Factors of variation of Amblyomma variegatum infestation on Creole cattle in Guadeloupe.

The level of infestation of "Creole" beef cattle of Guadeloupe by the tick Amblyomma variegatum was recorded during a long-term survey in an experimental farm: 61 steers and 83 cows were distributed in different lots according to sex and management. They grazed continuously either on irrigated Digitaria decumbens pastures or on dry native savannahs. Tick numbers as well as animal weights were registered monthly. Climatic data were also recorded. Different acaricide treatments were tested during the survey. But in order to minimize their effect in the data analysis, only tick counts over an average of 5 adult ticks per cattle were taken into account. The level of infestation is analyzed with respect to environmental factors (season, management) and individual factors (sex, weight, physiological stage, genetic effect). The effects of these factors are discussed with regard to alternative tick-control methods, such as the selection of resistant hosts.

Detection of genomic polymorphisms among isolates of the intracellular bacterium Cowdria ruminantium by random amplified polymorphic DNA and Southern blotting.

Sixteen primers were successfully used in a RAPD assay to generate reproducible fingerprints for six isolates of Cowdria ruminantium, a tick-transmitted rickettsia of ruminants. Distinction between stocks was possible by using one or at most two primers. Two stocks were very similar although originating from widely distant geographical regions. A genetic distance tree was constructed by analysing 108 fragments in pairwise comparison between stocks. Three amplification fragments probed with C. ruminantium genomic DNA determined a restriction fragment length polymorphism which allowed the distinction between stocks except for the two stocks that had similar RAPD patterns. The potential of RAPD to determine the extent of genetic diversity of C. ruminantium and to develop probes or PCR primers for diagnostic purposes is discussed.

Protection of goats against heartwater acquired by immunisation with inactivated elementary bodies of Cowdria ruminantium.

In two experiments, four and five goats were vaccinated by giving two subcutaneous injections of a preparation of inactivated elementary bodies of Cowdria ruminantium (Gardel stock) mixed with Freund's adjuvant. All vaccinated animals together with four naive controls were challenged intravenously with 5 ml of supernatant of a culture of bovine endothelial cells infected with the same stock of Cowdria. All goats developed a high temperature. Two out of four, and four out of five vaccinated goats survived the challenge whereas all naive control animals died within 7-12 days. Vaccinated goats which died survived longer than the controls. No difference in antibody titres was observed between protected and non-protected vaccinated goats. Moreover, immune sera from surviving goats, whether heat inactivated or not, were unable to neutralize the infection of bovine endothelial cells by Cowdria in vitro. Mechanisms conferring protection on the immunized goats are unknown at the moment but the hypothesis that T-helper lymphocyte populations have been elicited seems to be likely. This method of immunization with dead organisms will help in the search for protective antigens against cowdriosis.

Comparative efficacy of Freund's and Montanide ISA50 adjuvants for the immunisation of goats against heartwater with inactivated Cowdria ruminantium.

Two vaccines, based on inactivated elementary bodies of Cowdria ruminantium, one formulated in Montanide ISA50, the other in Freund's adjuvant, were compared in goats. Administered twice subcutaneously with an interval of 81 days, both protected three out of five goats against a very severe challenge, lethal for all 14 control goats, 3.5 months after the second injection. Both vaccines elicited similar antibody levels. The protection afforded by the Montanide ISA50 vaccine was tested 15 and 17 months after the second injection of the vaccine. Three out of six and five out of six goats, respectively, survived a challenge which killed all four control goats used on each occasion. Antibodies were still detectable in the immunised goats. The level of protection appears to be influenced by the dose of virulent C. ruminantium used for the challenge. As any stock of C. ruminantium can be incorporated in order to cover the antigenic repertoire of the organism, this kind of inactivated vaccine can now be tested in the field.

In vitro infection of bovine brain endothelial cells by Cowdria ruminantium.

Endothelial cells from bovine brain capillaries (BBEC) were successfully infected with the Gardel stock of Cowdria ruminantium. Growth conditions of cowdria in BBEC and bovine umbilical endothelial cells (BUE) were identical with average intervals between passages of 8.6 and 8.9 days in BBEC (17 passages) and BUE (28 passages), respectively. The time required to complete the life cycle and the morphology of the parasite were identical in both types of cells. The demonstration that BBEC may be infected with C ruminantium offers the means to study the mechanism of infection of the blood brain barrier endothelium by this parasite.

Detection of antibodies to Cowdria ruminantium in the serum of domestic ruminants by indirect ELISA.

A solid phase enzyme immunoassay for the detection of antibodies to Cowdria ruminantium in the serum of domestic ruminants was developed by using microorganisms cultivated on bovine umbilical endothelial cells as antigen. When the culture showed 90% lysis, the supernatant was centrifuged, sonicated and coated on polystyrene microtiter plates. Antibodies were detected as early as 9 days after experimental immunization of goats. The sensitivity of the ELISA calculated with 73 ruminant sera ranged between 97.3% and 98.6%. The overall specificity of the test was 97% (N = 2925). However, the specificity was far lower for sheep (94.4%, N = 881) than for goats (98.6%, N = 651) and cattle (97.8%, N = 1393). Crossreactivity which could explain some of the false positive reactions, was found between Cowdria antigen and sera raised against Ehrlichia bovis (1 bovine positive out of 2 inoculated) or E. ovina (2 sheep out of 2 inoculated became positive) but not with E. phagocytophila. The intra-assay and inter-assay variability were 7.5% and 7.8% respectively, indicating a good reproducibility of the ELISA.

[Dermatophilus congolensis bovine dermatophilosis in the French West Indies. III. A comparison between infected and non-infected cattle]. / La dermatophilose des bovins à Dermatophilus congolensis dans les Antilles françaises. III. Comparaisons entre élevages infectés et indemnes.

The determination of the distinctive features between infected and uninfected cattle by Dermatophilus has been performed among 93 bovine stock in La Martinique, and 120 in La Guadeloupe and Saint-Martin Islands. Nearly half of the herds had been or were infected. Morbidity rates reach 33 per cent in La Martinique and 29 per cent in La Guadeloupe with a mortality ratio of 15 per cent and 5 per cent respectively. No frequency difference could be observed with respect to the season or the geographical area. Similarly, differences are little or nil as regards animal exposure toward aggressive factors such as sun, soil moisture, thorns, etc. On the other hand, the presence of Amblyomma variegatum is a determining factor considering the prevailing situation in La Martinique, where all breeds of cattle are sensitive to the disease. In this case, 63 per cent of the tick infested cattle develops dermatophilosis versus 2.5 per cent for the still uninfected animals. Conversely, in La Guadeloupe were Creole zebus are highly resistant (as a consequence of their natural selection), only 27 per cent of the infected stock suffers from this illness. In these two islands, regular dipping measures have proved to be beneficial in that it reduces the disease frequency. The study also reveals an underlying typology of the various rearing systems, thus allowing their classification as for their behaviour and response when meeting illness. In La Martinique, whatever they may be, quite none of them is infected in the tick free area. In the tick infested zones, the traditional small breeders rearing a sensitive cattle and who do not care with prophylactic rules, are mostly stricken, while more structured ones have succeeded in controlling the disease, owning to some appropriate measures together with a fair animal management. On the other hand, in La Guadeloupe where this tick is cosmopolitan, the small breeders who run a resistant Creole cattle, are not concerned, despite a rudimentary prophylactic behaviour. Conversely, the intensive rearing system, based upon improved imported breeds, meets great difficulties to master the infection.

Development of an in vitro cloning method for Cowdria ruminantium.

Cowdria ruminantium is a tick-borne rickettsia which causes severe disease in ruminants. All studies with C. ruminantium reported so far were carried out with stocks consisting of infective blood collected from reacting animals or from the same stocks propagated in vitro. Cloned isolates are needed to conduct studies on immune response of the host, on genetic diversity of the parasite, and on mechanisms of attenuation and the development of vaccines. A method of cloning based on the particular chlamydia life cycle of Cowdria was developed. Instead of cloning extracellular elementary bodies, it appeared more convenient to clone endothelial cells infected by one morula resulting from the infection of the cell by one elementary body of Cowdria. Two hundred and sixteen clones were obtained by limiting dilution of infected cells. The method was experimentally validated by comparing randomly amplified polymorphic DNA fingerprints from individual clones obtained from endothelial cell cultures coinfected with two different stocks of C. ruminantium.

Characterization of variable immunodominant antigens of Cowdria ruminantium by ELISA and immunoblots.

Cross-immunization experiments have revealed a significant antigenic diversity of the isolate of Cowdria ruminantium which needs to be characterized for the development of vaccines. We identified polymorphic immunodominant antigens by ELISA and immunoblot. Using serum from a goat immune to the Gardel stock of Cowdria (isolated in Guadeloupe) adsorbed on antigen of the Senegal stock of this pathogen, distinct serogroups were revealed by ELISA among six isolates from different geographical origins. Furthermore, a goat serum directed against the Senegal stock and adsorbed on Gardel antigens was shown to be specific for the Senegal stock, thus confirming the existence of serotypes in Cowdria. The Major Antigenic Protein 1 (MAP1) of Cowdria was shown to have variable antigenic determinants. Also in a group of variable proteins ranging from 23 to 29 kDa, one antigen of 26-27 kDa had a determinant specific for the Gardel isolate. These polymorphic antigens may be relevant components of Cowdria ruminantium for a vaccine as the sera revealing these antigens originated from a goal surviving a lethal challenge. However, the presence of T-cell epitopes and the ability of the these antigens to confer protection to ruminants remain to be investigated. The production of a rabbit antiserum against this group of polypeptides will be of great use for their purification and for the screening of expression libraries.

Infection of bovine brain microvessel endothelial cells with Cowdria ruminantium elicits IL-1 beta, -6, and -8 mRNA production and expression of an unusual MHC class II DQ alpha transcript.

Cowdria ruminantium is a bacterial parasite that infects ruminants, causing an acute and often fatal disease. These obligate intracellular Gram-negative bacteria preferentially infect neutrophils and vascular endothelial cells, especially in the brain. The present study was performed with bovine brain microvessel endothelial cells in culture, infected by C. ruminantium in the presence or absence of IFN-gamma. Infection induced the production of IL-1 beta, -6, and -8 mRNAs, and this effect was potentiated by IFN-gamma. A semi-quantitative PCR analysis indicated that similar amounts of IL-1 beta and IL-6 mRNAs were produced in response to C. ruminantium infection and to treatment with 30 to 40 ng/ml LPS. In addition, although IFN-gamma induced the synthesis of an MHC class II DQ alpha transcript (1.3 kb), an unusual transcript (1.5 kb) was induced by infection and not after LPS treatment. Infection did not affect MHC class I, class II DQ beta, and invariant chain mRNA levels. The present results suggest that C. ruminantium infection raises the immune activity of brain endothelial cells in vitro and that only part of this response can be attributed to LPS. One can hypothesize that cerebral endothelium in vivo efficiently contributes, by MHC Ag expression and production of ILs, to the activation and/or recruitment of leukocytes to the brain and thus plays an active role in the pathogenesis of cowdriosis and in the immune response to this pathogen.