Problems of establishing anaplasmosis-free herds in Montana.
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Two black-tailed jackrabbits (Lepus californicus), 1 splenectomized and 1 intact, were inoculated with 0.2 ml of a 1:5 dilution of a Florida Anaplasma marginale stabilate. Five months later, both hares were inoculated with 1 ml of whole blood from a calf with acute anaplasmosis. Neither hare developed any signs of clinical anaplasmosis. Pooled blood (7 ml) from these jackrabbits which was inoculated into 2 Anaplasma-susceptible, splenectomized calves failed to induce hematologic or serologic signs of anaplasmosis for at least 90 days. Two susceptible, splenectomized calves were inoculated with 35 ml of pooled whole blood from 9 wild-collected black-tailed jackrabbits from a known anaplasmosis enzootic area. Both steers remained free of anaplasmosis signs for 90 days.
The most important ticks transmitting diseases to ruminants in the Caribbean islands are: Amblyomma variegatum, vector of cowdriosis and associated with acute dermatophilosis; Amblyomma cajennense, potential vector of cowdriosis; Boophilus microplus, vector of babesiosis and anaplasmosis. At the present time, Amblyomma variegatum is widespread in the Lesser Antilles from Barbados to Puerto Rico. The intensity of the tick control program varies in the different islands and the tick infestation in each island ranges from limited foci to widespread infestations. The vector situation appeared stable since 1988 in the Lesser Antilles but the outbreak in 1992 in Puerto Rico emphasized the risk of spread to other islands and to the American mainland, particularly with cattle egrets. Cowdriosis remains apparently limited to Guadeloupe and Antigua. Amblyomma cajennense, experimental vector of cowdriosis, is present in Cuba, Jamaica and Trinidad but also in most American countries. Boophilus microplus is widespread throughout the Caribbean. Its presence and importance are often indirectly estimated by the clinical and serological diagnosis of babesiosis and anaplasmosis. There is no correlation between the intensity of Amblyomma variegatum control in each island of the Lesser Antilles and the effect on the Boophilus microplus population measured by the seroprevalence to babesiosis and anaplasmosis, but within the island of Martinique a regular Amblyomma variegatum control decreases the seroprevalences to babesiosis and anaplasmosis.
Anaplasmosis is a hemolytic disease of cattle caused by the ehrlichial tick-borne pathogen Anaplasma marginale. Killed vaccines used for control of anaplasmosis in the US used antigen harvested from infected bovine erythrocytes which was often contaminated with bovine cells and other pathogens. In this study, we performed an initial cattle trial to test A. marginale harvested from tick cell culture as an immunogen for cattle. Eleven yearling Holstein cattle were immunized with the cell culture-derived A. marginale and 11 cattle were non-immunized contact controls. Each vaccine dose contained approximately 2 x 10(10) A. marginale in an oil-based adjuvant. Two immunizations were administered subcutaneously 4 weeks apart and the cattle were challenge-exposed 10 weeks after the second immunization with A. marginale infected blood. Maximum antibody levels as determined by an A. marginale specific competitive ELISA were observed 2 weeks after the last immunization. Antibody responses against major surface proteins (MSPs) 1a and 1beta1 were also characterized and immunized cattle demonstrated a preferential recognition for MSP1beta1. Cattle immunized with the cell culture-derived A. marginale had a significantly lower percent reduction in the packed cell volume (P<0.05) after challenge exposure as compared with the controls and did not display clinical anaplasmosis. The cell culture-derived A. marginale shows promise for use as antigen in development of a new killed vaccine for anaplasmosis.
Anaplasmosis, a tick-borne cattle disease caused by the rickettsia Anaplasma marginale, is endemic in tropical and subtropical areas of the world. The disease causes considerable economic loss to both the dairy and beef industries worldwide. Analyses of 16S rRNA, groESL, and surface proteins have resulted in the recent reclassification of the order Rickettsiales. The genus Anaplasma, of which A. marginale is the type species, now also includes A. bovis, A. platys, and A. phagocytophilum, which were previously known as Ehrlichia bovis, E. platys, and the E. phagocytophila group (which causes human granulocytic ehrlichiosis), respectively. Live and killed vaccines have been used for control of anaplasmosis, and both types of vaccines have advantages and disadvantages. These vaccines have been effective in preventing clinical anaplasmosis in cattle but have not blocked A. marginale infection. Thus, persistently infected cattle serve as a reservoir of infective blood for both mechanical transmission and infection of ticks. Advances in biochemical, immunologic, and molecular technologies during the last decade have been applied to research of A. marginale and related organisms. The recent development of a cell culture system for A. marginale provides a potential source of antigen for the development of improved killed and live vaccines, and the availability of cell culture-derived antigen would eliminate the use of cattle in vaccine production. Increased knowledge of A. marginale antigen repertoires and an improved understanding of bovine cellular and humoral immune responses to A. marginale, combined with the new technologies, should contribute to the development of more effective vaccines for control and prevention of anaplasmosis.
An evaluation was made of the protection induced by an attenuated Anaplasma marginale vaccine in young purebred cattle against the challenge exposure of naturally transmitted anaplasmosis in enzootic areas of Mexico. The cattle, which were raised in isolation units free of arthropods, consisted of 10 Brown Swiss calves (1 to 13 months of age) and 8 Holstein calves (5 to 7 months of age). They were paired by breed, age, and body weight, and allotted to 2 equal groups. Calves in 1 group were vaccinated, and at 6 weeks after vaccinations were done, calves in both groups were placed in the field where they were raised for approximately 1 year. Two Holstein and 3 Brown Swiss calves of the nonvaccinated group (group 2) developed clinical anaplasmosis, and the remaining calves of this group had hematologic evidence of the disease during the 2 to 4 months after introduction to the field. The vaccinated group, which remained free of anaplasmosis, showed consistently greater weight gain than did the controls. Among the Holstein calves, the maximum weight difference in favor of the vaccinated group was 50 kg/head at 5.5 months after field exposure, and among the Brown Swiss calves, the differences in weight gain in favor of vaccinated calves at the end of the 12-month period was between 11 and 30 percent. It is concluded that the vaccine provided a means for safe adaptation of high-quality young cattle to the tropics by protecting them against what appears to be the major obstacle to this practice, anaplasmosis.
The sheep ked Melophagus ovinus was evaluated as a possible vector of Anaplasma ovis. In 4 tests, 45 to 293 keds were transferred from sheep with acute anaplasmosis, low level parasitemia, or carrier state of anaplasmosis to individual splenectomized ewes. Keds were confined in stockinette cages attached to the dorsal midline of the recipient hosts for 10 days. Throughout the 90-day observation periods after the keds were transferred, no clinical, serologic, or hematologic evidence of anaplasmosis was detected in any of the ked-recipient ewes. Results indicate that sheep keds probably are not mechanical or biological vectors of the ovine anaplasmosis organism.
Laboratory-reared Dermacentor andersoni ticks experimentally infected as nymphs with Anaplasma marginale were allowed to feed as adults from 1 to 9 days on susceptible, splenectomized calves to determine when, during feeding, the hematozoan was transmitted from ticks to cattle. In experiment 1, ticks were allowed to feed on calves for 1, 2, 3, 4, 5, or 6 days and anaplasmosis did not result. The same calves were used for experiment 2, and ticks were allowed to feed for 1, 3, 6, 7, 8, or 9 days and anaplasmosis occurred in all calves on which ticks fed for greater than or equal to 6 days. In 2 trials in experiment 3, ticks were allowed to feed on calves for 1 to 9 days. Anaplasmosis developed only in calves on which ticks fed for 7, 8, or 9 days. The prepatent periods shortened with longer tick feeding, and linear regression analysis of combined prepatent periods of both trials of experiment 3 indicated a significant (P = 0.05) slope with an estimated daily decrease of 7.75 days from day 7 to 9 of feeding. There was no apparent correlation between length of tick feeding and severity of clinical signs in those calves that developed anaplasmosis. Seemingly, A marginale can be transmitted to cattle by adult D andersoni ticks no earlier than the 6th or 7th day of feeding.
Blood samples from 16 anaplasmosis complement-fixation (CF) positive sheep from a flock near Cambridge, Idaho, were inoculated into 3 intact sheep and 3 splenectomized calves. The 3 sheep became CF positive, and 2 developed parasitemia. None of the 3 calves had signs of anaplasmosis, but all became ill when they were subsequently challenge exposed with blood from known carriers of Anaplasma marginale. After a 75-day observation period, blood from the recipient intact sheep was subinoculated into 3 splenectomized sheep. These splenectomized sheep developed clinical anaplasmosis and became CF positive. In only 1 sheep was there a positive rapid card agglutination test. Therefore, the rapid card agglutination test was not considered to be diagnostic for anaplasmosis carrier status in sheep. Electron microscopic studies were performed on infected sheep erythrocytes to determine the morphologic structure of the organism involved. The causative organism was determined to be Anaplasma ovis.
Twenty four Hereford heifers free of anaplasmosis were allotted into three groups of eight animals each and inoculated three times with adjuvant in Puck saline as control or 50 microg and 100 microg of total protein of Anaplasma marginale initial bodies from three Mexican strains which share the same variable region of msp1alpha and msp4. Inoculation with the adjuvant or the immunogen at either of the two protein doses did not induce any undesirable changes attributable to inoculation in vaccinates or controls. On day 78 post vaccination animals were released in a ranch where bovine Anaplasmosis is endemic. The A. marginale strain prevalent in this ranch shares some of the msp1alpha tandem repeats with and the strains used in the vaccine. After release, all animals became infested with Boophilus microplus ticks and flies. During the challenge period, between days 279 and 300, loss of PCV due to clinical anaplasmosis in control animals was statistically higher from vaccinated animals. Likewise, controls mean peak rickettsemia was also significantly higher (p< or =0.01) than vaccinates' rickettsemias. The antibody responses of all vaccinates after the third vaccination reached OD values above 2.0 on day 49 and were different from controls (p<0.01). IgG(2) responses from both groups of vaccinates were different from controls (p<0.01). Vaccinates which required treatment, also showed the lowest IgG(2) and substantial IgG(1) responses. After contact with the rickettsia, controls developed clinical disease and 7 out of 8 required treatment, while vaccinates in general showed no substantial changes in hematocrit or rickettsemia and only one animal in each group required treatment. Our present results show that vaccination with either 50 microg or 100 microg of protein from purified IB derived from three strains induced protection to resist the challenge with the a field strain that shares some of the tandem repeats of MSP1a.
An indirect enzyme linked immunosorbent assay (iELISA) for diagnosis of anaplasmosis using undiluted individual milk samples from dairy cows was developed. The recombinant 19 kDa major surface protein 5 (rMSP5) of Anaplasma marginale was used as antigen. A monoclonal antibody against bovine IgG1 conjugated with peroxidase and the chromogen 3,5,3',5'-tetramethylbenzidine were used in the test. Strong and weak, positive and negative milk samples were set up as reference controls. Results were expressed as percentage of positivity (PP) contrasting with the strongest positive control. The test was evaluated in two groups (G1 and G2) of lactating dairy cows from herds located in A. marginale non-endemic areas of Argentina. The infection status of both groups, G1 (n=128) sampled after anaplasmosis outbreak, and G2 (n=216) free of anaplasmosis was established by polymerase chain reaction (PCR). Serum samples of cows from G1 and G2 were analyzed by card agglutination test (CAT) and competitive ELISA (cELISA), while the novel iELISA was evaluated in their corresponding milk samples. At a cutoff of 42 PP, the ELISA has 98% sensitivity and 95% specificity. A significant difference (P<0.0001) was found between the mean PP value of negative samples from G1 (17.4+/-14.9), and G2 (8.6+/-7.1). The agreement and kappa (kappa) value between iELISA and PCR was 96%, kappa=0.919; between iELISA and CAT was 97%, kappa=0.880; and between iELISA and cELISA was 97%, kappa=0.899. These results strongly support the usefulness of iELISA to detect A. marginale antibodies in milk. Additional studies are necessary to define the ability of the milk iELISA to detect not only acutely infected, but also carrier cattle.
An observational longitudinal study was carried out on 92 randomly selected smallholder farms in two coastal lowland zones of Kwale District in Kenya between December 1997 and November 1999. The objective was to estimate the incidence of the main vector-transmitted diseases in pre-weaned calves. From an initial 41 pure or cross-bred Bos taurus calves which were less than 2 months and whose birth and disease histories were known, study calves were recruited progressively and monitored until they were weaned at around 146 days. Overall, 130 calves in 67 farms were monitored and these contributed a total risk period of 30,062 days. Disease parameters were analysed and compared as true annual and age-specific incidence rates. The incidences of East Coast fever (ECF) (23.1%) and trypanosomosis (29.1%) were the highest among the vector-borne diseases. The corresponding mortality incidence rates of ECF and trypanosomosis were 10.9 and 3.6%, respectively. The annual incidence rates of anaplasmosis and babesiosis were 10.9 and 1.2%, respectively. There was no mortality arising specifically from anaplasmosis or babesiosis. Analysis of survival times to natural infection indicated that the field challenge resulting to cases of trypanosomosis was much higher compared to the risk of either ECF or anaplasmosis. It was concluded that these vector-borne diseases constrain production of replacement stock in this coastal lowlands region of Kenya.