Variation in haplotypes in single cysts of assemblages C and D, but not of assemblage E of Giardia duodenalis.

BACKGROUND: Giardia duodenalis, a single-celled intestinal parasite, is divided into eight assemblages (A-H), with differences in host specificity. Giardia duodenalis reproduces asexually and cycles between the binucleated trophozoite (4 N) and the infectious cyst with four nuclei (16 N). Interaction between the nuclei is limited. Therefore, genetic drift causes differences in genetic make-up between the non-daughter nuclei; the allelic sequence heterozygosity (ASH). The ASH is low (0.01%-0.0023%) for the related assemblages A and E, higher (0.43-0.53) for assemblage B and much higher (0.74% -0.89%) for the assemblage C and D at the root of the phylogenetic tree. The heterozygosity in assemblage F, in the same clade as assemblage A and E, was unknown. The heterozygosity in the sequences of the gdh and dis3 genes was used as proxy for the ASH and whole genome amplification of single cysts followed by cloning and Sanger sequencing of dis3 fragment could reveal the genetic variation within the cyst. The aim of the study was to determine the level of heterozygosity within pooled and single cysts of different assemblages. RESULTS: The heterozygosity in gdh and dis3 was determined in pooled cysts of the assemblages A to F. Heterozygosity in the isolates of the assemblages C (n = 2) and D (n = 1) ranged from 0.41% to 0.82% for gdh and dis3 and no heterozygosity was found in the isolates of the assemblages A (n = 4), E (n = 3) and F (n = 3). The heterozygosity in assemblage B (n = 7) was intermediate (0% to 0.62%). Next, the number of haplotypes of dis3 was determined for single cysts of assemblages C, D and E. In the assemblages C and D, two to four haplotypes were found per cyst, while in assemblage E only one haplotype was identified. CONCLUSIONS: Having high heterozygosity is characteristic for the assemblages C and D, while having a low heterozygosity is characteristic for the clade with the assemblages A, E and F. Presence of more than 1 haplotype per cyst in assemblage C and D suggests differences between the non-daughter nuclei, in contrast to the one haplotype in assemblage E.

Giardia duodenalis multi-locus genotypes in dogs with different levels of synanthropism and clinical signs.

BACKGROUND: In dogs, infections with Giardia duodenalis are mainly caused by assemblages C and D, but also by the potentially zoonotic assemblages A and B. The aims of this study were to assess differences in assemblages (i) between dogs living mainly in close proximity to humans (synanthropic dogs) versus dogs living mainly among other dogs, (ii) between samples of dogs with or without loose stool, and (iii) related to the amount of cysts shedding. METHODS: One hundred eighty-nine qPCR Giardia positive fecal samples of dogs originating from four groups (household, sheltered, hunting, and dogs for which a veterinarian sent a fecal sample to a diagnostic laboratory) were used for genotyping. For this, multi-locus genotyping of beta-giardin, triose phosphate isomerase, and glutamate dehydrogenase and genotyping of SSU rDNA gene fragments were performed. Fecal consistency was scored (loose or non-loose stool), and cysts per gram of feces were determined with qPCR. RESULTS: Assemblage D was the most prevalent in all groups, followed by the other canid assemblage C. Also, mixed C/D was common. In two (synanthropic) household dogs, the potentially zoonotic assemblage AI was present. Although occurrence of assemblage AI in household dogs was not significantly different from dogs living among other dogs (sheltered and hunting dogs), it was significantly higher compared to dogs for which a sample was sent to a diagnostic laboratory. Dogs with assemblage D shed significantly more cysts than dogs with other assemblages (except for mixed C/D results) or dogs in which no assemblage could be determined. None of the assemblages was significantly associated with loose stool. CONCLUSION: Not only do dogs mainly shed the canid Giardia duodenalis assemblages D and/or C, the numbers of cysts per gram for the canid assemblage D were also higher than for the potential zoonotic assemblage AI. Based on the assemblages shed by dogs, the risk to public health posed by dogs is estimated to be low, even though the dogs that shed AI were synanthropic household dogs. Loose stool in infected dogs was not associated with any particular Giardia assemblage.

Whole-genome sequencing of dog-specific assemblages C and D of Giardia duodenalis from single and pooled cysts indicates host-associated genes.

Giardia duodenalis (syn. Giardia intestinalis or Giardia lamblia) infSAects over 280 million people each year and numerous animals. G. duodenalis can be subdivided into eight assemblages with different host specificity. Unculturable assemblages have so far resisted genome sequencing efforts. In this study, we isolated single and pooled cysts of assemblages C and D from dog faeces by FACS, and sequenced them using multiple displacement amplification and Illumina paired-end sequencing. The genomes of assemblages C and D were compared with genomes of assemblages A and B from humans and assemblage E from ruminants and pigs. The genomes obtained from the pooled cysts and from the single cysts were considered complete (>99 % marker genes observed) and the allelic sequence heterozygosity (ASH) values of assemblages C and D were 0.89 and 0.74 %, respectively. These ASH values were slightly higher than for assemblage B (>0.43 %) and much higher than for assemblages A and E, which ranged from 0.002 to 0.037 %. The flavohaemoglobin and 4Fe-4S binding domain family encoding genes involved in O2 and NO detoxification were only present in assemblages A, B and E. Cathepsin B orthologs were found in all genomes. Six clades of cathepsin B orthologs contained one gene of each genome, while in three clades not all assemblages were represented. We conclude that whole-genome sequencing from a single Giardia cyst results in complete draft genomes, making the genomes of unculturable Giardia assemblages accessible. Observed differences between the genomes of assemblages C and D on one hand and the assemblages A, B and E on the other hand are possibly associated with host specificity.

Host factors associated with Giardia duodenalis infection in dogs across multiple diagnostic tests.

BACKGROUND: The aim of this study was to assess potential associations between Giardia duodenalis infection in dogs, as determined by three diagnostic tests, and dog's group of origin, fecal consistency, age, sex, neuter status, and co-infections with other gastrointestinal parasites. METHODS: Fecal samples from 1291 dogs from four groups (household, shelter, hunting and clinical dogs) were tested with qPCR, rapid enzyme immunochromatographic assay (IDEXX SNAP® Giardia), and direct immunofluorescence (DFA, Merifluor) for presence of G. duodenalis. Moreover, fecal samples were tested with centrifugation sedimentation flotation (CSF) coproscopical analysis for presence of gastrointestinal parasites. Associations were expressed as odds ratios (ORs). RESULTS: Several significant associations were found, of which a few were consistent for all three tests and Giardia positivity in general (positive with at least one of these tests). Dogs older than one year were significantly less likely to test positive for Giardia than younger dogs. Group-housed dogs, especially hunting dogs, were significantly more likely to test positive for Giardia compared to household and clinical dogs. A consistently significant association with Trichuris appeared to be driven by the high prevalence in hunting dogs. Although there was no significant association between loose stool and Giardia infection in the overall population, household dogs were significantly more likely to test Giardia-positive when having loose stool. Overall, Giardia-positive dogs with loose stool shed significantly more cysts, both determined semi-quantitatively with CSF and quantitatively by qPCR, than positive dogs with no loose stool. When other gastrointestinal parasites were present, significantly fewer cysts were detected with CSF, but this was not confirmed with qPCR. CONCLUSION: Giardia is the most common gastrointestinal parasite in Dutch dogs, except for hunting dogs, in which Trichuris and strongyle-type eggs (hookworms) prevailed. Giardia infection was not significantly associated with loose stool, except for household dogs. Young dogs and group-housed dogs were significantly more often Giardia-positive. These associations were consistent across diagnostic tests. Young dogs, clinical dogs and dogs with loose stool shed Giardia cysts in the highest numbers. If another gastrointestinal parasite was present lower numbers of cysts were observed by microscope (CSF), but not with a molecular method (qPCR).

Comparing four diagnostic tests for Giardia duodenalis in dogs using latent class analysis.

BACKGROUND: To accurately diagnose giardiosis in dogs, knowledge of diagnostic test characteristics and expected prevalence are required. The aim of this work was to estimate test characteristics (sensitivity and specificity) of four commonly used diagnostic tests for detection of Giardia duodenalis in dogs. METHODS: Fecal samples from 573 dogs originating from four populations (household dogs, shelter dogs, hunting dogs and clinical dogs) were examined with centrifugation sedimentation flotation (CSF) coproscopical analysis, direct immunofluorescence assay (DFA, Merifluor Cryptosporidium/Giardia®), a rapid enzyme immunochromatographic assay (IDEXX SNAP Giardia®) and qPCR (SSU rDNA) for presence of G. duodenalis. Bayesian latent class analysis was used to determine test performance characteristics and to estimate G. duodenalis prevalence of each of the four dog populations. RESULTS: All tests were highly specific. IDEXX SNAP Giardia® showed the highest specificity (99.6%) and qPCR the lowest (85.6%). The sensitivities were much more variable, with qPCR showing the highest (97.0%) and CSF the lowest (48.2%) sensitivity. DFA was more sensitive than IDEXX SNAP Giardia®, but slightly less specific. Prevalences of G. duodenalis differed substantially between populations, with the hunting dogs showing the highest G. duodenalis prevalence (64.9%) and the household dogs the lowest (7.9%). CONCLUSIONS: This study identifies qPCR as a valuable screening tool because of its high sensitivity, whereas methods using microscopy for cyst identification or cyst wall detection should be used in situations where high specificity is required. G. duodenalis is a prevalent gastro-intestinal parasite in Dutch dogs, especially in dogs living in groups (hunting and shelter dogs) and clinical dogs.

Immune recognition of salivary proteins from the cattle tick Rhipicephalus microplus differs according to the genotype of the bovine host.

BACKGROUND: Males of the cattle tick Rhipicephalus microplus produce salivary immunoglobulin-binding proteins and allotypic variations in IgG are associated with tick loads in bovines. These findings indicate that antibody responses may be essential to control tick infestations. Infestation loads with cattle ticks are heritable: some breeds carry high loads of reproductively successful ticks, in others, few ticks feed and they reproduce inefficiently. Different patterns of humoral immunity against tick salivary proteins may explain these phenotypes. METHODS: We describe the profiles of humoral responses against tick salivary proteins elicited during repeated artificial infestations of bovines of a tick-resistant (Nelore) and a tick-susceptible (Holstein) breed. We measured serum levels of total IgG1, IgG2 and IgE immunoglobulins and of IgG1 and IgG2 antibodies specific for tick salivary proteins. With liquid chromatography followed by mass spectrometry we identified tick salivary proteins that were differentially recognized by serum antibodies from tick-resistant and tick-susceptible bovines in immunoblots of tick salivary proteins separated by two-dimensional electrophoresis. RESULTS: Baseline levels of total IgG1 and IgG2 were significantly higher in tick-susceptible Holsteins compared with resistant Nelores. Significant increases in levels of total IgG1, but not of IgG2 accompanied successive infestations in both breeds. Resistant Nelores presented with significantly higher levels of salivary-specific antibodies before and at the first challenge with tick larvae; however, by the third challenge, tick-susceptible Holsteins presented with significantly higher levels of IgG1 and IgG2 tick salivary protein-specific antibodies. Importantly, sera from tick-resistant Nelores reacted with 39 tick salivary proteins in immunoblots of salivary proteins separated in two dimensions by electrophoresis versus only 21 spots reacting with sera from tick-susceptible Holsteins. CONCLUSIONS: Levels of tick saliva-specific antibodies were not directly correlated with infestation phenotypes. However, in spite of receiving apparently lower amounts of tick saliva, tick-resistant bovines recognized more tick salivary proteins. These reactive salivary proteins are putatively involved in several functions of parasitism and blood-feeding. Our results indicate that neutralization by host antibodies of tick salivary proteins involved in parasitism is essential to control tick infestations.

Identification of a thrombospondin-like immunodominant and phosphorylcholine-containing glycoprotein (GP300) in Dictyocaulus viviparus and related nematodes.

GP300 is a high molecular weight glycoprotein of the bovine lungworm Dictyocaulus viviparus. The N-linked glycans are substituted with phosphorylcholine (PC) giving it immunomodulatory potential. GP300 is highly immunogenic and its recognition by IgE antibodies is correlated with protection against infection. Here we identified and characterized the protein backbone of GP300. Mass spectrometric analysis on purified GP300 and DNA sequencing of the corresponding gene indicated that GP300 is a thrombospondin-like protein with 7 thrombospondin domains, 6 kunitz domains and 15 putative N-glycosylation sites. Purified GP300 display protease inhibitory activity. The protein was located in the brushborder of the gut, but also in muscles, hypodermis and the lining of the uterus. Analysis of GP300 orthologues in Haemonchus contortus and Cooperia oncophora revealed that these proteins also contain PC-substituted N-glycans and showed immunological cross-reactive responses. These data suggest the existence in nematodes of a GP300 protein family that is characterized by PC-substituted N-linked glycans attached to a thrombospondin-like protein backbone. This finding is of particular interest considering the immunomodulatory and vaccine potential of members of the GP300 family.

Serum antibody responses in Creole kids experimentally infected with Haemonchus contortus.

The objective of this study was to evaluate the relationship of parasite-specific serum antibodies with the resistance status of Creole kids. The average breeding values on egg output predicted in a context of natural infection at 11 months of age were distant of 1.07 genetic standard deviation between resistant and susceptible animals. After drenching the animals were maintained worm-free during 1 month until experimental infection with 10,000 Haemonchus contortus infective larvae (L3). Enzyme-linked immunosorbent assay was carried out in serum samples to determine the level of IgG, IgA and IgE anti-H. contortus L3 crude extracts and adult excretion/secretion products (ESP). Parasitological and blood immunological parameters were measured on the 2 extreme groups. Despite the absence of any typical signs of haemonchosis, susceptible kids had more than 11 times higher faecal egg counts (FEC) at 35 days post-infection (d.p.i.) than resistant kids had. Levels of immunoglobulin against H. contortus L3 and ESP increased significantly after infection in both groups. However, no difference in the host immune response mediated by immunoglobulin against H. contortus was evidenced between groups. This finding suggests that, in goats previously infected by H. contortus, a degree of protection occurred and the phenotypic and genetic segregation in resistant and susceptible animals were not related to the humoral immune response. The correlation coefficients between FEC and IgE anti-ESP (r=0.593; P<0.05 was significant in both resistant and susceptible animals. Such correlation suggesting a hypersensitivity reaction dependent on worm prolificacy has never been described. This result needs further studies to understand the mechanisms underlying this observation.

Antibodies elicited by the bovine lungworm, Dictyocaulus viviparus, cross-react with platelet-activating factor.

Parasite N-glycans may play an important role in helminth infections. As antibodies from Dictyocaulus viviparus-infected calves strongly react with N-glycans, we investigated the characteristics of the major immunodominant glycoprotein (GP300) of this parasite. Probing of worm extracts with various lectins demonstrated unique binding of GP300 to wheat germ agglutinin. Analysis of lectin-purified GP300 revealed that the glycan was substituted with phosphorylcholine and reacted with the phosphorylcholine-specific antibody TEPC-15. Competitive enzyme-linked immunosorbent assay with GP300-coated plates and GP300-specific immunoglobulin G (IgG) in conjunction with free phosphorylcholine or TEPC-15 demonstrated that antibodies from infected calves recognized phosphorylcholine on GP300. Additional assays showed that these antibodies cross-reacted with the phosphorylcholine moiety present on platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), a proinflammatory mediator of the host. Heavily infected calves contained high levels of serum GP300-specific IgG1 but low levels of IgA and IgG2 and showed a reduced influx of eosinophils in the lungs, all consistent with a neutralization of PAF activity. In conclusion, we demonstrated that D. viviparus infection elicits GP300-specific antibodies that cross-react with PAF and may neutralize PAF function, thus limiting the development of a protective response as well as parasite-induced host pathology.

Vaccination-induced protection of lambs against the parasitic nematode Haemonchus contortus correlates with high IgG antibody responses to the LDNF glycan antigen.

Lambs respond to vaccination against bacteria and viruses but have a poor immunological response to nematodes. Here we report that they are protected against the parasitic nematode Haemonchus contortus after vaccination with excretory/secretory (ES) glycoproteins using Alhydrogel as an adjuvant. Lambs immunized with ES in Alhydrogel and challenged with 300 L3 larvae/kg body weight had a reduction in cumulative egg output of 89% and an increased percentage protection of 54% compared with the adjuvant control group. Compared to the adjuvant dimethyl dioctadecyl ammonium bromide, Alhydrogel induced earlier onset and significantly higher ES- specific IgG, IgA, and IgE antibody responses. In all vaccinated groups a substantial proportion of the antibody response was directed against glycan epitopes, irrespective of the adjuvant used. In lambs vaccinated with ES in Alhydrogel but not in any other group a significant increase was found in antibody levels against the GalNAcbeta1,4 (Fucalpha1,3)GlcNAc (fucosylated LacdiNAc, LDNF) antigen, a carbohydrate antigen that is also involved in the host defense against the human parasite Schistosoma mansoni. In lambs the LDNF-specific response increased from the first immunization onward and was significantly higher in protected lambs. In addition, an isotype switch from LDNF-specific IgM to IgG was induced that correlated with protection. These data demonstrate that hyporesponsiveness of lambs to H. contortus can be overcome by vaccination with ES glycoproteins in a strong T-helper 2 type response-inducing aluminum adjuvant. This combination generated high and specific antiglycan antibody responses that may contribute to the vaccination-induced protection.