladorsagia circumcincta and subjected to detailed characterization. A cDNA representing Tci-mif-1 was isolated following its identification in third-stage larvae (L3)-enriched cDNA population. Sequencing of the cDNA indicated a 348-bp open reading frame (ORF) with the closest orthologue being a MIF derived from the human hookworm Ancylostoma ceylanicum. Messenger RNA (mRNA) representing the Tci-MIF-1 transcript was detected in eggs, L3 and adult stages of T. circumcincta. The transcript was also present, but to a lesser extent in fourth-stage larvae (L4). Detection of Tci-MIF-1 protein in T. circumcincta developmental stages reflected the transcript levels identified by reverse transcriptase-PCR. Using immunohistochemistry, the Tci-MIF-1 protein was shown to have a diffuse distribution in L3 tissue, and in L4 and adult stages, the protein was localized to the nematode gut. A recombinant version of Tci-MIF-1 was produced, and enzymic assays indicated that this recombinant protein and a somatic extract of L3 possessed dopachrome tautomerase activity as has been observed previously in other MIF-like molecules. Neither native, purified Tci-MIF nor recombinant Tci-MIF-1 dramatically influenced the in vitro migration of sheep monocytes.

daf-7-related TGF-beta homologues from Trichostrongyloid nematodes show contrasting life-cycle expression patterns.

The transforming growth factor-beta (TGF-beta) gene family regulates critical processes in animal development, and plays a crucial role in regulating the mammalian immune response. We aimed to identify TGF-beta homologues from 2 laboratory model nematodes (Heligmosomoides polygyrus and Nippostrongylus brasiliensis) and 2 major parasites of ruminant livestock (Haemonchus contortus and Teladorsagia circumcincta). Parasite cDNA was used as a template for gene-specific PCR and RACE. Homologues of the TGH-2 subfamily were isolated, and found to differ in length (301, 152, 349 and 305 amino acids respectively), with variably truncated N-terminal pre-proteins. All contained conserved C-terminal active domains (>85% identical over 115 amino acids) containing 9 cysteine residues, as in C. elegans DAF-7, Brugia malayi TGH-2 and mammalian TGF-beta. Surprisingly, only the H. contortus homologue retained a conventional signal sequence, absent from shorter proteins of other species. RT-PCR assays of transcription showed that in H. contortus and N. brasiliensis expression was maximal in the infective larval stage, and very low in adult worms. In contrast, in H. polygyrus and T. circumcincta, tgh-2 transcription is higher in adults than infective larvae. The molecular evolution of this gene family in parasitic nematodes has diversified the pre-protein and life-cycle expression patterns of TGF-beta homologues while conserving the structure of the active domain.

Immunomodulatory effects of dietary protein during Nippostrongylus brasiliensis re-infection in lactating rats.

Periparturient relaxation of immunity (PPRI) to secondary infection with nematodes is believed to have a nutritional basis due to differential partitioning of scarce nutrient resources, particularly protein, to reproductive rather than immune functions. At times of protein scarcity, an increase in protein supply has been reported to assuage this phenomenon. The Nippostrongylus brasiliensis reinfected lactating rat model is now being utilized to investigate the immune reactions underlying the modifying role of dietary protein on PPRI. Herein, we demonstrate that lactating rats reinfected with N. brasiliensis under high protein (HP) dietary conditions exhibit decreased worm burdens and reduced colon egg counts compared to their low protein (LP) counterparts. These reductions correlated with increased mastocytosis and greater goblet cell hyperplasia. Additionally, the local antibody profile revealed that HP reinfected lactating rats developed a stronger antigen specific IgG2b response earlier in infection in comparison with their LP counterparts. Our study provides evidence that increased dietary protein content reduces the PPRI to N. brasiliensis re-infection in the lactating rat through improved mucosal immune responses.

Molecular characterization, expression and localization of a peroxiredoxin from the sheep scab mite, Psoroptes ovis.

The sheep scab mite, Psoroptes ovis, induces an intensely pruritic exudative dermatitis which is responsible for restlessness, loss of appetite and weight loss. Within the first 24 h of infection, there is a rapid inflammatory influx of eosinophils and apoptosis of the keratinocytes at the site of infection. The former cell type is capable of a sustained respiratory burst, toxic products of which may directly damage the mite and also contribute to lesion formation. Analysis of a P. ovis expressed sequence tag (EST) database identified a number of antioxidant enzyme-encoding sequences, including peroxiredoxin (thioredoxin peroxidase EC 1.11.1.15), all of which may help the mite endure the potentially toxic skin environment. A full length sequence encoding Po-TPx, a protein of 206 amino acids which showed high homology to a peroxiredoxin from the salivary gland of the tick Ixodes scapularis, was amplified from P. ovis cDNA. Recombinant Po-TPx was expressed in bacteria and antiserum to this protein was used to localize native Po-TPx in mite sections. Peroxiredoxin was localized, amongst other sites, to a subpharyngeal region in mite sections. The recombinant protein was recognized by sera from sheep infested with the mite suggesting that it may be secreted or excreted by the mite and interact with the host immune response.

Immune recognition of the surface associated antigen, Tc-SAA-1, from infective larvae of Teladorsagia circumcincta.

A cDNA encoding a surface-associated antigen was amplified by reverse transcriptase polymerase chain reaction (PCR) from RNA extracted from Teladorsagia circumcincta exsheathed third stage larvae (xL3). The protein encoded by this cDNA, Tc-SAA-1, displays 77% identity over 162 amino acid residues to a surface associated antigen from Ancylostoma caninum (Ac-SAA-1). Antiserum raised against a bacterially-expressed recombinant form of Tc-SAA-1 reacted with a native protein in somatic and surface extracts of xL3 but not with L4 or adult parasites. Limited binding of anti-Tc-SAA-1 antibody was observed on the cuticular surface of xL3 s, however, regions of localization underlying the cuticle were observed. Incubation of xL3 T. circumcincta with anti-SAA rabbit serum failed to significantly inhibit penetration of the abomasal mucosa in vitro. IgA in abomasal mucus derived from sheep that had received a trickle infection of T. circumcincta bound recombinant Tc-SAA-1.

Analysis of the transthyretin-like (TTL) gene family in Ostertagia ostertagi--comparison with other strongylid nematodes and Caenorhabditis elegans.

The transthyretin-like (ttl) gene family is one of the largest conserved nematode-specific gene families, coding for a group of proteins with significant sequence similarity to transthyretins (TTR) and transthyretin-related proteins (TRP). In the present study, we investigated the ttl family in Ostertagia ostertagi (a nematode of the abomasum of cattle). Mining of expressed sequence tag (EST) databases revealed the presence of at least 18 ttl genes in O. ostertagi (Oo-ttl), most of which are constitutively transcribed from the free-living, third larval stage onwards. The full-length cDNA of one of these genes (Oo-ttl-1) was amplified and cloned for recombinant expression. Western blot analysis using a specific antiserum showed that the native protein Oo-TTL-1 was highly present in the excretory-secretory (ES) products of adults of O. ostertagi. The protein was immunolocalized to the pseudocoelomic fluid of adult worms. A phylogenetic-bioinformatic analysis of all amino acid sequence data for TTL proteins from a range of strongylid nematodes showed that they could be divided into at least five different classes. This classification was based on conserved amino acids in the first TTL signature domain and the number and location of cysteine residues. The biological role(s) of the TTLs in nematode biology is still unclear. A theoretical three-dimensional model of Oo-TTL-1 indicated that it had a similar structure to TTRs (i.e., containing ß-sheets, arranged in a ß-sandwich). In contrast to TTRs, competitive binding studies using recombinant Oo-TTL-1 indicated that the protein was devoid of any hydrophobic ligand- or thyroid hormone-binding properties. Finally, combinatorial analysis by double-stranded RNA interference of five ttl genes in the free-living nematode Caenorhabditis elegans did not reveal any visible phenotypes. More information on the transcription profile and tissue distribution of TTLs in nematodes is needed to provide new insights into the biological role of this gene family.

Control of parasitic disease using vaccines: an answer to drug resistance?

Antiparasitic drugs have been used successfully to control parasitic diseases in animals for many years, as they are safe, cheap and effective against a broad spectrum of parasites. One drawback of this success appears to be the emergence of drug resistance in many target parasites. Moreover, issues of residues in the food chain and environment have arisen, which threaten their sustained use. Control methods in which vaccines would have a central role provide attractive alternatives. However, while attenuated parasite vaccines have been successful, sub-unit vaccines are still rare. The advent of new techniques in molecular biology allows the elucidation of entire parasite genomes and the identification of individual genes. It is envisaged that a further understanding of parasite genes and the role of their products in parasite biology may lead to the identification of useful antigens, which could then be produced in recombinant systems. However, for this aim to be realised, continued investment in basic research on the complex interplay between parasite and host will be necessary.

The effect of vaccination with a recombinant Nippostrongylus brasiliensis acetylcholinesterase on infection outcome in the rat.

Nippostrongylus brasiliensis, the rodent hookworm, is a commonly used model of gastrointestinal nematode infection. This parasite, hookworms and several livestock nematode parasites of importance secrete distinct forms of acetylcholinesterases (AChE) that have been ascribed a putative parasite protective function. We tested the hypothesis that vaccination with the secreted enzyme would be deleterious to the parasite. Rats were immunised with a recombinant AChE isoform B via the subcutaneous, intra-peritoneal and intra-nasal routes using different adjuvants dependent on the mode of delivery and subsequently challenged with N. brasiliensis. Rats immunised via the subcutaneous and intra-nasal routes showed a modest but significant decrease in egg output of between 23 and 48%. This was mirrored by differences in the titre of specific antibody isotypes in the serum and mucosa following infection and serum from vaccinated animals was demonstrated to inhibit the activity of recombinant and native AChE. The utility of this model for future development of hookworm and veterinary nematode vaccines is discussed.

Proteinase inhibitors and helminth parasite infection.

The concept that parasites may utilize proteinase inhibitors to survive within the host has been with us for 100 years. Given that we now know that proteinases are involved in key areas of the host anti-parasite immune response including antigen presentation, effector cell function and tissue dissolution and remodelling, it is somewhat surprising that the proteinase inhibitors of parasite origin have not generally been the subject of intense research effort. There is now substantial evidence to show that nematode parasites utilize these inhibitors to protect themselves from degradation by host proteinases, to facilitate feeding and to manipulate the host response to the parasite. The diversity of the parasite-derived inhibitors is also being revealed and they target the four major proteinase classes, namely serine, cysteine, aspartic and metallo-proteinases. This review summarizes the information available on nematode-derived proteinase inhibitors and what is known of their putative functions. Their potential as targets for immunological control is also addressed.

Efficacy and specificity of RNA interference in larval life-stages of Ostertagia ostertagi.

RNA interference (RNAi) on parasitic nematodes has been described as successful and useful for the identification of novel drug and vaccine candidates. In this study we have evaluated this technology on the cattle parasite Ostertagia ostertagi. Eight different genes were targeted in L1 and L3 O. ostertagi larvae, by electroporation and soaking in dsRNA respectively. Down-regulation of target transcript levels was evaluated by semi-quantitative reverse transcriptase (RT) PCR. In L3 larvae, variable decreases in mRNA levels were observed for 5 genes, ranging from a complete knock down (tropomyosin, beta-tubulin) to a minor decrease (ATPsynthase, superoxide dismutase, polyprotein allergen). However, repeated experiments indicated that effects were sometimes difficult to reproduce. RNAi for ubiquitin, a transthyretin-like protein and a 17 kDa excretion secretion (ES) protein never resulted in a knock down of the transcript. The mRNA levels of 7 non-target genes showed no difference between larvae soaked in C. elegans control dsRNA versus O. ostertagi tropomyosin dsRNA, supporting that the observed reductions are specific for the target gene. Electroporation of L1 larvae proved to be less effective. Reductions in mRNA levels were only noticed for 2 genes and were not reproducible. In conclusion, the results indicate that the RNAi pathway is probably present in O. ostertagi but that the current RNAi techniques can not be used as a reliable screening method.

Identification and characterization of an asparaginyl proteinase (legumain) from the parasitic nematode, Haemonchus contortus.

Asparaginyl proteinases (or legumains) are a recently identified, novel class of cysteine proteinase which specifically hydrolyse peptide bonds after asparagine residues. Legumains have been implicated in the activation of cysteine proteases, particularly cathepsin B-like proteinases which are thought to help degrade the bloodmeal in blood-feeding helminths such as schistosomes, hookworms and other nematode species. An EST sequence representing a full-length legumain was identified from the Haemonchus contortus dataset. This encoded a protein with a predicted Mr of 49 kDa, the amino acid sequence of which showed good homology (34-40% identity) to legumains from Schistosoma mansoni, human and rat and contained a legumain-like active site. RT-PCR indicated that the legumain transcript was expressed from the L4 life-cycle stage onwards. The coding sequence was expressed in E. coli and antibodies to the resultant recombinant protein indicated that the enzyme was expressed in the microvillar surface of the intestinal cells. Legumain activity was detected in extracts of the adult parasite but not the host protective Thiol-Sepharose-binding fraction, although it was detectable in the latter by immunoblot. Activity was relatively insensitive to E64, an inhibitor of cysteine proteinases and completely inhibited by the alkylating agent, N-ethylmaleimide, consistent with inhibitor effects on previously characterized legumains.

Further protection studies using recombinant forms of Haemonchus contortus cysteine proteinases.

Significant levels of protection against Haemonchus contortus have been achieved in sheep by vaccination with a cysteine proteinase-enriched fraction (TSBP) isolated from the gut of adult parasites. Protection is associated with three cathepsin B-like cysteine proteinases (hmcp 1, 4 & 6). Lambs vaccinated with these proteinases, expressed in bacteria as glutathione S-transferase fusion proteins, had significantly reduced (38%) worm burdens compared to challenge controls although, intriguingly, egg output was unaffected. Here, a repeat trial with similar results is reported and protection obtained compared to that induced by vaccination with the predicted mature forms of hmcp1, 4 and 6 expressed in bacteria as non-fusion proteins. Sheep immunized with a cocktail of these non-fusion proteins had reduced faecal egg counts of 27% (P = 0.17) and worm burdens of 29% (P = 0.01) compared to controls. High levels of host serum IgG were detected in GST-hmcp and non-fusion hmcp-immunized animals, although no correlation with protection could be determined. Sera from these groups bound to the microvillar surface of the gut of H. contortus.

A preliminary proteomic survey of the in vitro excretory/secretory products of fourth-stage larval and adult Teladorsagia circumcincta.

The nature of the proteins which comprise the in vitro excretory/secretory products (ES) of the fourth-stage larva (L4) and adult Teladorsagia circumcincta are largely undefined, despite the fact that this nematode induces profound changes, in part related to parasite ES, in the cellular architecture of the glands lining the abomasal surface of infected sheep and goats. In this study, the protein components of L4 and adult ES were fractionated using 1D gel electrophoresis and the major protein bands, detected by Coomassie blue staining, excised from the gel and subjected to tryptic digest and subsequent mass spectrometric analysis. The resultant peptide mass fingerprints were used to identify 15 L4 and 13 adult ES proteins. Several proteins, such as globin and some metabolic enzymes, were present in both ES. L4 ES alone contained thioredoxin peroxidase, an enzyme that can detoxify free radicals resulting from host inflammatory responses to the parasite, a cysteine proteinase which may aid penetration of the gastric mucosa and 2 different galectins which may influence cell differentiation and morphogenesis. Adult ES contained a nucleoside diphosphate kinase homologue, an enzyme which has been linked to cellular changes and can affect liquid secretion and goblet cell degranulation.

An immunogenic cathepsin F secreted by the parasitic stages of Teladorsagia circumcincta.

Teladorsagia circumcincta is a common, pathogenic abomasal nematode of sheep. In order to improve disease control in parasite isolates resistant to several anthelmintics, alternative methods must be sought. Sheep develop acquired immunity to T. circumcincta so vaccination is a valid option for control. For this reason, we are investigating parasite excretory/secretory products for molecules, which have potential to invoke protective immunity against T. circumcincta. Here, we describe experiments in which we identified a novel, immunogenic cathepsin F secreted by L4 T. circumcincta. This protease, initially identified by mass spectrometry analysis, is the most abundant molecule in excretory/secretory products released in vitro by T. circumcincta harvested at 5, 6 or 9 days p.i. and is a target of specific, local IgA responses in sheep which are immune to challenge infection. The full-length cDNA encoding this secreted protease was isolated. Sequence and phylogenetic analyses indicated that the protease (designated T. circumcincta cathepsin F-1, Tci-CF-1) belongs to the cathepsin F class and exhibits greatest identity (>60%) to expressed sequence tags present in the Ostertagia ostertagi and Haemonchus contortus expressed sequence tag databases. Tci-CF-1 also displays high identity to hypothetical proteins identified in the genomes of Caenorhabditis elegans and Caenorhabditis briggsae, both proteins having been described as cathepsin F enzymes. Specific inhibitor binding assay of larval excretory/secretory products confirmed the classification of this excretory/secretory component as a cathepsin F. Reverse transcription-PCR studies indicated that Tci-cf-1 is developmentally regulated and is particular to the host parasitic stages of T. circumcincta. The abundance, immunogenicity and temporal expression pattern of Tci-CF-1 make this a potential vaccine candidate for teladorsagiosis.