Ovine IgA-reactive proteins from Teladorsagia circumcincta infective larvae

Infection of small ruminants with Teladorsagia circumcincta has, until now, been controlled using a combination of pasture management and frequent anthelmintic treatments. Resistance to the commonly used anthelmintics has driven research into the development of a subunit vaccine, encouraged by the demonstration of development of protective immunity in sheep following exposure to this parasite. Local immune effectors in the abomasum, in particular IgA, are thought to play important roles in naturally- and experimentally-acquired immunity. L3s represent the first contact of this pathogen with the host immune system and, herein, the presence of L3 antigen-specific IgA was demonstrated in abomasal mucus from immune sheep. This antibody source was used to immunoaffinity purify and identify IgA-reactive molecules present in L3s. We identified 155 different proteins in this way, including a number of activation-associated secretory proteins, venom allergen-like-type proteins, detoxifying enzymes, galectins and a suite of other potential vaccine candidate molecules. Levels of immunoaffinity-enriched L3 antigen-specific IgA in gastric lymph from previously-infected sheep were statistically significantly higher (P = 0.004) than those measured in helminth-free sheep and a statistically significant negative correlation (P = 0.005, r_s = −0.565) was identified between immunoaffinity-enriched L3 antigen-specific IgA levels in efferent gastric lymph and total T. circumcincta burden measured at necropsy. In addition, a statistically significant positive correlation (P = 0.007, r_s = 0.534) was measured between immunoaffinity-enriched L3 antigen-specific IgA levels in efferent gastric lymph and the percentage of inhibited L4s enumerated at necropsy. These results indicate that the purified antigens contain components that could be strongly considered as vaccine candidates.     

Nitrogen excretion by the sheep abomasal parasite Teladorsagia circumcincta

Excretion of nitrogenous substances by Teladorsagia circumcincta was investigated during incubation of L3 in phosphate buffer for up to 30 h and adult worms for 4-6 h. Ammonia was the main excretory product, with about 20% urea. For the first 4-6 h, ammonia excretion by L3 was temperature dependent, directly proportional to the number of larvae, but independent of the pH or strength of the phosphate buffer. Later, ammonia excretion slowed markedly in L3 and adults and reversed to net uptake in L3 by 30 h. An initial external ammonia concentration of 600 μM did not alter the pattern or magnitude of excretion. Re-uptake of ammonia did not occur at extremes of pH or low buffer strength and was slightly reduced at the highest external concentrations. Ammonium transporters and enzymes of glutamate metabolism, including glutamate dehydrogenase, glutamine synthetase and possibly glutamate synthase, are worthy of further investigation as anthelmintic targets.     

Teladorsagia circumcincta: Survival of adults in vitro is enhanced by the presence of a mammalian cell line

Adult Teladorsagia circumcincta survival and motility in vitro was examined in a range of different cell culture media, supplements and gas mixes. Under optimum conditions, worms survived for 14 days, exhibiting high motility for 9 days and egg production for 72 h. Optimum conditions involved co-culture of worms with a HeLa cell line in a supplemented cell medium (CEM) and an atmosphere containing 10% CO₂, 5% O₂ 85% N₂, 65% humidity at 37 °C. The incubation medium consisted of Minimum Essential Medium with 10% fetal calf serum, 1% non-essential amino acids, 1% glutamax and 1% penicillin-neomycin-streptomycin cocktail mix. Compared with optimum conditions, incubation in CEM alone, cell conditioned CEM, RPMI alone, Medium 199 alone, reduced CO₂ or O₂, or when cells were replaced with Escherichia coli, both survival and motility were reduced. Optimum conditions for adult T. circumcincta maintenance for culture, anthelmintic testing or generation of excretory/secretory products are described.     

Benzimidazole resistance allele haplotype diversity in United Kingdom isolates of Teladorsagia circumcincta supports a hypothesis of multiple origins of resistance by recurrent mutation

Polymorphisms in the isotype I β-tubulin gene are important genetic determinants of benzimidazole (BZ) resistance in a number of parasitic nematode species including Teladorsagia circumcincta, a major gastrointestinal nematode of sheep. This study investigates the genetic diversity at this locus in a BZ-resistant isolate of T. circumcincta (MTci5) derived from a sheep farm in the United Kingdom (UK) that was open to animal, and therefore parasite, migration. Pyrosequencing was used to determine the frequency of single nucleotide polymorphisms (SNPs) known to be associated with BZ resistance. This was followed by a combination of single strand conformation polymorphism (SSCP) analysis and nucleotide sequencing to sample allelic diversity in a 276 bp fragment immediately surrounding the isotype I β-tubulin F200Y mutation. The genetic diversity at this locus was extremely high, with seven different haplotypes found to contain the resistant F200Y polymorphism in this single resistant isolate. Genotyping by SSCP interfaced with pyrosequencing demonstrated that the P200^Y mutation is also present on multiple haplotypes in two other BZ-resistant T. circumcincta isolates from the UK. This contrasts with much lower levels of haplotype diversity in BZ-resistant alleles present in T. circumcincta isolates from French goat farms that are closed to parasite migration. Taken together with our knowledge of T. circumcincta population genetic structure, these results are most consistent with multiple independent origins of resistance and mixing of alleles due to the large amount of livestock movement in the UK.     

Giardia duodenalis: Kinetics of cyst elimination and the systemic humoral and intestinal secretory immune responses in gerbils (Meriones unguiculatus) experimentally infected

This study aimed to determine the pre-patent period and to evaluate the kinetics of cyst elimination and the systemic humoral (IgA, IgG₁, IgG_2a, IgM, IgE) and intestinal secretory (IgA) immune responses in gerbils (Meriones unguiculatus) experimentally innoculated with different doses of Giardia duodenalis trophozoites. Forty-eight animals aged 6-8 weeks were used, equally distributed among six groups, five groups innoculated with different doses of trophozoites (10¹, 10², 10³, 10⁴, 10⁵) and one control (non-infected) group. Coproparasitological examinations were carried out daily up to 91 days after inoculation (d.a.i.) to determine the pre-patent period and the kinetics of cyst elimination. Blood and stool samples were weekly collected for antibody assays. The pre-patent period was observed from the 9 d.a.i. onwards, with intermittent elimination of variable quantities of cysts up to 27 d.a.i.. All infected gerbils, irrespective of the dose received, were able to mount systemic humoral immune responses as evidenced by specific IgM titers from 7 to 28 d.a.i., corresponding to the peak of cyst elimination, followed by high and persistent IgG1 titers. Intestinal secretory responses were also seen with two peaks of fecal IgA titers, corresponding to IgM and IgG1response peaks, respectively. In conclusion, systemic and intestinal humoral immune responses were related to the control of giardiasis in this experimental model.     

Phenotypic analysis of host-parasite interactions in lambs infected with Teladorsagia circumcincta

Female Blackface lambs expected to exhibit genetic variability for resistance to gastrointestinal nematodes, were either exposed to continuous experimental infections of Teladorsagia circumcincta or were sham-dosed to monitor phenotypic responses to infection. As a measure of parasitism and host response, worm-eggs in faeces (faecal egg count, FEC) were counted over a 3-month period and worm burdens were ascertained at post-mortem. The host response to the infection was also measured by differential counts of white blood cells, anti-T. circumcincta IgA antibody levels and body weight. Results suggest that nematode abundance (mean number of parasites per host) and prevalence (proportion of infected animals) were maximal shortly after the beginning of infection (21 days p.i.) when virtually all the infected animals were shedding worm eggs. Increasing anti-T. circumcincta IgA antibody and eosinophil concentrations were associated with a reduction in total numbers of adult worms and an increase in the frequency of early L4s. The data also suggest that genetic selection for an enhanced anti-T. circumcincta IgA response might complement selection based on a reduced FEC as a strategy to select for resistance to gastrointestinal nematodes.     

Leishmania infantum chagasi: A genome-based approach to identification of excreted/secreted proteins

The parasitic protozoan, Leishmania, survives in harsh environments within its mammalian and sand fly hosts. Secreted proteins likely play critical roles in the parasite’s interactions with its environment. As a preliminary identification of the spectrum of potential excreted/secreted (ES) proteins of Leishmania infantum chagasi (Lic), a causative agent of visceral leishmaniasis, we used standard algorithms to screen the annotated L. infantum genome for genes whose predicted protein products have an N-terminal signal peptide and lack transmembrane domains and membrane anchors. A suite of 181 candidate ES proteins were identified. These included several that were documented in the literature to be released by other Leishmania spp. Six candidate ES proteins were selected for further validation of their expression and release by different parasite stages. We found both amastigote-specific and promastigote-specific released proteins. The ES proteins of Lic are candidates for future studies of parasite virulence determinants and host protective immunity.     

Toxocara canis: Potential activity of natural products against second-stage larvae in vitro and in vivo

The anthelmintic activity of extracts from Chenopodiumambrosioides, Pycnanthusangolensis and Nutridesintox® was in vitro and in vivo investigated, against Toxocaracanis larvae. The in vitro assays results showed that the aqueous extract of Nutridesintox® was the most effective, followed by C. ambrosioides extracts, hexane, dichloromethane and the infusion. P. angolensis extracts showed a lower anthelmintic activity compared to the other natural products. For the in vivo assays, Nutridesintox®, the hexane extract and the infusion of C. ambrosioides were administered orally to T. canis-infected mice, in single doses, during three consecutive days. The efficacy was evaluated on the 17th day post-infection, not only by counting T. canis larvae in the tissues but also by ELISA detection of IgM and IgG antibodies and histological analysis of liver and lungs. The different treatments did not reduce the larvae burden and had no influence on the antibodies dynamic. Interestingly, a reduction on the inflammatory infiltrates was observed in the liver and lung sections of the group treated with the hexane extract of C. ambrosioides. In conclusion, the hexane extract of C. ambrosioides is of further research interest, as it showed an anthelmintic activity in vitro and a reduction on the inflammatory reaction produced by the infection of T. canis larvae in vivo.     

The zinc finger homeodomain-2 gene of Drosophila controls Notch targets and regulates apoptosis in the tarsal segments

The development of the Drosophila leg is a good model to study processes of pattern formation, cell death and segmentation. Such processes require the coordinate activity of different genes and signaling pathways that progressively subdivide the leg territory into smaller domains. One of the main pathways needed for leg development is the Notch pathway, required for determining the proximo-distal axis of the leg and for the formation of the joints that separate different leg segments. The mechanisms required to coordinate such events are largely unknown. We describe here that the zinc finger homeodomain-2 (zfh-2) gene is highly expressed in cells that will form the leg joints and needed to establish a correct size and pattern in the distal leg. There is an early requirement of zfh-2 to establish the correct proximo-distal axis, but zfh-2 is also needed at late third instar to form the joint between the fourth and fifth tarsal segments. The expression of zfh-2 requires Notch activity but zfh-2 is necessary, in turn, to activate Notch targets such as Enhancer of split and big brain. zfh-2 is controlled by the Drosophila activator protein 2 gene and regulates the late expression of tarsal-less. In the absence of zfh-2 many cells ectopically express the pro-apoptotic gene head involution defective, activate caspase-3 and are positive for acridine orange, indicating they undergo apoptosis. Our results demonstrate the key role of zfh-2 in the control of cell death and Notch signaling during leg development.     

Local immune response against larvae of Rhipicephalus (Boophilus) microplus in Bos taurus indicus and Bos taurus taurus cattle

Bos taurus indicus cattle are less susceptible to infestation with Rhipicephalus (Boophilus) microplus than Bos taurus taurus cattle but the immunological basis of this difference is not understood. We compared the dynamics of leukocyte infiltrations (T cell subsets, B cells, major histocompatibility complex (MHC) class II-expressing cells, granulocytes) in the skin near the mouthparts of larvae of R. microplus in B. t. indicus and B. t. taurus cattle. Previously naïve cattle were infested with 50,000 larvae (B. t. indicus) or 10,000 larvae (B. t. taurus) weekly for 6 weeks. One week after the last infestation all of the animals were infested with 20,000 larvae of R. microplus. Skin punch biopsies were taken from all animals on the day before the primary infestation and from sites of larval attachment on the day after the first, second, fourth and final infestations. Infiltrations with CD3⁺, CD4⁺, CD8⁺ and γδ T cells followed the same pattern in both breeds, showing relatively little change during the first four weekly infestations, followed by substantial increases at 7 weeks post-primary infestation. There was a tendency for more of all cell types except granulocytes to be observed in the skin of B. t. indicus cattle but the differences between the two breeds were consistently significant only for γδ T cells. Granulocyte infiltrations increased more rapidly from the day after infestation and were higher in B. t. taurus cattle than in B. t. indicus. Granulocytes and MHC class II-expressing cells infiltrated the areas closest to the mouthparts of larvae. A large volume of granulocyte antigens was seen in the gut of attached, feeding larvae.     

Characterisation of novel protein families secreted by muscle stage larvae of Trichinella spiralis

Proteins secreted by Trichinella spiralis have a potential role in remodelling host skeletal muscle. However, whilst many parasite-secreted proteins have been identified, it has rarely been demonstrated that these are secreted into the nurse cell. Using an informatics-based analysis, we have searched the T. spiralis expressed sequence tag (EST) datasets for cDNAs encoding potential secreted proteins. Here we describe the characterisation of three of the top candidates isolated from our analysis, termed secreted from muscle stage larvae (SML)-1, -2 and -3. All three proteins were demonstrated to be secreted by muscle stage larvae, and immunohistochemical analysis established that SML-1 and -2 are secreted into developing nurse cells. We also show that SML-2 is processed from a precursor into smaller peptides by a metalloprotease contained within T. spiralis-secreted products. With the identification of these and other secreted proteins, we now have molecules to test in functional assays designed to dissect molecular features of the developing nurse cell.     

Trypanosoma evansi: Hematologic changes in experimentally infected cats

This study aimed at evaluating hemogram and erythropoietic changes in cats experimentally infected with Trypanosoma evansi. Thirteen adult female non-breeding Felix catus were separated into two groups: seven animals were infected with 10⁸ trypomastigotes each, and six animals were used as negative controls. Animals were kept in air-conditioned rooms and blood smears were performed daily for 49 days. Blood samples were collected from the jugular vein at days 0, 7, 21, 35 and 49 and stored in blood-collecting tubes containing anticoagulant. Bone marrow was collected from the proximal epiphysis of the right femur at days 14 and 42 post-inoculation (PI). Total erythrocyte count, hematocrit and hemoglobin showed statistical differences among groups from the seventh day PI onwards (P < 0.05). The mean corpuscular volume and the mean corpuscular hemoglobin concentration remained normal, characterizing a normocytic-normochromic anemia. Reticulocyte count increased in the infected group from the 21st day onwards, but remained near normal values suggesting a mild regenerative anemia. Moreover, the myeloid:erythroid ratio significantly reduced at day 42 PI, evidencing a bone marrow hematopoietic response. Based on these results we conclude that cats infected with T. evansi have normocytic, normochromic, regenerative anemia.     

Induction of apoptosis in densovirus infected Aedes aegypti mosquitoes

The mechanism of death in densovirus infected mosquitoes remains unexplored. This study investigated the cellular consequences of densovirus infection in Aedes aegypti mosquitoes after a second generation challenge with a densovirus isolated from adult Aedes albopictus mosquitoes in Thailand (AThDNV). Specimens were analyzed by TUNEL assay, fluorescent in situ hybridization (FISH) and a calorimic assay to detect activation of caspase 3-like activity. After challenge, moribund mosquitoes showed considerable evidence of TUNEL positive cells. The caspase 3-like activity assay showed that the presence of TUNEL positive cells was associated with increased levels of activated caspase 3-like activity in AThDNV infected mosquitoes.     

Impaired pro-inflammatory cytokine production and increased Th2-biasing ability of dendritic cells exposed to Taenia excreted/secreted antigens: A critical role for carbohydrates but not for STAT6 signaling

In cysticercosis, a parasitic disease caused by cestodes, the details of early interactions between parasite antigens and innate cells from the host are not well understood. In this study, the role of cestode-conditioned dendritic cells (DCs) in priming Th1 versus Th2 responses to bystander antigen was examined by using CD11c⁺ DCs as antigen-presenting cells and naive CD4⁺ DO11.10 lymphocytes specific to ovalbumin (OVA) as responding cells. No conventional maturation was induced in DCs exposed to Taenia crassiceps excreted/secreted antigens (TcES). The ability of TcES to affect Toll-like receptor (TLR)-mediated maturation and the pro-inflammatory response was analyzed by co-pulsing DCs with TcES and TLR ligands. DCs exposed to TcES blocked TLR4, TLR9 and Toxoplasma soluble antigen-induced phenotypic maturation. TcES-exposed DCs also blocked secretion of pro-inflammatory cytokines and alloreactive T cell proliferation, while preserving IL-10 production. DCs pulsed with TcES + OVA suppressed IFN-γ, whereas they induced greater IL-4 production by CD4⁺ DO11.10 cells. TcES with chemically-altered glycans failed to modulate TLR-mediated activation of DCs and their Th1-inhibitng ability, which was STAT6-independent. Our results reflect the capacity of TcES glyco-antigens to modulate Th1-type and inflammatory responses mediated through DC activation.     

Litomosoides sigmodontis: A simple method to infect mice with L3 larvae obtained from the pleural space of recently infected jirds (Meriones unguiculatus)

Litomosoides sigmodontis is a filarial nematode that is used as a mouse model for human filarial infections. The life cycle of L. sigmodontis comprises rodents as definitive hosts and tropical rat mites as alternate hosts. Here, we describe a method of infecting mice with third stage larvae (L3) extracted from the pleural space of recently infected jirds (Meriones unguiculatus). This method enables infection of mice with a known number of L3 larvae without the time-consuming dissection of L3 larvae from mites and results in higher worm recovery and patency rates than conventional methods. Additionally, this method allows for geographical separation of the facility maintaining the L. sigmodontis life cycle from the institution at which mice are infected.