Transstadial and transovarial persistence of Babesia divergens DNA in Ixodes ricinus ticks fed on infected blood in a new skin-feeding technique.

Although Babesia divergens is the the principal confirmed zoonotic Babesia sp. in Europe, there are gaps in our knowledge of its biology and transmission by the tick Ixodes ricinus. In order to reproduce the part of the parasite cycle that occurs in the vector, an in vitro animal skin feeding technique on blood containing in vitro cultivated B. divergens was developed. Parasite DNA was detected in all samples of salivary glands of nymphs and adults that had fed on parasitized blood as larvae and nymphs, respectively, indicating acquisition as well as a transtadial persistence of B. divergens. PCR performed on eggs and larvae produced by females that had fed on parasitized blood demonstrated the existence of a transovarial transmission of the parasite. Gorging B. divergens infected larvae on non-infected gerbils showed persistance of the parasite over moulting into the resulting nymphs. These results indicate that the parasitic stages infective for the vector (i.e. the sexual stages) can be produced in vitro. To our knowledge, this is the first report of artificial feeding of I. ricinus via membrane as well as in vitro transmission of B. divergens to its vector. The opportunities offered by the use of such a transmission model of a pathogen by I. ricinus are discussed.

Myocastor coypus as a reservoir host of Fasciola hepatica in France.

To clarify the role of the nutria Myocastor coypus in the epidemiology of domestic fasciolosis in Loire-Atlantique (department of western France), 438 nutrias were trapped in 9 humid areas of the department and 304 nutrias were trapped in 3 farms where Fasciola hepatica was present; all animals were necropsied. Liver flukes were found in 160 nutrias: 38 nutrias randomly taken in the department (8.7%) and 122 trapped in fasciolosis areas (40.1%). The average parasitic burden was 5.7 flukes per nutria. Sixty-five percent of the liver flukes measured more than 18 mm (size of sexual maturity). The coproscopic examinations carried out on 144 infected nutrias showed that 90% of the infected nutrias shed fluke eggs. The hatching rate was 39.6%. Two groups of 100 Lymnaea truncatula snails, originating from 2 different populations, were exposed to F. hepatica miracidiae hatched from eggs collected from infected nutrias. The prevalence of the infection was 74% and 58.6% in the 2 groups of snails. The average redial burden was 6.2 rediae per snail. The total number of metacercariae was 72.4 metacercariae per snail producing cercariae. Two groups of 5 sheep were orally infected by 150 metacercariae of nutria or sheep origin, respectively. The installation rates of F. hepatica in sheep were respectively 31.6% and 29.6% for the two groups. Specific antibody kinetics of sheep were similar whether the metacercariae were of nutria or sheep origin. M. coypus allows the complete development of F. hepatica and releases parasitic elements that are infective for domestic ruminants. Because of its eco-ethologic characteristics, the nutria could be a potential wild reservoir of F. hepatica in France.

Inventory of wild rodents and lagomorphs as natural hosts of Fasciola hepatica on a farm located in a humid area in Loire Atlantique (France).

With the objective of studying the role of wild fauna in the epidemiology of fasciolosis disease, a definitive wild-host inventory was carried out in a french farm where infected domestic hosts (cows) cohabit with wild potential ones. Liver flukes, faecal eggs and antibodies were looked for in lagomorphs (Oryctolagus cuniculus) and rodents (Myocastor coypus, Ondatra zybethicus, Rattus norvegicus, Arvicola sapidus and micromammal species) trapped in the study area. Presence of Fasciola hepatica was detected in two species: O. cuniculus and M. coypus. Infection rates were respectively 34% (42/124) and 55% (106/193). Liver flukes were found in 78 M. coypus (n = 192) and 11 O. cuniculus (n = 35). No other species was infected by F. hepatica. The number of animals shedding fluke eggs was higher in M. coypus (49 out of 127 sampled; 38.6%) than in O. cuniculus (two out of 17 sampled; 11.7%). The results indicate that M. coypus may play a role in the maintenance and the dissemination of F. hepatica in various environments and open a discussion on the role of other natural wild hosts.

Babesia divergens in France: descriptive and analytical epidemiology.

Babesia divergens cause of bovine babesiosis transmitted by Ixodes ricinus, is widely spread especially in West, Central and South-West parts of France. It occurs with two annual peaks, in spring and autumn. The study was carried out during a period of two years (1991-1993) in four farms in the Sarthe area, in order to know the distribution and the ecology of I. ricinus, and to show the presence of B. divergens. Cattle are parasitised as early as March essentially by adult ticks, according to a seasonal distribution (spring, autumn). The flag method allows to catch essentially the larvae and the nymphs on the pastures; nymphs appear as early as March, and larvae one to two months later. Ectoparasite collection on trapped micromammals (Apodemus sylvaticus, Clethrionomys glareolus, etc.) in pastures hedges, is used to detect small I. ricinus populations, mostly larvae. A new ELISA method has been used for the study of the kinetics of anti-B. divergens antibodies in 236 cattle during two years. Most of the animals (60%) show a high antibody level, essentially at the end of the pasture season; the percentage of positive animals decreases during winter and increases again during the pasture season. Calves become seropositive since their first months on pastures. Adults show asymptomatic infections several times along the year, mostly during spring and autumn; only three clinical babesiosis cases have been observed during the whole study, in animals exhibiting nevertheless a high specific antibody level.

Assessment of the risk of infestation of pastures by Ixodes ricinus due to their phyto-ecological characteristics.

This study aims to assess the relationships between the phyto-ecological characteristics of grazing pastures and their risk of being infested by the hard tick Ixodes ricinus. To that purpose, 128 pastures belonging to 20 dairy farms were observed from April to July 1994. The farms were located in western France. Assessment of the tick population was made by four monthly, blanket-dragging sessions. The average infestation rate was 40.2%. Six types of pastures were identified according to their basic phyto-ecological characteristics using a factorial analysis of correspondences followed by a hierarchical ascending classification. These identified types were significantly related to the infestation rates, thereby making it possible to provide a predictive value in risk assessment. The infestation rates were high (96% on average) in two types of pastures characterized by their proximity to woods, and low (13%) in two other types characterized by seeded grass species located at some distance from woods, and intermediate (39% on average) in the last two types.

Survey of Babesia divergens antibody kinetics in cattle in western France.

Data collected from a longitudinal survey carried out over a 2-year period, in four dairy herds in western France, were used to assess Babesia divergens antibody kinetics. Farms were visited once a month. The total number of animals monitored was 236 including calves, heifers and cows of the Holstein and Normande breeds. An ELISA was used to follow the humoral response levels against Babesia divergens. When the study began, in the autumn of 1991 (200 animals), half of the animals were already seropositive (57.5%). In all four herds, the percentage of positive animals decreased during winter, and increased during spring. Antibody levels remained stable in 49 animals, high for some, very low or negative for others. Most seropositive animals showed at least one antibody peak at some time during the 2-year period, but some presented two to five. Among the calves, 61.3% showed seroconversion during the first pasture season. Similarly, 60% of the newly purchased cows showed increases in antibody levels during the 3 months after their introduction into a new herd, regardless of the initial antibody level. Only three dairy cows had expressed a clinical babesiosis, these cows were already seropositive. Clinical incidence is low in the four farms, nevertheless serological prevalence and incidence are high.

Seasonal incidence of Ixodes ricinus ticks (Acari:ixodidae) on rodents in western France.

Data collected from a longitudinal survey carried out over 2 years on four farms in western France were used to assess the incidence and infestation of Ixodes ricinus on rodents. Once a month, on each farm, 25 Sherman live traps were set in hedges bordering selected pastures. A total of 799 micromammals were examined, including Apodemus sylvaticus, Clethrionomys glareolus, Microtus agrestis, Microtus arvalis, and Crocidura spp. Larvae and nymphs of I. ricinus were found. Small numbers of Ixodes (Exopalpiger) trianguliceps were also recovered from each farm. The mean infestation rate of the I. ricinus larvae (1.6-5.9) among all animals examined varied between farms Most animals were infested by only a single tick, but one M. agrestis harboured 43 I. ricinus larvae. Larvae or nymphs were found throughout the year, with peaks from March to October.

Female Ixodes ricinus (Acari, Ixodidae) in cattle of western France: infestation level and seasonality.

Data collected from a longitudinal survey carried out over a 2 year period in four herds located in western France were used to assess the infestation level in cattle by female ixodes ricinus and to describe its seasonality. Farms were visited once a month. The population studied consisted of calves, heifers and dairy cows (of Holstein and Norman de breeds). The total number of animals monitored varied from 207 to 173 throughout the study, because of the culling of dairy cows and birth of calves. The infestation level by l ricinus varied from one herd to another. Heifers were found to have a greater level of infestation than dairy cows and calves. The infestation rate appeared to be low, ie, most animals (between 47 and 83%) were infested by only one tick in the axilla. The seasonal distribution of ticks showed two peaks, one in spring and a second lower one in autumn, with slight fluctuations between herds and years. The number of ticks on cattle and the percentage of infested animals showed a parallel seasonality.

Babesia divergens: an ELISA with soluble parasite antigen for monitoring the epidemiology of bovine babesiosis.

An enzyme linked immunosorbent assay (ELISA) for bovine babesiosis caused by Babesia divergens was developed to analyse the evolution of the serological status of cattle living in an enzootic area. The antigen used was a soluble extract of B. divergens obtained from in vitro culture. Specificity, evaluated with negative sera, was 96.6%. The ELISA was compared to indirect immunofluorescence analysis (IFA) on naturally or experimentally infected animals. It appeared that IFA was positive for at least seven or eight weeks; on the contrary, B. divergens-specific antibodies were only transitorily detected by ELISA. Furthermore, the ELISA was more sensitive than the IFA for the detection of post-infection antibody increases, particularly when infection-pressure was low. These results suggest that IFA and ELISA could be complementary tools in epidemiological surveys; indeed, this ELISA could be the most efficient tool to detect new infections in cohort monitoring.

Seasonal incidence and ecology of the tick Ixodes ricinus (Acari: Ixodidae) on grazing pastures in western France.

A longitudinal survey was carried out during a 2 year period in Western France to assess the infestation level of grazing pastures by Ixodes ricinus ticks. Four farms were visited once a month and each of the grazing pastures was sampled in the centre and at the border using the blanket dragging method. A total of 3562 I. ricinus (34 adults, 900 nymphs and 2628 larvae) were collected and the infestation was significantly higher during the first year (p < 0.0001). The infestation level by I. ricinus varied between grazing pastures and farms. Grazing pastures in the vicinity of forest were more infested than the others, all through the study. The seasonal distribution of ticks showed peaks, with low fluctuations between farms, years and stages. Tick abundance could not be related to vegetation, but only to the vicinity of woods.

Large scale survey of bovine babesiosis due to Babesia divergens in France.

Data on the epidemiology of bovine babesiosis in France were collected from two sources: a questionnaire sent to large animal veterinary surgeons and blood samples collected from cattle treated for clinical babesiosis. Babesia divergens was common and widespread. B major was found in only one sample. The disease was particularly widespread in north west, south west and central France. The mean clinical incidence was 0.4 per cent. The bimodal seasonality of the clinical cases was correlated with that of the tick vector (Ixodes ricinus). Of the affected animals 72.6 per cent were more than three years old.

Sites of attachment and density assessment of female Ixodes ricinus (acari: ixodidae) on dairy cows.

A sample of 110 lactating dairy cows was examined to describe the attachment sites of female Ixodes ricinus ticks and to assess whether counts on one or two body areas can predict infestation and its level. Seventy per cent of the cows were infested by I. ricinus and the average tick burden on the infested cows was 15, ranging from 1 to 136. Preferred attachment sites were the axilla, udder/groin, neck, dewlap and flank. Udder/groin and axilla carried respectively 35.3% and 44.1% of the total tick burden. To predict infestation (yes/no), sensitivity of counts on axilla, on udder/groin and on both areas was respectively, 76.6%, 80.6% and 94.8%. Total burden prediction equations based on count on axilla and udder/groin are presented.

A framework for animal health management.

In modern production systems, the most economically limiting diseases are multifactorial enzootic health disorders. Relevant decisions in health management have to be based on rational procedures. A first-level approach implies the use of quantitative information on the economic impact of the disease. A more relevant approach of decisions regarding health problems consists of evaluating the economic worth of the interventions in terms of cost-effectiveness. Decision analysis and decision support are of interest today, particularly for herd health control schemes regarding enzootic disorders.

Cellular and humoral immune responses induced in cattle by vaccination with Babesia divergens culture-derived exoantigens correlate with protection.

Previous results with the Babesia divergens gerbil vaccination model were extended in studies with cattle. Two calves were vaccinated with culture-derived B. divergens exoantigens, and two others were treated with control supernatant; both preparations were adjuvanted with Quil-A saponin. A parasite-specific humoral response was observed after the first vaccine injection and was boosted by two succeeding vaccine injections. Sera from the two vaccinated calves immunoprecipitated eight major parasitic proteins (with molecular masses ranging between 17 and 110 kDa) whose patterns were close to those observed in gerbil vaccine assays. The cellular immune response, monitored by lymphoproliferation assays, was slightly delayed in comparison with the humoral response; a significant proliferation occurred only after the second vaccine injection. Mononuclear cell proliferation was dose dependent in the presence of (i) lysates of B. divergens-parasitized erythrocytes, (ii) exoantigens of the whole supernatant, or (iii) protective exoantigens of two low-molecular-mass fractions obtained after supernatant gel filtration chromatography. An infectious challenge was administered 3 weeks after the third vaccine injection, with 3.6 x 10(10) B. divergens-parasitized erythrocytes. Erythrocyte count, rectal temperature, and parasitemia of the animals were monitored daily until they returned to initial values. All parameters indicated that the exoantigens induced protection from B. divergens infection for the two vaccinated calves.

Cytological and immunological responses to Babesia divergens in different hosts: ox, gerbil, man.

A continuous in vitro culture system for Babesia divergens was initiated from a human isolate. It was maintained through 305 subcultures for 3 years using a low concentration of serum and a low haematocrit, with no decrease in the initial virulence. This in vitro system enabled the routine culture of all human and bovine B. divergens isolates thus far tested, with a mean parasitaemia level of 30%-40%. Different cytological aspects observed in the same isolate by optical and electron microscopy were described in parasitized ox, gerbil and human erythrocytes. The sequence of B. divergens antibody responses was determined in man and ox, enabling the precise identification of major B. divergens antigens as candidates for vaccines.

Identification of major Babesia divergens polypeptides that induce protection against homologous challenge in gerbils.

[35S]methionine-radiolabeled proteins from the Babesia divergens Rouen 1987 isolate were immunoprecipitated with immune sera from three potential hosts: human, ox, and gerbil. The results showed a constant humoral response against major babesial antigens. Similarly, immunoprecipitation of radiolabeled in vitro culture supernatant demonstrated that the exoantigens of 37, 46, 70, and 90 kDA were the immunodominant polypeptides, whatever the host. The effects of vaccination with concentrated supernatant from B. divergens Rouen 1987 in vitro cultures (30 to 40% parasitemia) were examined in gerbils inoculated with the homologous B. divergens isolate. Gerbils having received two or three injections of a whole vaccine dose (1.5 ml of parasitized culture supernatant equivalent [PCSE]) or of a 1:5 diluted vaccine dose (0.3 ml of PCSE) showed 100% survival after intraperitoneal challenge with 10(6) B. divergens-infected gerbil erythrocytes. Moreover, two or three injections of a 1:25 diluted vaccine dose (0.06 ml of PCSE) or 9% NaCl or 1.5 ml of unparasitized culture supernatant equivalent resulted in a mortality rate of 80 to 90% of the infected gerbils. Immunoprecipitation and immunofluorescence assays performed with antisera from vaccinated and control gerbils demonstrated that a single vaccine injection induced a humoral response, which increased slightly after the second or third injection. After challenge, antibody levels increased significantly, although the immunoprecipitation did not display any modification of Babesia antigen patterns.