Evaluation of diagnostic tests for the detection of classical swine fever in the field without a gold standard.

Knowledge of the sensitivity of diagnostic tests for infectious diseases under field conditions can be used to design a surveillance program that increases the effectiveness of the control policy. In this study, the sensitivity of tests for the detection of classical swine fever (CSF) virus (CSFV) under field conditions was estimated without knowledge of the true disease status of the animals tested. During the CSF epidemic of 1997-1998 in The Netherlands, tonsil samples from pigs of CSF suspect farms were collected for laboratory diagnosis of CSE These specimens were tested in a fluorescence antibody test (FAT1) for the presence of CSFV antigen. When at least 1 specimen in a particular sample series from a farm was positive, this farm was declared CSFV infected. Specimens of that series, either FAT1 negative (98) or FAT1 positive (127), were subsequently tested again (FAT2). After that, a suspension was made of the remaining tissue, and this suspension was evaluated with a virus isolation test. In total, 225 tonsil specimens were examined. A statistical model was formulated, and the sensitivity of the 3 tests and the prevalence of positive specimens in the sample were estimated by the method of maximum likelihood. The sensitivity of the FAT1, the test that was used for confirmation of CSFV infection in a pig herd, was approximately 78% (95% confidence interval [CI] = 62-92%). The effectiveness of the selection process of animals on the farm by the veterinarian was estimated to be 77% (64-87%). The sensitivity of the combination of FAT1 and FAT2 (60%) indicates that at least 5 animals should be selected on a CSF-suspect farm to gain a detection probability for CSFV of 99%.     

Validation of a multiplex PCR assay to detect Babesia spp. and Anaplasma marginale in cattle in Uruguay in the absence of a gold standard test

Detection of bovine Babesia spp. and Anaplasma marginale is based on the reading of Giemsa-stained blood or organ smears, which can have low sensitivity. Our aim was to improve the detection of bovine Babesia spp. and A. marginale by validating a multiplex PCR (mPCR). We used 466 samples of blood and/or organs of animals with signs and presumptive autopsy findings of babesiosis or anaplasmosis. The primers in our mPCR amplified the rap-1a gene region of Babesia bovis and B. bigemina, and the msp-5 region of A. marginale. We used a Bayesian model with a non-informative priori distribution for the prevalence estimate and informative priori distribution for estimation of sensitivity and specificity. The sensitivity and specificity for smear detection of Babesia spp. were 68.6% and 99.1%, and for A. marginale 85.6% and 98.8%, respectively. Sensitivity and specificity for mPCR detection for Babesia spp. were 94.2% and 97.1%, and for A. marginale 95.2% and 92.7%, respectively. Our mPCR had good accuracy in detecting Babesia spp. and A. marginale, and would be a reliable test for veterinarians to choose the correct treatment for each agent.     

Assessing the convergence of Markov Chain Monte Carlo methods: an example from evaluation of diagnostic tests in absence of a gold standard

The accessibility of Markov Chain Monte Carlo (MCMC) methods for statistical inference have improved with the advent of general purpose software. This enables researchers with limited statistical skills to perform Bayesian analysis. Using MCMC sampling to do statistical inference requires convergence of the MCMC chain to its stationary distribution. There is no certain way to prove convergence; it is only possible to ascertain when convergence definitely has not been achieved. These methods are rather subjective and not implemented as automatic safeguards in general MCMC software. This paper considers a pragmatic approach towards assessing the convergence of MCMC methods illustrated by a Bayesian analysis of the Hui–Walter model for evaluating diagnostic tests in the absence of a gold standard. The Hui–Walter model has two optimal solutions, a property which causes problems with convergence when the solutions are sufficiently close in the parameter space. Using simulated data we demonstrate tools to assess the convergence and mixing of MCMC chains using examples with and without convergence. Suggestions to remedy the situation when the MCMC sampler fails to converge are given. The epidemiological implications of the two solutions of the Hui–Walter model are discussed.     

Diagnosing diagnostic tests: evaluating the assumptions underlying the estimation of sensitivity and specificity in the absence of a gold standard

Latent class analysis to assess the sensitivity and specificity of a diagnostic test can be carried out under different assumptions. An often applied set of assumptions is known as the Hui-Walter paradigm, which essentially states that: (i) the population is divided into two or more populations in which two or more tests are evaluated under assumption that (ii) sensitivity and specificity of the tests are the same in all populations; and (iii) the tests are conditionally independent given the disease status. This study explores the implications of these assumptions. Through simulation studies, it is shown how the size of the difference between disease prevalences within the populations influences the precision of the estimates. It is also illustrated by a simulation study how a difference in a test sensitivity between populations may result in estimates that are biased towards the sensitivity of the test in the population with highest disease prevalence, since that population estimate is supported by most of the data. It is shown that the assumption of conditional independence between tests in general cannot be ignored in latent class models. Failure to impose conditional independence will result in a model that lacks identifiability in a way that cannot be handled by adding more tests or dividing the sample into more populations.     

Evaluation of sequential coinfection with Anaplasma phagocytophilum and Anaplasma marginale in cattle

OBJECTIVE: To determine whether sequelae of infection differed among single versus double infection with Anaplasma phagocytophilum or Anaplasma marginale, with and without tick salivary extract, in cattle. ANIMALS: Eighteen 13-month old steers. PROCEDURES: Treatment groups of 3 cattle each included A marginale inoculated ID followed on day 35 by A phagocytophilum without tick saliva, A phagocytophilum followed on day 10 by A marginale without tick saliva, A marginale followed on day 35 by A phagocytophilum with tick saliva, A phagocytophilum followed on day 10 by A marginale with tick saliva, tissue culture control injection, and tick saliva control injection. Infection was monitored via clinical observations, CBC, serologic testing, and PCR analysis of blood and tissues. RESULTS: Infected cattle had significantly reduced weight gain. Anemia occurred 25 to 32 days after A marginale infection, which was attenuated by tick saliva. Parasitism was greater if cattle had not previously been inoculated with A phagocytophilum. Nine of the 12 treated cattle had positive results of PCR analysis for A phagocytophilum from at least 1 blood sample. Five tissue samples had positive results of PCR analysis for A phagocytophilum; PCR results for A marginale were positive in spleen, lung, lymph node, heart, and ear skin of infected cattle. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated an important biological interaction between A marginale and A phagocytophilum infection as well as with tick saliva in disease kinetics and severity in cattle, which may be important for interpretation of diagnostic tests and management of disease in areas where both pathogens occur.     

Concomitant infection of cattle with the vaccine strain Anaplasma marginale ss centrale and field strains of A. marginale

Bovine anaplasmosis, caused by Anaplasma marginale, the intraerythrocytic rickettsia, is controlled by vaccination with live Anaplasma marginale ss centrale (A. centrale), a subspecies of relatively low pathogenicity. We have experimentally demonstrated that an animal primarily infected with A. marginale, or with the related vaccine subspecies A. centrale can be infected with the heterologous subspecies, and carries both bacteria. The co-infection was detected in experimentally cross-infected calves for up to 3 months after the last inoculation with the heterologous subspecies. The occurrence of characteristic cyclic rickettsemia of A. centrale and A. marginale was observed by examination of Giemsa-stained blood smears, or by the presence of specific rickettsial DNA confirmed in PCR assays based on specific msp1a and msp4 for A. marginale, and on specifically designed msp3 and msp4 primers for A. centrale. Sequence analysis of msp4-specific fragments for each subspecies revealed the presence of dual infection in both calves on days 30 and 60 after cross-inoculation with the heterologous Anaplasma subspecies. The experimental cross-infection of calves clearly demonstrated that the concept of "infection exclusion" does not apply to Anaplasma infection in cattle; as there was no infection exclusion of A. marginale in A. centrale-infected cattle, and vice versa. The present results confirmed our previous findings that cattle grazing in an anaplasmosis-endemic field were subject to concomitant infection with both the vaccine A. centrale and the field A. marginale strains.     

Efficacy of attenuated Anaplasma marginale vaccine under laboratory and field conditions in Colombia

Four-month-old Holstein-Friesian calves were inoculated with 3 different doses (1, 2, and 3 ml) of attenuated Anaplasma marginale vaccine. Vaccinated calves showed mild anaplasma parasitemia, slight decrease in packed cell volume, low serologic conversion, and no clinical illness. An artificial challenge exposure of vaccinated and unvaccinated calves with virulent Colombian A marginale showed that the vaccine provided protection against clinical signs of the disease, including parasitemia and anemia. The volume of the vaccinal dose did not alter the degree of protection provided. A 2nd group of 8- to 9-month-old Holstein-Friesian calves was then inoculated with 3 ml of anaplasma vaccine and premunized with both Babesia bigemina and Babesia argentina while being housed in an area free of these diseases. Calves were moved to an enzootic region heavily infested with various arthropods, including ticks, for natural field challenge exposure. Control calves, which were not given anaplasma vaccine, suffered clinical illness manifested by severe anemia and an average weight loss of 50.6 kg due to anaplasma field challenge exposure. In contrast, vaccinated calves did not show anemia and their weight loss was 3.9 kg.     

Sample size determination for estimation of the accuracy of two conditionally independent tests in the absence of a gold standard

We developed an Excel spreadsheet template (available at http://www.epi.ucdavis.edu/diagnostictests/) to calculate sample sizes to estimate sensitivity and specificity with desired precision in the absence of a gold standard. Calculations are predicated on the use of two conditionally independent tests for screening animals from two populations and are based on the methods of Hui and Walter(1980). Sample size calculations rely on asymptotic normality of maximum likelihood (ML) estimates of parameters. Spreadsheets for calculating standard errors for the parameter estimates and for providing ML estimates using cross-tabulated data also are included. An example of application of the methods to bovine paratuberculosis is presented.     

Antigenic characterization of morphologically distinct Anaplasma marginale isolates using a panel of monoclonal antibodies

The present study, describes the antigenic characterization of a Brazilian isolate of Anaplasma marginale with appendage (tail). A panel of monoclonal antibodies (McAbs) was produced and tested by the indirect fluorescent antibody test (IFAT), ELISA and Western blotting, and used to characterize two isolates of A. marginale (one with appendage and another without appendage). Among the clones produced, eight recognized antigenic proteins, with molecular weights varying from 18.4 to 66kDa. In Western blotting, the McAb reacted against a 45kDa antigen, which was shown, by the IFAT, to be located in the tail. Immunocytochemistry confirmed the tail specificity of the monoclonal reacting against the 45kDa antigen. The panel of McAb produced has a potential use in discriminating morphologically distinct A. marginale isolates. The present study, demonstrates the occurrence of antigenic diversity among Brazilian isolates of A. marginale.     

Detection of antibodies against Anaplasma marginale in milk using a recombinant MSP5 indirect ELISA

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.     

Evaluation of a commercially available ELISA kit for detection of antibodies to Anaplasma marginale and Anaplasma centrale in cattle in Australia and Zimbabwe

A newly available competitive inhibition ELISA kit for the serological diagnosis of anaplasmosis was evaluated in Australia and Zimbabwe. In Australia the performance of the test was compared with the card agglutination test (CAT).The assay was evaluated using negative sera collected from Anaplasma-free herds, positive sera from experimentally infected cattle and sera from Anaplasma marginale-endemic herds. The sensitivity and specificity of the ELISA in Australia were 100 % and 83,3 %, respectively, and the sensitivity and specificity of the CAT were both 100%. The agreement between the ELISA and CAT in the sera from endemic herds was 86,4 % (kappa = 0,718). The specificity of the ELISA in Zimbabwe was 100%. No meaningful estimate of sensitivity was possible in Zimbabwe because few known positive sera were available for testing, but all eight known positive sera that were available were clearly positive. We conclude that the ELISA is a useful alternative to the CAT for epidemiological studies.The ELISA kits have advantages over the CAT in that the ELISA is more robust and reagents are better standardized, but the kits are expensive.     

Cultivation of Anaplasma marginale from cattle in a Dermacentor cell line

A tick cell line derived from Dermacentor variabilis (RML-15) was inoculated with bovine RBC infected with Anaplasma marginale. Two hours after inoculation, numerous RBC were phagocytized by the tick cells. After one passage of the cell culture, numerous groups of Anaplasma-like particles were seen in the tick cell cytoplasm. Increased numbers of Anaplasma-like particles also were present. Seemingly, Anaplasma can multiply in tick cells.     

Identification of antigens of two isolates of Anaplasma marginale, using a western blot technique

Antigens of the Illinois (IAM) and Florida (FAM) isolates of Anaplasma marginale were analyzed, using the western blot technique and antiserum from A marginale-infected calves. Crude antigens were prepared from the parasitemic blood of each. Antiserum was collected after the primary and recrudescent parasitemias. Antigens were separated, using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Antigens were then transferred onto nitrocellulose membranes and exposed to test sera. Antibodies attached to the membrane-bound antigens were detected, using an avidin/biotin peroxidase assay and biotinylated rabbit anti-goat immunoglobulin G. Antigens detected were of a high molecular weight group (108 to 91 kilodaltons [kd]) or of a low molecular weight group (47 to 27 kd). The IAM antigens were 100 kd, 96 kd, 47 kd, 38 to 43 kd, and 27 kd; these antigens were detected, using anti-IAM and anti-FAM antibodies, but the anti-FAM antibodies had a strong reaction to only the 100-kd and 38- to 43-kd antigens of IAM. The FAM antigens were 108 kd, 91 kd, 47 kd, 38 to 43 kd, and 27 kd; these antigens were detected, using anti-FAM antibodies and, except the 91 kd antigen, anti-IAM antibodies. Because the 91-kd antigen was detected only in the FAM antigen and detected only by sera from FAM-infected calves, this isolate-specific antigen may be associated with the ability of FAM to induce disease in an IAM-immune animal. Sheep anti-A ovis antibodies reacted only to the 38- to 43-kd antigens of each isolate, indicating that these antigens may be genus-specific.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of world strains of Anaplasma marginale using major surface protein 1a repeat sequences

Anaplasma marginale is a tick-borne pathogen of cattle that causes the disease bovine anaplasmosis worldwide. Major surface proteins (MSPs) are involved in host-pathogen and tick-pathogen interactions and have been used as markers for the genetic characterization of A. marginale strains and phylogenetic studies. MSP1a is involved in the adhesion and transmission of A. marginale by ticks and varies among geographic strains in the number and sequence of amino-terminal tandem repeats. The aim of this study was to characterize the genetic diversity of A. marginale strains collected from countries in North and South America, Europe, Asia, Africa and Australia, inclusive of all continents. In this study, we characterized 131 strains of A. marginale using 79 MSP1a repeat sequences. These results corroborated the genetic heterogeneity of A. marginale strains in endemic regions worldwide. The phylogenetic analyses of MSP1a repeat sequences did not result in clusters according to the geographic origin of A. marginale strains but provided phylogeographic information. Seventy-eight percent of the MSP1a repeat sequences were present in strains from a single geographic region. Strong (> or =80%) support was found for clusters containing sequences from Italian, Spanish, Chinese, Argentinean and South American strains. The phylogenetic analyses of MSP1a repeat sequences suggested tick-pathogen co-evolution and provided evidence of multiple introductions of A. marginale strains from various geographic locations worldwide. These results contribute to the understanding of the genetic diversity and evolution of A. marginale and tick-pathogen interactions.     

Comparison of a DNA probe, complement-fixation and indirect immunofluorescence tests for diagnosing Anaplasma marginale in suspected carrier cattle

Most estimates of the prevalence of anaplasmosis have been based on serologic data using the complement-fixation (CF) and/or card agglutination tests. Since these tests are considered to be only about 50 percent reliable for detecting carrier cattle in enzootically stable herds, the need for more sensitive diagnostic tests is widely recognized. The objective in the present study was to compare the sensitivity of the CF test with that of the indirect immunofluorescence (IIF) test and a recently developed DNA probe in determining the prevalence of Anaplasma marginale infection in cattle from an enzootic area. The study herd consisted of 52 8-month-old steers and 13 3-year-old cows of mixed beef breed. All cattle were initially tested for this comparative purpose. All but one animal (one that was a positive reactor as assessed by all three tests, and served as a positive control), were treated with long-acting oxytetracycline in an attempt to clear any carrier infections. Each animal was then retested at 1 month and 2 months post-treatment (PT), in an effort to determine if the DNA probe could be used to evaluate the effectiveness of the drug. Six of the 65 (9.2%) initial serum samples were CF positive. In contrast, 60 (92.3%) and 64 (98.5%) of the samples were positive as assessed with the IIF test and the DNA probe, respectively. The DNA hybridization reactions varied in intensity within the sample population indicating different individual levels of infection. The DNA probe hybridized with two samples taken at 1 month PT, and with two different samples taken at 2 months PT. The mean IIF titers were reduced at both the 1 month and 2 month sampling times. These results suggest that the drug did not eliminate infections in all cattle. Some may have been cleared, but, in any event, the drug did reduce the level of infections below the sensitivity of the DNA probe and interrupted continuity of stimulation of antibody. Therefore, the DNA probe and the IIF test appear to be considerably more sensitive in detecting carrier infections than the CF test, and should be considered in future epidemiologic studies.     

Frozen and fresh Anaplasma centrale vaccines in the protection of cattle against Anaplasma marginale infection

The immunity induced by frozen and fresh Anaplasma centrale vaccines against anaplasmosis caused by A. marginale was tested in 12-month old Friesian steers. A. centrale parasitaemia occurred in all cattle inoculated with both types of vaccine. The average maximal decrease in PCV for the frozen and fresh vaccines was 41.0 and 40.3% respectively. All cattle recovered spontaneously. Vaccinated and control steers of the same age were challenged six months later with doses of 10(6), 10(7) or 10(8) A. marginale organisms. Vaccinated cattle showed average maximal A. marginale parasitemia of 1.2-4.0 versus 10.3-12.0% in control cattle. The average maximal decrease in packed cell volume (PCV) was 33.1 and 30.0% for steers vaccinated with frozen or fresh vaccine, respectively, and 57.4% for the non-vaccinated steers. All vaccinated cattle recovered spontaneously from the A. marginale infection while 7 out of 8 control steers required specific treatment. It thus appears that both frozen and fresh A. centrale vaccines are equally capable of inducing partial protection against infection with A. marginale and of preventing severe red blood cell destruction.     

Genetic diversity of Anaplasma marginale strains from an outbreak of bovine anaplasmosis in an endemic area

Anaplasma marginale is a tick-borne pathogen of cattle that causes the disease bovine anaplasmosis worldwide. Major surface proteins (MSPs) are involved in host-pathogen and tick-pathogen interactions and have been used as markers for the genetic characterization of A. marginale strains. A. marginale genotypes are highly variable in endemic areas worldwide. The genetic composition of A. marginale strains during anaplasmosis outbreaks has been characterized in one study only which reported a single msp1alpha genotype in infected cattle. However, more information is required to characterize whether a single genotype is responsible for an anaplasmosis outbreak or whether multiple genotypes can cause disease in na?ve cattle within a single herd in endemic areas. The aim of this study was to characterize the genetic diversity of A. marginale strains from an outbreak of bovine anaplasmosis in the State of Tamaulipas, Mexico. A. marginale genotypes were characterized at the molecular level using msp4 and msp1alpha gene sequences. The results revealed that several A. marginale genotypes are present in cattle during acute anaplasmosis outbreaks, thus suggesting that mechanical transmission or stochastic biological transmission through equally efficient independent transmission events may explain A. marginale genotype frequency in a cattle herd during acute bovine anaplasmosis outbreaks in endemic areas. The results reported herein corroborated the genetic heterogeneity of A. marginale strains in endemic regions worldwide. The development and implementation of anaplasmosis control measures is dependent upon understanding the epidemiology of A. marginale in endemic regions, including the characterization of the genetic diversity of strains that produce outbreaks of bovine anaplasmosis.     

Investigation of endothelial cells as an in vivo nidus of Anaplasma marginale infection in cattle

Continuous culture of Anaplasma marginale in endothelial cells and the potential implications for vaccine development heightened interest in determining the importance of endothelial cells in the A. marginale life cycle. A. marginale-infection trials were performed to determine if endothelial cells are an in vivo host cell in cattle and if A. marginale from in vitro endothelial cells were infective to cattle. Adult, immunocompetent steers were infected by tick-feeding transmission and were euthanized at different points in the parasitemic cycle. Based on quantitative PCR, the tissue distribution of A. marginale DNA during peak and trough parasitemia was variable with higher quantities observed in spleen, lung, hemal nodes, and abomasum. A. marginale was not conclusively identified in tissue endothelial cells from the steers' tick-bitten dermis or post-mortem tissues using three microscopy techniques (dual indirect immunofluorescence, transmission electron microscopy, and in situ DNA target-primed rolling-circle amplification of a padlock probe). Intravenous inoculation of spleen-intact or splenectomized calves with endothelial cell culture-derived VA isolate A. marginale did not cause seroconversion or clinical anaplasmosis regardless of whether the endothelial culture-derived bacteria were inoculated as host cell-free organisms or within endothelial cells and regardless of the type of endothelial cell culture used - RF/6A primate endothelial cells or primary bovine testicular vein endothelial cells. Data presented here suggest that endothelial cells are likely not a pivotal component of the A. marginale life cycle in vivo.     

Fatal bovine anaplasmosis in a herd with new genotypes of Anaplasma marginale, Anaplasma ovis and concurrent haemoplasmosis

Haematological and molecular analysis of blood samples was carried out during an outbreak of bovine anaplasmosis in Hungary. Acute disease was observed in five animals, two of which died. Anaplasma-carrier state was diagnosed in 69 (92%) of cattle. Further evaluation of 24 blood samples revealed concurrent infections with Mycoplasma wenyonii and 'CandidatusM. haemobos' in 22 and 21 animals, respectively. In addition, two cows were identified with rickettsaemia. Regarding molecular investigation of potential hard tick vectors, Haemaphysalis inermis and Dermacentor marginatus males collected from the animals were PCR-negative. However, in one pool (out of 18) of Ixodesricinus males, and in six pools (out of 18) of D. reticulatus males the msp4 gene of Anaplasma marginale was detected. In the same I. ricinus pool Anaplasma ovis was also identified. All ticks were negative for haemoplasmas. Anaplasma sequences yielded 97-99% homology to sequences deposited in the Genbank. This is the first report of fatal bovine anaplasmosis associated with divergent A. marginale genotypes and concurrent 'CandidatusM. haemobos' infection, as well as of an A. ovis strain in ticks collected from cattle.     

Establishment and characterization of an Oklahoma isolate of Anaplasma marginale in cultured Ixodes scapularis cells

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.