Search

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.

Anaplasma marginale (A. marginale) is a tick-borne ehrlichial pathogen of cattle that causes the disease anaplasmosis. Six major surface proteins (MSPs) have been identified on A. marginale from cattle and ticks of which three, MSP1a, MSP4 and MSP5, are from single genes and do not vary within isolates. The other three, MSP1b, MSP2 and MSP3, are from multigene families and may vary antigenically in persistently infected cattle. Several geographic isolates have been identified in the United States which differ in morphology, protein sequence and antigenic properties. An identifying characteristic of A. marginale isolates is the molecular weight of MSP1a which varies in size among isolates due to different numbers of tandemly repeated 28-29 amino acid peptides. For these studies, genes coding for A. marginale MSP1a and MSP4, msp1alpha and msp4, respectively, from nine North American isolates were sequenced for phylogenetic analysis. The phylogenetic analysis strongly supports the existence of a south-eastern clade of A. marginale comprised of Virginia and Florida isolates. Analysis of 16S rDNA fragment sequences from the A. marginale tick vector, Dermacentor variabilis, from various areas of the United States was used to evaluate possible vector-parasite co-evolution. Our phylogenetic analysis supports identity between the most parsimonious tree from the A. marginale MSP gene data and the tree that reflected the western and eastern clades of D. variabilis. These phylogenetic analyses provide information that may be important to consider when developing control strategies for anaplasmosis in the United States.

Anaplasma marginale is a tick-borne hemoparasite of cattle worldwide. The Virginia isolate of A. marginale was propagated previously in a cell line derived from embryos of the tick, Ixodes scapularis. The cultured Anaplasma (VA-tc) was passaged continuously for over 4 years and retained its infectivity for cattle and antigenic stability. We report herein the continuous in vitro cultivation of a second isolate of A. marginale derived from a naturally infected cow in Oklahoma (OK-tc). Blood from the infected cow was subinoculated into a splenectomized calf and blood collected at peak parasitemia was frozen, thawed and used as inoculum on confluent tick cell monolayers. Colonies of Anaplasma were apparent in low numbers at 9 days post exposure (PE) and infection in monolayers reached 100% by 4-5 weeks PE. Cultures were passaged by placing supernatant onto fresh tick cell monolayers at a dilution of 1:5 or 1:10. By the third passage development of the OK-tc was similar to that of the VA-tc and a 1:5 dilution resulted in 100% infection in 10-12 days. Inoculation of OK-tc into a splenectomized calf caused clinical anaplasmosis and Dermacentor ticks that fed on this calf transmitted the organism to a second susceptible calf. Major surface proteins (MSPs) 1-5 of the OK-tc were compared with homologous proteins present on VA-tc and the erythrocytic stage of the Oklahoma isolate. The MSPs 1, 2, 4, 5 were conserved on the OK-tc but there was evidence for structural variation in MSP3 between the cultured and erythrocytic stage of Anaplasma. MSP2 and MSP3 were the major proteins recognized by serum from infected cattle. Two-dimensional gels also identified positional differences between VA-tc and OK-tc in MSP2 and MSP3. The OK-tc may have potential to be used as antigen for development of an improved vaccine for anaplasmosis in the South Central United States.

Four trials were conducted in which laboratory-reared Dermacentor variabilis nymphs were exposed to Ehrlichia canis by feeding on experimentally infected dogs as soon as classical morulae were detected in peripheral blood monocytes. After molting 25, 50 or 90 adult tick pairs were permitted to feed on 7 Ehrlichia-naive dogs. Transmission occurred in trials 1 (1/1 dog), 3 (1/1 dog) and 4 (2/2 dogs) but not in trial 2 (0/3 dogs), with 4 of 7 dogs becoming infected. Successful transstadial transmission was demonstrated by detection of morulae in peripheral blood lymphocytes and by seroconversion to Ehrlichia canis 30 d post-exposure. Incubation periods ranged between 17 and 22 days (mean = 19). Clinical signs, typical of ehrlichiosis, included mucopurulent ocular discharge, lymphadenopathy and malaise with accompanying pyrexia, leukopenia and thrombocytopenia. Pyrexia, thrombocytopenia and erythrophagocytosis and vacuolization of the cytoplasm of monocytic cells were observed 1-4 d prior to detection of morulae. This is the first demonstration that a tick other than Rhipicephalus sanguineus is capable of transstadial transmission of this important pathogen of dogs.

A mixed-breed pup approximately 3 months old obtained in north central Oklahoma by the Laboratory Animal Resources Unit of Oklahoma State University presented with platelet inclusions. The dog developed severe thrombocytopenia (< 10,000 microliters-1) following the appearance of inclusions. Blood films were monitored daily and when about 75% of platelets had inclusions, samples were collected in EDTA and processed for electron microscopic (EM) studies and polymerase chain reaction (PCR). EM studies on glutaraldehyde-fixed buffy coat revealed rickettsia-like inclusions in numerous platelets. Serologic examination, using Ehrlichia platys antigen, showed high titre suggestive of E. platys infection. PCR primers derived from a highly variable region of the 16S rRNA gene sequence of E. platys were used to specifically amplify that region of the parasite's DNA. Sequencing of the PCR product obtained by general Ehrlichia primers showed one nucleotide difference from the published sequence for E. platys which suggests possible strain variation of this intracellular parasite. Our results indicate that PCR may be a useful tool in the diagnosis of E. platys infection and that, like other Ehrlichia spp., E. platys isolates may vary.

Development in and transmission of hemoparasites by tick vectors are phenomena closely synchronized with the tick feeding cycle. In all known life cycles, initial infection of tick tissues occurs in midgut epithelial cells and transmission is effected as ticks feed after parasites have developed and multiplied in salivary glands. Many factors reviewed affect development and transmission of hemoparasites by ticks including age of ticks, artificial temperature, climate and/or season, tick stage or sex, hemoparasite variation, concurrent infection of ticks with other pathogens, host cell susceptibility, transovarial transmission, effect of hemoparasites on tick biology, and the effect of infecting parasitemia level in cattle on infection rates in ticks. Four hemoparasites of cattle, Anaplasma marginale, Cowdria ruminantium, Theileria parva, and Babesia spp., are all dependent on ticks for biological transmission. Babesia is transmitted transovarially whereas the other three are transmitted transstadially. Mechanical transfer of infective blood via fomites and mouthparts of biting arthropods is also a major means of transmission for Anaplasma marginale but not of the others. Potential control methods for hemoparasites that target parasites as they are developing in their respective tick hosts include tick control, vaccines (against ticks and parasites), and drugs (against ticks and parasites). Successful application of control strategies will be dependent upon thorough understanding of parasite developmental cycles, biology of the tick vectors and the immune response of cattle to ticks and to hemoparasites. The most effective control measures will be those that are targeted against both ticks and the hemoparasites they vector.

Current methods and commercial test systems for the diagnosis of parasitic infections in both animals and humans are reviewed. Lists of test kits and their manufacturers are provided along with ordering information: the only commercially available test kits are for the diagnosis of toxoplasmosis in humans or animals and dirofilariasis (heartworm) in dogs. A partial list of diagnostic laboratories and the parasite tests they perform is also provided. Complete lists of diagnostic tests that could be obtained in the private sector are not available but would be useful. Two microfluorometric solid-phase assay systems are reviewed, and adaptations to custom assays for several kinds of parasites are briefly described. User problems in performing tests and interpreting results are stressed with emphasis placed on diagnosis of dirofilariasis in dogs. False-positive serology in dogs without heartworms and negative antibody responses in micro-filariae-positive animals are discussed with respect to proper interpretation of results.