Prevalence and seasonal dynamic of gastrointestinal parasites in equids in France during two years.

Grazing equids are constantly exposed to three clinically important gastrointestinal parasites (small strongyles/cyathostomins, Anoplocephala spp. and Parascaris spp.). Knowledge of the local seasonal dynamic of these parasitic infections is important for constructing a sustainable parasite control program with a rational number of anthelmintic treatments. However, studies describing these patterns are sparse in France. In this context, a two-year study was carried out to assess i) the seasonal dynamic and variability of strongyle faecal egg counts (FEC) and infective larvae (L3) counts on pastures, and ii) the prevalence of Anoplocephala spp. and Parascaris spp. and the dynamic evolution of their presence. During 2021 and 2022 grazing seasons, monthly individual faecal egg counts (FEC) and diarrhea scores (DS) were determined on 428 equids divided into 33 groups. A monthly body condition score (BCS) was also attributed to animals ≥3 years old and a monthly bodyweight was estimated for each animal <3 years old. At the group level, the strongyle L3 counts on grazed pastures were carried out at least in spring, summer and autumn. Eggs of strongyles were observed in 97% of equids. In 64% of the groups, the peaks of FEC were noted in September and October. At the individual level, the maximum strongyle FEC was related to age, group of breeds, number of grazed plots and number of anthelmintic treatments. No negative association was observed between strongyle FEC and BCS or average daily weight gain. In the pastures, cyathostomin larvae were found almost exclusively. Over the two years, the peaks of cyathostomin L3 counts occurred in 87% of the groups between September and November and ranged from 635 to 87,500 L3 kg-1 dry herbage. The variability of the maximum cyathostomin L3 count in each group was explained by the year and the number of grazed plots. Eggs of Anoplocephala spp. were observed in 12% of equids. Eggs of Parascaris spp. were noted in 34% of one year-old animals, 9% of two years-olds and 2% of olders. Anoplocephala spp. and Parascaris spp. eggs were observed every month with a peak in the percentage of shedders in groups in October for Anoplocephala spp. and May-June for Parascaris spp.This study highlights the prevalence of each parasite, the variability in cyathostomin egg excretion and L3 counts amongst groups and individuals and the factors involved in this variation These local epidemiological data will help us to re-think a newer strategy against these parasites.

Glucose Sensing Mediated by Portal Glucagon-Like Peptide 1 Receptor Is Markedly Impaired in Insulin-Resistant Obese Animals.

The glucose portal sensor informs the brain of changes in glucose inflow through vagal afferents that require an activated glucagon-like peptide 1 receptor (GLP-1r). The GLP-1 system is known to be impaired in insulin-resistant conditions, and we sought to understand the consequences of GLP-1 resistance on glucose portal signaling. GLP-1-dependent portal glucose signaling was identified, in vivo, using a novel 68Ga-labeled GLP-1r positron-emitting probe that supplied a quantitative in situ tridimensional representation of the portal sensor with specific reference to the receptor density expressed in binding potential units. It also served as a map for single-neuron electrophysiology driven by an image-based abdominal navigation. We determined that in insulin-resistant animals, portal vagal afferents failed to inhibit their spiking activity during glucose infusion, a GLP-1r-dependent function. This reflected a reduction in portal GLP-1r binding potential, particularly between the splenic vein and the entrance of the liver. We propose that insulin resistance, through a reduction in GLP-1r density, leads to functional portal desensitization with a consequent suppression of vagal sensitivity to portal glucose.

Pancreatic GLP-1r binding potential is reduced in insulin-resistant pigs.

INTRODUCTION: The insulinotropic capacity of exogenous glucagon like peptide-1 (GLP-1) is reduced in type 2 diabetes and the insulin-resistant obese. We have tested the hypothesis that this response is the consequence of a reduced pancreatic GLP-1 receptor (GLP-1r) density in insulin-resistant obese animals. RESEARCH DESIGN AND METHODS: GLP-1r density was measured in lean and insulin-resistant adult miniature pigs after the administration of a 68Ga-labeled GLP-1r agonist. The effect of hyperinsulinemia on GLP-1r was assessed using sequential positron emission tomography (PET), both in the fasted state and during a clamp. The impact of tissue perfusion, which could account for changes in GLP-1r agonist uptake, was also investigated using 68Ga-DOTA imaging. RESULTS: GLP-1r binding potential in the obese pancreas was reduced by 75% compared with lean animals. Similar reductions were evident for fat tissue, but not for the duodenum. In the lean group, induced hyperinsulinemia reduced pancreatic GLP-1r density to a level comparable with that of the obese group. The reduction in blood to tissue transfer of the GLP-1r ligand paralleled that of tissue perfusion estimated using 68Ga-DOTA. CONCLUSIONS: These observations establish that a reduction in abdominal tissue perfusion and a lower GLP-1r density account for the diminished insulinotropic effect of GLP-1 agonists in type 2 diabetes.

Use of Predicted Behavior from Accelerometer Data Combined with GPS Data to Explore the Relationship between Dairy Cow Behavior and Pasture Characteristics.

Our aim in this study was to investigate whether the behaviors of dairy cows on pasture, predicted with accelerometer data and combined with GPS data, can be used to better understand the relationship between behaviors and pasture characteristics. During spring 2018, 26 Holstein cows were equipped with a 3D-accelerometer and a GPS sensor fixed on a neck-collar for five days. The cows grazed alternatively in permanent and in temporary grasslands. The structural elements, soil moisture, slope and botanical characteristics were identified. Behaviors were predicted every 10 s from the accelerometer data and combined with the GPS data. The time-budgets expressed in each characterized zone of 8 m × 8 m were calculated. The relation between the time-budgets and pasture characteristics was explored with a linear mixed model. In the permanent grassland, dairy cows spent more time under a tree to ruminate (p < 0.001) and to rest (p < 0.001) and more time to graze in areas with Holcus lanatus (p < 0.001). In the temporary grassland, behavior was influenced by the external environment (presence of other animals on the farm; p < 0.05). Thus, this methodology seems relevant to better understand the relationship between the behaviors of dairy cows and grazing conditions to develop precision grazing.

Locomotion and eating behavior changes in Yucatan minipigs after unilateral radio-induced ablation of the caudate nucleus.

The functional roles of the Caudate nucleus (Cd) are well known. Selective Cd lesions can be found in neurological disorders. However, little is known about the dynamics of the behavioral changes during progressive Cd ablation. Current stereotactic radiosurgery technologies allow the progressive ablation of a brain region with limited adverse effects in surrounding normal tissues. This could be of high interest for the study of the modified behavioral functions in relation with the degree of impairment of the brain structures. Using hypofractionated stereotactic radiotherapy combined with synchrotron microbeam radiation, we investigated, during one year after irradiation, the effects of unilateral radio-ablation of the right Cd on the behavior of Yucatan minipigs. The right Cd was irradiated to a minimal dose of 35.5 Gy delivered in three fractions. MRI-based morphological brain integrity and behavioral functions, i.e. locomotion, motivation/hedonism were assessed. We detected a progressive radio-necrosis leading to a quasi-total ablation one year after irradiation, with an additional alteration of surrounding areas. Transitory changes in the motivation/hedonism were firstly detected, then on locomotion, suggesting the influence of different compensatory mechanisms depending on the functions related to Cd and possibly some surrounding areas. We concluded that early behavioral changes related to eating functions are relevant markers for the early detection of ongoing lesions occurring in Cd-related neurological disorders.

Rapid assessment of faecal egg count and faecal egg count reduction through composite sampling in cattle.

BACKGROUND: Faecal egg counts (FEC) and the FEC reduction test (FECRT) for assessing gastrointestinal nematode (GIN) infection and efficacy of anthelmintics are rarely carried out on ruminant farms because of the cost of individual analyses. The use of pooled faecal samples is a promising method to reduce time and costs, but few studies are available for cattle, especially on the evaluation of different pool sizes and FECRT application. METHODS: A study was conducted to assess FEC strategies based on pooled faecal samples using different pool sizes and to evaluate the pen-side use of a portable FEC-kit for the assessment of FEC on cattle farms. A total of 19 farms representing 29 groups of cattle were investigated in Italy and France. On each farm, individual faecal samples from heifers were collected before (D0) and two weeks after (D14) anthelmintic treatment with ivermectin or benzimidazoles. FEC were determined individually and as pooled samples using the Mini-FLOTAC technique. Four different pool sizes were used: 5 individual samples, 10 individual samples, global and global on-farm. Correlations and agreements between individual and pooled results were estimated with Spearman's correlation coefficient and Lin's concordance correlation coefficients, respectively. RESULTS: High correlation and agreement coefficients were found between the mean of individual FEC and the mean of FEC of the different pool sizes when considering all FEC obtained at D0 and D14. However, these parameters were lower for FECR calculation due to a poorer estimate of FEC at D14 from the faecal pools. When using FEC from pooled samples only at D0, higher correlation and agreement coefficients were found between FECR data, the better results being obtained with pools of 5 samples. Interestingly, FEC obtained on pooled samples by the portable FEC-kit on-farm showed high correlation and agreement with FEC obtained on individual samples in the laboratory. This field approach has to be validated on a larger scale to assess its feasibility and reliability. CONCLUSIONS: The present study highlights that the pooling strategy and the use of portable FEC-kits on-farm are rapid and cost-effective procedures for the assessment of GIN egg excretion and can be used cautiously for FECR calculation following the administration of anthelmintics in cattle.

Epidemiology and genetic diversity of Anaplasma ovis in goats in Corsica, France.

BACKGROUND: Anaplasma ovis is a major cause of small ruminant anaplasmosis, a tick-borne disease mainly affecting small ruminants in tropical and subtropical regions of the world. Due to health and production problems in dairy goat flocks in Corsica, France, and the demonstration of A. ovis infection in some animals, an extensive survey was conducted in the island in spring 2016. The aim of the survey was to determine the prevalence and geographical distribution of A. ovis infections in goats and ticks as well as possible relationships with anaemia and other health indicators. In addition, the genetic diversity of A. ovis was evaluated. METHODS: Blood and faecal samples were collected in 55 clinically healthy flocks (10 goats per flock) for A. ovis qPCR, haematocrit determination, paratuberculosis ELISA seropositivity and gastrointestinal nematode egg excretion quantification. Ticks were collected, identified and processed for A. ovis DNA detection. RESULTS: A high prevalence of A. ovis DNA detection was found at the individual (52.0%) and flock levels (83.6%) with a within-flock prevalence ranging between 0-100%. Rhipicephalus bursa was the only tick species collected on goats (n = 355) and the detection rate of A. ovis DNA in ticks was 20.3%. Anaplasma ovis DNA prevalence was higher in flocks located at an altitude above 168 m, in goats of Corsican/crossbred breed and in goats > 3 years-old. No relationship was found between A. ovis DNA detection at the individual or flock level and haematocrit, paratuberculosis seropositivity or gastrointestinal parasites. Positive A. ovis goat samples were used for amplification of gltA and msp4 genes for species confirmation and strain identification, respectively. Sequence and phylogenetic analysis of these genes confirmed the detection of A. ovis and allowed identification of six different strains of this pathogen (named Corsica 1-6 (COR1-6). While the msp4 sequence of strain COR1 had 100% identity with strains previously reported, COR2 to 6 were found to be novel strains. The strain COR1 was the most represented, corresponding to 94.6% of the msp4 sequences obtained. CONCLUSIONS: The results showed a relatively high genetic diversity of A. ovis associated with high bacterial prevalence in goats.

Significance of anti-CarLA salivary IgA antibody in first grazing season cattle naturally infected with gastrointestinal nematodes.

A carbohydrate larval surface antigen (CarLA) present on infective larvae of all trichostrongylid nematodes is a target antigen for host immunoglobulins (Ig). Levels of anti-CarLA salivary IgA antibody (CarLA-IgA) have been shown to be correlated to the level of protective immunity to GIN in sheep and deer but no information is available in cattle. The first objective of this study was to assess the pattern of CarLA-IgA response in 7 groups (G1-G7) of first grazing season cattle (FGSC) naturally infected with gastrointestinal nematodes. The second objective was to assess the phenotypic correlations between CarLA-IgA level, 3 parasitological indicators (faecal egg count-FEC, pepsinogen level, serum anti-O. ostertagi IgG antibody level-OstertagiaIgG), a clinical indicator (diarrhea score) and average daily weight gain (ADWG). Overall, CarLA-IgA response gradually increased over grazing season and showed large variations in speed and magnitude both between and within groups. Based on the mean group CarLA-IgA response pattern, the 7 groups could be allocated to 3 different classes: (i) 'Late High' class characterized by a high response at housing (G1 and G2); (ii) 'Low' class with a low response over time (G3, G4 and G5); and (iii) 'Early' class with an high initial then stable response (G6 and G7). This classification was consistent with the grazing management practices. In the 'Late High' class, the mean CarLA-IgA at housing was 6.05units/mL and negatively correlated with FEC while no correlation was seen with the other indicators nor ADWG. In the 'Low' class, CarLA response at housing was low (1.95units/mL) and mainly positively correlated with OstertagiaIgG. In the 'Early' class, mean CarLA-IgA ranged from 1.32 to 1.86units/mL during the grazing season and positive correlations were seen with parasitological and clinical indicators. These results suggest that, according to the intensity of larval challenge occurring during the first grazing season, CarLA-IgA response in cattle could be either an indicator of the early manifestation of immunity (FEC decreases) or the reflection of exposure to GIN.

Design and evaluation of multi-indicator profiles for targeted-selective treatment against gastrointestinal nematodes at housing in adult dairy cows.

Targeted-selective treatments against gastrointestinal nematode (GIN) in adult dairy cows require the identification of "cows to treat", i.e. cows whose milk production (MP) would increase after treatment. This study aimed at quantifying the ability of multi-indicator profiles to identify such cows. A randomized controlled clinical trial was conducted at housing in 25 French pasturing dairy herds. In each herd, treated cows received fenbendazole orally, control cows remained untreated. Daily MP was recorded and the MP variation between the pre- and post-visit periods was calculated (ΔMP) for each cow. ΔMP was modelled with control cows data (n=412) (piecewise linear mixed model). Estimated parameters were applied to treated cows data (n=414) to predict the expected ΔMP in treated cows if they had not been treated. Treated cows with an observed ΔMP (with treatment) higher than the expected ΔMP (without treatment) were labelled as "cows to treat". Herds where at least 50% of the young cows were "cows to treat" were qualified as "herds to target". To characterize such cows and herds, the available candidate indicators were (i) at the cow-level: parity, stage of lactation and production level, faecal egg count (FEC), serum pepsinogen level and anti-Ostertagia antibody level (expressed as ODR); (ii) at the herd-level: bulk tank milk (BTM) Ostertagia ODR, Time of Effective Contact (TEC, in months) with GIN infective larvae before the first calving, and percentage of positive FEC. These indicators were tested one-by-one or in combination to assess their ability to characterize "herds to target" and "cows to treat" (Chi-square tests). 115 out of 414 treated cows (27.8%) were considered as "cows to treat", and 9 out of 22 herds were qualified as "herds to target". The indicators retained to profile such cows and herds were the parity, the production level, the BTM Ostertagia ODR and the TEC. Multi-indicator profiles were much more specific than single indicator profiles, induced lower treatment rates, thereby minimizing the selection pressure on parasite populations. Particularly, to target a herd, the specificity was better with the profile "high BTM Ostertagia ODR and low-TEC" than with the BTM ODR value taken into account alone. The targeted-selective treatment of "young cows, belonging to herds with a high BTM ODR at housing and a low TEC" appeared as a pertinent solution, enabling a global approach for the control of GIN infection in which GIN control in heifers is connected to GIN control in adult cows.

End-season daily weight gains as rationale for targeted selective treatment against gastrointestinal nematodes in highly exposed first-grazing season cattle.

A two-year study was carried out to assess the feasibility of a targeted selective treatment to control gastrointestinal nematodes (GIN) in 24 groups of first grazing season (FGS) cattle. A two-step procedure aiming at defining exposure risk at group level and at identifying the most infected individuals within groups through measurement of the average daily weight gain (ADWG) at housing was used. The first step was to define retrospectively, by grazing management practices (GMP) indicators, two levels of groups' exposure to GIN determined by anti O. ostertagi antibody ODR level (cut-off 0.7). For the low level of exposure, no relationship between parasitological parameters and heifer growth was seen, whereas for the high level ADWG was negatively correlated with increasing Ostertagia ODR values. The best classification was obtained with an expert system modelling the number of Ostertagia L3 generations on plots. GMP input for the expert system included standard data (turnout/housing data and supplementary feeding amount) combined with paddock rotation planning and monthly temperatures. The threshold of 3 successive generations of L3 or more on plots allowed identifying the groups according to low or high infection exposure level, except two groups that were misidentified as being highly exposed. In the second step, individual ADWG was found to be negatively associated with Ostertagia ODR in heifers from groups classified as highly exposed (≥3 generations of L3). In these groups, sensitivity and specificity of ADWG thresholds were calculated for several individual Ostertagia ODR thresholds. The best compromise between sensitivity (i.e., correctly treating the heifers that need to be treated) and specificity (i.e., not treating animals that should not be treated) was equivalent respectively to 76% and 56% (AUC≈0.7) and was reached using an end-season ADWG threshold of 683g/day to detect animals exhibiting an Ostertagia ODR cut-off at 0.93. Other ADWG thresholds were proposed taking into account the farmers' or the veterinarians' objectives: either maximizing the production through both an increase of the ADWG threshold and the sensitivity or keeping a significant nematode population in refugia with a corresponding limitation of anthelmintic treatments through a decrease of ADWG threshold and an increase of the specificity. Finally, a targeted selective treatment for FGS cattle based on GMP and flexible ADWG thresholds seems feasible at housing without laboratory analysis, accepting that some resilient animals with high Ostertagia ODR will not be treated due to their ability to perform under parasitic challenge.

Explaining variability in first grazing season heifer growth combining individually measured parasitological and clinical indicators with exposure to gastrointestinal nematode infection based on grazing management practice.

The objective of our study was to explain the variability of average daily weight gain (ADWG) due to gastrointestinal nematode (GIN) infection for 291 non treated first grazing season (FGS) heifers, from 12 independent groups in the western part of France, by combining parasitological and clinical indicators at individual level and grazing management indicators at group level. Parasitological indicators were faecal egg count (FEC), anti Ostertagia ostertagi antibody level (Ostertagia ODR), and pepsinogen level. Clinical indicators were diarrhea score (DISCO) and breech soiling score (BSS). At group level, grazing management practice (GMP), based on three variables (supplementation, month of turnout, grazing season duration), was clustered into three categories reflecting low, medium or high exposure (EXP) to GIN. Depending on the groups, turnout was from mid-March to early July and housing was from mid-October to late November, with a FGS duration ranging from 4 to 8.4 months. At turnout, the mean age of heifers was 8 months (range: 6-16 months) and they weighed between 175 and 268kg. In each GMP category, FEC significantly decreased between the mid-season and the housing, while Ostertagia ODR and pepsinogen level increased gradually throughout the grazing season. In contrast, clinical indicators did not show any seasonal variation. In a multivariate linear model, 22% of the ADWG variability was significantly explained by two individual indicators (Ostertagia ODR: 12.6%, DISCO: 4.8%) and by the group indicator (GMP category: 4.8%). ADWG losses due to GIN exposure (Ostertagia ODR) were estimated up to 39kg per heifer for the overall grazing season. For groups within the low EXP category the difference between animals with low (<697g/day) or high (>697g/day) ADWG was explained by the clinical indicator DISCO. In contrast, for groups within the medium and high EXP categories this difference was explained by a parasitological indicator (Ostertagia ODR). This study highlighted the value of combining both grazing management (group level) and parasitological (individual level) indicators to assess the impact of GIN on ADWG of FGS heifers. As a result, this combination might allow a better discrimination of animals or groups that may be in need of treatment in a targeting selective treatment approach.

Unexpected Decrease in Milk Production after Fenbendazole Treatment of Dairy Cows during Early Grazing Season.

Gastrointestinal nematodes (GIN) infection can impair milk production (MP) in dairy cows. To investigate whether MP would be optimized by spring targeted-selective anthelmintic treatment in grazing cows, we assessed (1) the effect on MP of an anthelmintic treatment applied 1.5 to 2 months after turn-out, and (2) herd and individual indicators associated with the post-treatment MP response. A randomized controlled clinical trial was conducted in 13 dairy farms (578 cows) in western France in spring 2012. In each herd, lactating cows of the treatment group received fenbendazole orally, control cows remained untreated. Daily cow MP was recorded from 2 weeks before until 15 weeks after treatment. Individual serum pepsinogen and anti-Ostertagia antibody levels (expressed as ODR), faecal egg count and bulk tank milk (BTM) Ostertagia ODR were measured at treatment time. Anthelmintic treatment applied during the previous housing period was recorded for each cow. In each herd, information regarding heifers' grazing and anthelmintic treatment history was collected to assess the Time of Effective Contact (TEC, in months) with GIN infective larvae before the first calving. The effect of treatment on weekly MP averages and its relationships with herd and individual indicators were studied using linear mixed models with two nested random effects (cow within herd). Unexpectedly, spring treatment had a significant detrimental effect on MP (-0.92 kg/cow/day on average). This negative MP response was particularly marked in high producing cows, in cows not treated during the previous housing period or with high pepsinogen levels, and in cows from herds with a high TEC or a high BTM ODR. This post-treatment decrease in MP may be associated with immuno-inflammatory mechanisms. Until further studies can assess whether this unexpected result can be generalized, non-persistent treatment of immunized adult dairy cows against GIN should not be recommended in early grazing season.

A member of the HSP90 family from ovine Babesia in China: molecular characterization, phylogenetic analysis and antigenicity.

Heat shock protein 90 (HSP90) is a key component of the molecular chaperone complex essential for activating many signalling proteins involved in the development and progression of pathogenic cellular transformation. A Hsp90 gene (BQHsp90) was cloned and characterized from Babesia sp. BQ1 (Lintan), an ovine Babesia isolate belonging to Babesia motasi-like group, by screening a cDNA expression library and performing rapid amplification of cDNA ends. The full-length cDNA of BQHsp90 is 2399 bp with an open reading frame of 2154 bp encoding a predicted 83 kDa polypeptide with 717 amino acid residues. It shows significant homology and similar structural characteristics to Hsp90 of other apicomplex organisms. Phylogenetic analysis, based on the HSP90 amino acid sequences, showed that the Babesia genus is clearly separated from other apicomplexa genera. Five Chinese ovine Babesia isolates were divided into 2 phylogenetic clusters, namely Babesia sp. Xinjiang (previously designated a new species) cluster and B. motasi-like cluster which could be further divided into 2 subclusters (Babesia sp. BQ1 (Lintan)/Babesia sp. Tianzhu and Babesia sp. BQ1 (Ningxian)/Babesia sp. Hebei). Finally, the antigenicity of rBQHSP90 protein from prokaryotic expression was also evaluated using western blot and enzyme-linked immunosorbent assay (ELISA).

Validation of BdCCp2 as a marker for Babesia divergens sexual stages in ticks.

Babesiosis is a tick-transmitted disease of mammalian hosts, caused by the intraerythrocytic protozoan parasites of the genus Babesia. Transmission of Babesia parasites from the vertebrate host to the tick is mediated by sexual stages, the gametocytes which are the only intraerythrocytic stages that survive and develop inside the vector. Very few data are available concerning these parasite stages and some markers are needed in order to refine our knowledge of Babesia life cycle inside the tick and to permit the monitoring of parasite transmission from vertebrate to vector. We previously identified some potential markers of the Babesia divergens gametocytes using an in silico post-genomic approach based on sequence identity between the available genomes of Plasmodium and Babesia spp. Here, one of the identified proteins, BdCCp2, was validated as a marker of sexual stages of B. divergens, in infected ticks challenged with antisera directed against recombinant BdCCp2 protein. The BdCCp2 protein was detected by Western blot in some infected ticks, as a discrete band of approximately 171 kDa, while no signal was detected in the laboratory-reared non-infected tick. BdCCp2 was also detected, by immunohistochemical analyses, in piriform or ovoid bodies, measuring 2.5-4.5 µm in diameter, in the gut of partially engorged ticks that were experimentally infected. This molecular marker can then be used in the future to characterize and analyze the biology of B. divergens gametocytes.

Antibody prevalence and molecular identification of Babesia spp. In roe deer in France.

In a region-wide serologic study carried out in 2004 on free-ranging hunted roe deer in various landscapes, we found that 58% of the animals (237 out of 406) were antibody positive for Babesia divergens antigen. Serologic and infection status was also analyzed for 327 roe deer live-trapped in two fenced forest areas over 5 yr (2004-08). For two consecutive years during this period, 92 and 94% of the deer in these closed populations were antibody-positive for B. divergens. Babesia spp. were isolated in autologous red blood cell culture for 131 of the trapped animals (40%). Molecular typing was done on 76 isolates with polymerase chain reaction (PCR)-restriction fragment length polymorphism methods targeted at the 18S ribosomal subunit gene (18 isolates) and the Bd37 gene coding for a merozoïte surface antigen implicated in a protective response (60 isolates). Results indicated continuous cocirculation of B. capreoli and B. venatorum in both forests and possible coinfection of animals with both species. No infection with B. divergens was detected. Fifteen isolates were confirmed to be B. capreoli by sequencing part of the 18S rRNA gene. Using PCR detection of the Bd37 gene, all nine isolates of B. venatorum in this study were negative, whereas the 15 confirmed and 50 putative B. capreoli isolates showed very variable restriction profiles, distinct from those known for Bd37 in B. divergens. Two isolates showed conflicting results, suggestive of mixed infection.

BQP35 is a novel member of the intrinsically unstructured protein (IUP) family which is a potential antigen for the sero-diagnosis of Babesia sp. BQ1 (Lintan) infection.

A new gene of Babesia sp. BQ1 (Lintan) (BQP35) was cloned by screening a merozoite cDNA expression library with infected sheep serum and using rapid amplification of cDNA ends (RACE). The nucleotide sequence of the cDNA was 1140bp with an open reading frame (ORF) of 936bp encoding a 35-kDa predicted polypeptide with 311 amino acid residues. Comparison of BQP35 cDNA and genomic DNA sequences showed that BQP35 does not possess an intron. Recombinant BQP35 (rBQP35), expressed in a prokaryotic expression system, showed abnormally slow migration on SDS-PAGE. Gel shifting, amino acid sequence and in silico disorder region prediction indicated that BQP35 protein has characteristics of intrinsically unstructured proteins (IUPs). This is the first description of such proteins in the Babesia genus. BQP35 induced antibodies production as early as one week after Babesia sp. BQ1 (Lintan) infection in sheep. No cross-reaction was observed with sera from sheep infected with other ovine piroplasms dominant in China, except with Babesia sp. Tianzhu. The interest of BQP35 as a diagnostic antigen is discussed.

The invasion process of bovine erythrocyte by Babesia divergens: knowledge from an in vitro assay.

Babesia divergens is a tick-transmitted apicomplexan parasite for which asexual multiplication in its vertebrate hosts is restricted to erythrocytes. Current knowledge of invasion of these target cells is limited. An efficient in vitro invasion assay was set up to gain access to this information. Parasites prepared from infected RBC, lysed by electroporation, and mixed with bovine RBC in a selected synthetic medium (RPMI 1640 supplemented with calcium) were able to establish subsequent cultures with parasitemia ranging from 6 to 14%. Free parasites remaining in the invasion medium could be eliminated by Percoll gradient and culture could be pursued with the freshly invaded erythrocytes. In this way, the invasion time window could be shortened to obtain a synchronised start of the culture or to study the kinetics of invasion. With this assay we demonstrate that 1) erythrocyte invasion by B. divergens is a rapid process since 70% of the invasion-competent parasites invaded the RBC in less than 45 s; 2) all invasion-competent parasites achieved invasion within 10 min of contact; 3) one erythrocyte could be invaded concomitantly by two merozoites; 4) despite a synchronous start, the parasite population evolved heterogeneously resulting in a progressive loss of synchronisation. Western blot analysis of proteins collected from invasion medium were performed with sera from animals experimentally infected with B. divergens and highlighted several proteins. The dose-dependent, inhibitory effects of these sera on B. divergens invasion suggest that these proteins might be involved in the invasion process. Further investigations are required for their characterisation.

Molecular cloning and genetic polymorphism of Babesia capreoli gene Bcp37/41, an ortholog of Babesia divergens merozoite surface antigen Bd37.

Babesia divergens and Babesia capreoli are closely related species with distinct host ranges, a zoonotic feature being described only for B. divergens. The two species are 99.8% similar in the 18S rDNA gene sequence and indistinguishable by morphological or serological means, leading to confusion as to their species status. The phylogenetic relatedness between the two species, and the frequent involvement of surface components in serological cross-reactions led us to postulate that an ortholog of Bd37, the merozoite surface antigen described for B. divergens, could also exist in B. capreoli. We were able to amplify a single partial PCR product from B. capreoli genomic DNA using primers specific for the B. divergens merozoite surface protein coding gene Bd37, and sequencing confirmed the presence of a Bd37 ortholog in B. capreoli, named Bcp37/41. The full sequences of the Bcp37/41 genes and their intron-exon structures were obtained for two cloned lines of B. capreoli. They suggest functional homologies between Bd37 and Bcp37/41 such as their surface localization, their role in immune escape mechanism and in the initial non-specific attachment to the erythrocyte. Restriction fragment length polymorphism (RFLP) analysis of the amplicons and partial sequencing revealed an extreme polymorphism within B. capreoli, greater than the one observed for its ortholog Bd37. Such a marker could thus be useful in epidemiological as well as phylogenetic studies.

Babesiosis in immunocompetent patients, Europe.

We report 2 cases of babesiosis in immunocompetent patients in France. A severe influenza-like disease developed in both patients 2 weeks after they had been bitten by ticks. Diagnosis was obtained from blood smears, and Babesia divergens was identified by PCR in 1 case. Babesiosis in Europe occurs in healthy patients, not only in splenectomized patients.

Analysis of thioredoxin peroxidase as a promising antigen for diagnosis of Fasciola gigantica infection: a preliminary study.

Buffalo fasciolosis induced by Fasciola gigantica causes important economic losses in tropical areas of Asia. Detection of prepatent infection is essential to control this disease. Classical tools such as coprology, necroscopy or ELISA based on crude extracts from F. gigantica are poorly sensitive or specific. Purified antigens could be used to increase these parameters. Western blot analysis and mass spectrometry of a fraction of F. gigantica excretory-secretory products obtained by gel filtration showed that thioredoxin peroxidase could be a potential antigen for serodiagnosis: it was recognized from the 2nd week after infection, by all buffalo experimentally or naturally infected with F. gigantica but not by healthy animals.