Comparisons of the complement-fixation, indirect fluorescent antibody, and card agglutination tests for the diagnosis of bovine anaplasmosis.

Results of complement-fixation (CF), indirect fluorescent antibody (IFA), and card agglutination (CT) tests were statistically compared, using 380 serum samples obtained from 140 cattle which were disease-free or naturally or experimentally infected with Anaplasma marginale of Colombian origin. The IFA test was significantly the most sensitive for detection of amimals infected with anaplasmosis (97%); the CT test and the CF test were less so (84% and 79%, respectively). However, the most efficient test for identifying noninfected animals was the CF test (100%), and the CT and the IFA tests were less efficient (98% and 90%). A linear regression analysis performed on the average IFA and CF titers of 10 calves artificially infected with A marginale during a 20-week period showed significant regression coefficients for both tests. The regression line for the CF titers decreased below the sensitivity threshold at 14 weeks after calves were inoculated, whereas the regression line for the IFA titers continued above the sensitivity threshold 20 weeks after inoculation. The CT test also detected antibodies until the end of the observation period.     

Comparison of three oxytetracycline regimes for the treatment of persistent Anaplasma marginale infections in beef cattle

Anaplasmosis, caused by the tick-borne rickettsia, Anaplasma marginale, is an economically important disease of cattle in the United States and worldwide. Cattle that recover from acute infection become carriers in which low or microscopically undetectable A. marginale rickettsemia persists. Tetracycline antimicrobials are currently the only drug used in the US for treatment of acute anaplasmosis. There are currently no drugs specifically licensed for elimination of persistent infections. This study tested the efficacy of three oxytetracycline treatment regimens to clear A. marginale from cattle that were persistently infected. Forty Angus x Simmental steers, aged 6-12 months were experimentally infected with A. marginale. After the steers recovered from acute infection, seroconverted, and were confirmed infected using nested PCR followed by DNA hybridization, the carrier status of each animal was ascertained by sub-inoculation of blood into a separate, splenectomized Holstein calf. The steers were then blocked by bodyweight and randomly assigned as follows to four treatment groups: Treatment A, 300 mg/ml solution of oxytetracycline (Tetradure LA-300, Merial Canada Inc.) administered at 30 mg/kg, by intramuscular (i.m.) injection on day 0; Treatment B, the same 300 mg/ml solution of oxytetracycline administered at 30 mg/kg, i.m. on day 0 and again on day 5; Treatment C, a 200 mg/ml solution of oxytetracycline (Liquamycin LA-200, Pfizer Animal Health) administered at 22 mg/kg, intravenously (i.v.), q 24 h for 5 days (a treatment dose that corresponds with current Office International des Epizooties (OIE) recommendations for treatment prior to export). The fourth group consisted of untreated infected control cattle. All steers were still nested PCR and cELISA positive at 60 days after treatment. Infection was confirmed by subinoculation of blood into a splenectomized Holstein calf. These results demonstrated that the treatment regimens tested failed to clear A. marginale infections in carrier cattle.     

Sensitivity and specificity of the complement fixation test for detection of cattle persistently infected with Anaplasma marginale.

The complement fixation (CF) test commonly is used to identify cattle infected with Anaplasma marginale prior to interstate or international movement. Estimates of the accuracy of the CF test in detecting animals persistently infected with A. marginale vary widely. In this study, the sensitivity and specificity of the CF test for detection of carrier animals was determined using serum from 232 cattle previously defined as A. marginale positive or negative by nested polymerase chain reaction methods and hybridization. Considering results from 2 independent laboratories and interpreting a 1:5 suspect reaction as positive, the best estimate of CF test sensitivity was 20%, with a specificity of 98%. Using a 1:10 cutoff, sensitivity decreased to 14% and specificity increased to 99%. Results of this study indicate that the CF test is ineffective for identifying cattle persistently infected with A. marginale and thus is inadequate for anaplasmosis regulatory and surveillance programs.     

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.     

Demonstration of vaccine-induced immunity to anaplasmosis without induction of persistent postvaccinal complement-fixing and agglutinating antibodies in yearling steers

The protective effect and anti-Anaplasma complement-fixing and agglutinating antibody responses induced in yearling steers by vaccination with an inactivated Anaplasma marginale vaccine were evaluated by challenge exposure. Eleven 12- to 14-month-old Hereford X Angus steers were randomly allotted into 6 principals and 5 controls. The principals were injected IM with 5 ml of vaccine on days 0 and 21. On day 70, the 11 steers were challenge exposed by IV inoculation of A marginale-infected blood. After a prolonged prepatent period, the vaccinated steers developed a significantly (P less than 0.01) lower A marginale parasitemia (8.0%) than did the nonvaccinated controls (26.3%). The persistence of a greater than or equal to 0.5% parasitemia was significantly (P less than 0.025) reduced from 34 days in the control to 21 days in the vaccinated group. The percentage reduction in PCV was significantly (P less than 0.025) less in the vaccinated group (34%) as compared with the controls (57%). Complement-fixing and agglutinating antibody responses induced by vaccination did not persist for more than 21 days after the 2nd vaccine injection and did not interfere with positive seroconversion resulting from challenge exposure.     

Prevalence of antibodies to bluetongue virus and Anaplasma marginale in Montana yearling cattle entering Alberta feedlots: Fall 2001

A serologic survey was conducted in yearling cattle imported into Alberta feedlots from Montana during October 2001 to estimate the prevalence of antibodies to bluetongue virus (BTV) and Anaplasma marginale in Montana yearling cattle. The apparent prevalence of antibodies to BTV when the competitive enzyme-linked immunosorbent assay (cELISA) was used was 0.37% (21/5608). Test positive cELISA samples were also all positive when tested by virus neutralization (VN) and they reacted to 1 or more BTV serotypes, including 2, 10, 11, 13, and 17. The apparent prevalence of antibodies to A. marginale when a recombinant cELISA (rcELISA) was used with a positive cutoff at 30% inhibition was 1.93% (108/5608). When the rcELISA positive cutoff was at 42% inhibition, the apparent prevalence was 0.73% (41/5608). After the reported sensitivity and specificity of the test had been accounted for, the A. marginale antibody results were consistent with a population that was either free of exposure or had a very low prevalence for A. marginale.     

Molecular and serological detection of A. centrale- and A. marginale-infected cattle grazing within an endemic area

A reverse line blot hybridization (RLB) one-stage nested PCR (nPCR) for Anaplasma centrale and a nested PCR for Anaplasma marginale were used to detect infected cattle grazing within an endemic region in Israel. A novel set of PCR primers and oligonucleotide probes based on a 16S ribosomal RNA gene was designed for RLB detection of both Anaplasma species, and the performance of the molecular assays compared. The immunofluorescent antibody test (IFA) was used to detect antibodies to both Anaplasma species, whereas, a highly sensitive and specific competitive enzyme-linked immunosorbent assay (cELISA) was used to detect antibodies in A. centrale-vaccinated cattle. The RLB and the nested PCR procedures showed bacteremia with sensitivity of 50 infected erythrocytes per milliliter. Up to 93% of the A. centrale vaccinates carried specific antibodies that were detected by cELISA, and up to 71% of the vaccinated cattle were found to be naturally infected with A. marginale according to the PCR and the RLB assays. Nevertheless, no severe outbreaks of A. marginale infection occurred among vaccinated herds in this endemic region. It appears that both, molecular tools and serology are useful for evaluation of the vaccine efficacy. In the light of wide natural field infection with A. marginale, strong recommendations to continue the A. centrale vaccination program regime will continue until a new generation of non-blood-based vaccine will be developed.     

Effect of breed of cattle on innate resistance to infection with Anaplasma marginale transmitted by Boophilus microplus

OBJECTIVE: To assess the innate resistance of and transmission in naive Bos taurus cross Bos indicus and purebred Bos indicus cattle when placed in a paddock with cattle infected with Anaplasma marginale and carrying Boophilus microplus ticks. DESIGN: A group of 49 purebred B indicus, and 48 B indicus cross B taurus (50%, F1 generation) 24-month-old steers were kept in the same paddock with cattle artificially infected with a virulent isolate of A marginale and Boophilus microplus. The cattle were seronegative for A marginale at the start of the trial but had previously been exposed to Babesia bovis and B bigemina. PROCEDURE: Cattle were inspected twice weekly for 118 days. Whole blood, blood smears and serum samples were collected from the cattle on day 37 after exposure and then at regular intervals to day 83 after exposure to measure packed-cell volumes, parasitaemias and antibody titres to A marginale. Any animals that met preset criteria were treated for anaplasmosis. On day 83 all cattle were treated with an acaricide and cattle infected with A marginale were removed from the rest of the group. RESULTS: A marginale was detected in blood smears from 14 crossbred and 9 B indicus steers between days 56 and 72 after exposure. Five and two of the infected crossbred and B indicus steers required treatment, respectively. One of the Bos indicus cattle died as a result of the A marginale infection despite treatment. Antibodies to A marginale were detected in the 23 infected cattle. The mean packed-cell volume depression was 40 and 37% in the affected crossbred and Bos indicus groups, respectively. There was no significant difference detected in susceptibility between these two groups. CONCLUSIONS: Innate resistance of purebred B indicus and crossbred cattle was not significantly different. The results confirm that purebred B indicus and crossbred cattle are sufficiently susceptible to warrant the use of vaccination against Anaplasma infections.     

A COMPARISON OF 4 SEROLOGICAL TESTS IN THE DETECTION OF HUMORAL ANTIBODIES TO ANAPLASMOSIS IN CATTLE

A capillary agglutination (CA), a complement fixation (CF), a plate agglutination (PT) and an indirect fluorescent antibody (IFA) test to detect humoral antibodies to Anaplasma marginale are described. Serums from 3, 4 or 5 groups of cattle were used to examine the efficiency of the tests. Agreement between all 4 tests was 86.6%. Agreement between pairs of tests was greater. The CF test was the most sensitive while the PT test was the least sensitive. However the PT could be carried out very rapidly and was suggested as the best screening test, providing improved antigen preparation techniques could increase sensitivity. The CA, CF and IFA tests all showed a stronger homologous antibody reaction when A. marginale antigen was tested against serums obtained from cattle infected with either A. marginale or A. centrale. Antibodies in the A. marginale serums were first detected by day 7 post-inoculation, rose to peak around day 29 and were still present on day 200. Antibodies in the A. centrale serums were first detected by day 29 rose to a peak around day 50 and had disappeared by day 150.     

A case of apparent suppression of Anaplasma marginale infection by eperythrozoonosis (Eperythrozoon teganodes)

The apparent suppression of Anaplasma marginale infection by Eperythrozoon teganodes in a splenectomized calf has been reported. A splenectomized calf, inoculated with 500 ml of blood having 23% erythrocytes infected with A. marginale, developed eperythrozoonosis on the fourth day post inoculation. A. marginale parasitaemia remained very low during the patent eperythrozoonosis. A. marginale parasites started to increase in number only after E. teganodes infection had been controlled with neoarsphenamine. A splenectomized calf treated identically, but not showing E. teganodes parasites in the peripheral blood, developed clinical anaplasmosis and fulminant parasitaemia within 3-4 days post inoculation.     

Procedurally similar competitive immunoassay systems for the serodiagnosis of Babesia equi, Babesia caballi, Trypanosoma equiperdum, and Burkholderia mallei infection in horses.

Procedurally similar competitive enzyme-linked immunoassay (cELISA) methods were developed for the serodiagnosis of Babesia equi and Babesia caballi (piroplasmosis), Trypanosoma equiperdum (dourine), and Burkholderia mallei (glanders) infections in horses. Apparent test specificities for the B. equi, B. caballi, T. equiperdum, and B. mallei cELISAs were 99.2%, 99.5%, 98.9%, and 98.9%, respectively. Concordances and kappa values between the complement fixation (CF) and the cELISA procedures for the serodiagnosis of B. equi, B. caballi, T. equiperdum, and B. mallei infections in experimentally exposed horses were 76% and 0.55, 89% and 0.78, 97% and 0.95, and 70% and 0.44, respectively. The cELISA method may be a technically more reproducible, objective, and convenient approach for piroplasmosis, dourine, and glanders serodiagnosis in qualifying animals for international movement and disease eradication programs than the CF systems currently in use. Use of the cELISA method also obviated the problems associated with testing hemolyzed or anticomplementary sera.     

A competitive ELISA for detection of antibodies to the group antigen of bluetongue virus.

A competitive enzyme-linked immunosorbent assay (cELISA) was developed to detect antibodies to the group antigen of bluetongue virus (BTV). The epitope recognized by the BTV-specific monoclonal antibody was confirmed, by immunofluorescence staining of monolayers of virus-infected Vero cells, to be present on BTV serotypes 2, 10, 11, 13, and 17 but not on epizootic hemorrhagic disease virus (EHDV) serotypes 1 and 2. Sera from BTV-inoculated ruminants and rabbits were used to evaluate the cELISA and to compare its specificity and sensitivity with that of the conventional BTV-specific agar gel immunodiffusion (AGID) and serum neutralization (SN) tests. Rabbit antisera to the 5 serotypes of BTV present in the United States had cELISA titers (inverse of the final dilution of serum that gave greater than 20% inhibition) that ranged from 32 to greater than 1.024. Seroconversion of the 8 calves and lambs inoculated with BTV was detected by all 3 serologic tests (SN, AGID, cELISA) by 6 weeks after inoculation. Specificity of the cELISA test was confirmed with bovine sera that contained neutralizing antibodies to EHDV but not to the 5 serotypes of BTV present in the United States; these sera gave positive results by AGID test but were negative by cELISA. The sensitivity and specificity of the cELISA test was further confirmed by analysis of a panel of bovine test sera supplied by the National Veterinary Services Laboratories, indicating that the cELISA is a superior test for detection of BTV group-specific antibodies in sera from ruminants in the United States.     

Molecular and serological prevalence of Anaplasma marginale in cattle of North Central Morocco

A cross sectional study was conducted to investigate the epidemiological distribution of Anaplasma marginale in North Central Morocco. Blood samples from five provinces of Morocco were collected from apparently healthy cattle (n=668) and simultaneously analyzed by a nested polymerase chain reaction (nPCR) assay and competitive enzyme-linked immunosorbent assay (cELISA). The overall prevalence of A. marginale was 21.9% by nPCR and 16.5% by cELISA. The Kappa coefficient between nPCR and cELISA indicated a modest level of agreement (0.54). The prevalence of A. marginale varied significantly according to the province and the month of sampling. However age, gender and breed did not have a significant effect on the prevalence of this pathogen. The highest prevalence of A. marginale was found in the Gharb, a sub-humid area while the lowest was reported in the Saiss, a semi-arid area. These results indicate that an A. marginale infection are widespread in the country and suggests that either or both techniques are excellent tools for epidemiological studies and control programs.     

Changes in antimicrobial susceptibility in a population of Escherichia coli isolated from feedlot cattle administered ceftiofur crystalline-free acid

OBJECTIVE: To determine effects of administration of ceftiofur crystalline-free acid (CCFA) on antimicrobial susceptibility of Escherichia coli in feedlot cattle. ANIMALS: 61 feedlot steers. PROCEDURES: A cohort study was conducted. Steers were housed in pens (5 pens with 10 steers and 1 pen with 11 steers). Five steers in each pen were administered CCFA, and 5 served as control steers (1 pen had 6 control steers). The CCFA administration included a single-dose regimen (6.6 mg/kg, SC, on day 0), two-thirds-dose regimen (4.4 mg/kg, SC, on day 0), and 3-dose regimen (6.6 mg/kg, SC, on days 0, 6, and 13). Fecal samples were collected on days 0, 2, 6, 9, 13, 16, 20, and 28. Fecal samples were collected immediately before CCFA administration. Minimum inhibitory concentrations of 15 antimicrobials were determined for 3 E coli isolates/fecal sample. Escherichia coli were enumerated by use of direct-plating techniques. RESULTS: Resistance to 1 or more antimicrobials was detected in 986 of 1,441 (68.4%) isolates recovered. Administration of CCFA was associated with a transient increase in the population of ceftiofur-resistant isolates. Susceptibility returned to day 0 values (ie, samples collected immediately before CCFA administration) approximately 2 weeks after completion of CCFA administration. Agreement between ceftiofur resistance and co-resistance to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, and tetracycline was almost perfect (kappa 0.97). We did not detect variation in susceptibility of E coli recovered from commingled control steers. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of CCFA provided selection pressure that favored transient expansion of multiple-resistant variants.     

Validation of an Anaplasma marginale cELISA for use in the diagnosis of A. ovis infections in domestic sheep and Anaplasma spp. in wild ungulates

A commercially available (cELISA) kit for diagnosing Anaplasma marginale infection in cattle was validated for diagnosing A ovis infection in sheep using the bovine serum controls as supplied by the manufacturer (BcELISA) and sheep serum controls from pathogen-free sheep (OcELISA). True positives were identified using two previously established assays, a nested PCR (nPCR) test and an indirect immunofluorescent assay (IFA). The BcELISA was also applied to sera from various species of wild ruminants, comparing the results with the IFA. Receiver operating characteristic (ROC) analysis indicated that the predicted threshold inhibition for the BcELISA was 19.2. The sensitivity for the BcELISA was 98.2% and the specificity was 96.3%. The predicted threshold inhibition decreased to 14.3 for the OcELISA; the sensitivity was 96.5% and the specificity was 98.1%. There was >/=90% concordance between IFA and nPCR, as well as between the BcELISA at 19% inhibition cutoff and either IFA or PCR. Concordance between the cELISA and IFA using sera from elk, mule deer, bighorn sheep, pronghorn antelope, and black-tailed deer ranged from 64% to 100%. This commercially available cELISA test kit can be used very effectively to test domestic sheep for infection with A. ovis using the kit-supplied controls (i.e. the BcELISA) and a 19% inhibition cutoff; the kit may also be useful for detecting intra-erythrocytic Anaplasma infections in wild ruminants.     

Evaluation of a competitive ELISA for antibody detection against avian influenza virus

Active serologic surveillance is necessary to control the spread of the avian influenza virus (AIV). In this study, we evaluated a commercially-available cELISA in terms of its ability to detect AIV antibodies in the sera of 3,358 animals from twelve species. cELISA detected antibodies against reference H1- through H15-subtype AIV strains without cross reactivity. Furthermore, the cELISA was able to detect antibodies produced following a challenge of the AIV H9N2 subtype in chickens, or following vaccination of the AIV H9 or H5 subtypes in chickens, ducks and geese. Next, we tested the sensitivity and specificity of the cELISA with sera from twelve different animal species, and compared these results with those obtained by the hemagglutination-inhibition (HI) test, the "gold standard" in AIV sera surveillance, a second commercially-available cELISA (IZS ELISA), or the agar gel precipitation (AGP) test. Compared with the HI test, the sensitivities and specificities of cELISA were 95% and 96% in chicken, 86% and 88% in duck, 97% and 100% in turkey, 100% and 87% in goose, and 91% and 97% in swine, respectively. The sensitivities and specificities of the cELISA in this study were higher than those of IZS ELISA for the duck, turkey, goose, and grey partridge sera samples. The results of AGP test against duck and turkey sera also showed significant correlation with the results of cELISA (R-value >0.9). In terms of flock sensitivity, the cELISA correlated better with the HI test than with commercially-available indirect ELISAs, with 100% flock sensitivity.     

Oral vesicular lesions in horses without evidence of vesicular stomatitis virus infection

OBJECTIVE: To report clinical and serologic findings in horses with oral vesicular lesions that were consistent with vesicular stomatitis (VS) but apparently were not associated with VS virus (VSV) infection. DESIGN: Serial case study. ANIMALS: 8 horses. PROCEDURE: Horses were quarantined after appearance of oral lesions typical of VS. Severity of clinical signs was scored every 2 to 5 days for 3 months. Serum samples were tested for antibodies by use of competitive ELISA (cELISA), capture ELISA for IgM, serum neutralization, and complement fixation (CF). Virus isolation was attempted from swab specimens of active lesions. RESULTS: 2 horses with oral vesicular lesions on day 1 had antibodies (cELISA and CF) against VSV; however, results of CF were negative by day 19. Five of the 6 remaining horses were seronegative but developed oral lesions by day 23. Virus isolation was unsuccessful for all horses. CONCLUSIONS AND CLINICAL RELEVANCE: Horses were quarantined for 75 days in compliance with state and federal regulations. However, evidence suggests that oral lesions were apparently not associated with VSV infection. The occurrence in livestock of a vesicular disease that is not caused by VSV could confound efforts to improve control of VS in the United States and could impact foreign trade. Vesicular stomatitis is of substantial economic and regulatory concern.     

Seroprevalence of anaplasmosis among cattle in Switzerland in 1998 and 2003: no evidence of an emerging disease

Anaplasma marginale infection in Europe has been limited to the Mediterranean and eastern countries, to Austria and to very sporadic cases in Switzerland. There are no reports of its occurrence in the countries north of Switzerland. A severe outbreak of anaplasmosis in August 2002 in a cattle farm in the canton Grisons, Switzerland, north of the Alps, with more than 300 cattle that had to be culled, came unexpected and gave reason to hypothesize presence of an increased yet undetected prevalence of A. marginale in Switzerland. Randomly selected bovine serum samples collected in 1998 and 2003 were tested using a competitive inhibitory ELISA (cELISA) to test the hypothesis. Our validation of the diagnostic sensitivity and specificity of this test, done in the outbreak herd, yielded 99.2 and 83.3%, respectively, probably underestimating the true specificity. The true seroprevalence of anaplasmosis in Swiss cattle determined by cELISA was likely to be zero with upper 95% confidence limits of 2.49% in the canton Grisons and 1.17% in the rest of Switzerland, respectively, in 1998. For 2003, these estimates were even lower. There was no significant difference in apparent prevalences between 1998 and 2003. In search of a possible reservoir, three chamoises out of 46 free ranging wild ruminants from the Swiss National Park, Grisons, tested positive in the cELISA. This reaction is in accordance with A. marginale or a cross reacting agent such as Anaplasma ovis. From our results we conclude that the hypothesis of an increased prevalence of anaplasmosis in cattle in Switzerland must be rejected.     

Antibodies to Anaplasma marginale major surface proteins 1a and 1b inhibit infectivity for cultured tick cells

Major surface protein 1 (MSP1) of the cattle pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) is a complex of two proteins, MSP1a and MSP1b. Previous studies demonstrated that MSP1a and MSP1b are adhesins for bovine erythrocytes, while only MSP1a proved to be an adhesin for tick cells. In this study, a tick cell culture system for propagation of A. marginale was used to develop an infection inhibition assay for testing the ability of antisera to block infection of A. marginale for cultured tick cells. A. marginale derived from cell culture was incubated with various antisera prior to inoculation onto cell monolayers. The monolayers were harvested 7 days post-inoculation and A. marginale in the cultures was quantified using an antigen detection ELISA. Antisera tested in the infection inhibition assay were derived from persistently infected cattle, from cattle immunized with A. marginale purified from bovine erythrocytes, and from rabbits and cattle that were immunized with the recombinant MSP1a, MSP1b and MSP1 complex. Antibodies from cattle persistently infected with A. marginale, cattle immunized with A. marginale from bovine erythrocytes or cattle immunized with the recombinant MSP1 complex did not inhibit the infectivity of A. marginale for tick cells. Antiserum from rabbits immunized with MSP1a and MSP1b (individually or combined) reduced infection of both the Virginia and Oklahoma isolates of A. marginale for tick cells by 25-70%. Likewise, antisera from cattle immunized with recombinant MSP1a or MSP1b inhibited infection of tick cells by 26-37%. These results further confirm the role of MSP1 complex proteins in infection of tick cells. Lack of inhibition of infection by antisera from naturally infected cattle or cattle immunized with whole organisms suggests that the bovine immune response is not directed toward blocking infection of A. marginale for tick cells and may contribute to the continued infectivity of the pathogen for ticks.     

Development of an ELISA system for detection of anti-Anaplasma marginale antibodies in cattle in Brazil

An ELISA test was developed for detecting antibodies against Anaplasma marginale in bovine sera. Four antigenic preparations were produced from infected red blood cells. Some aliquots of this preparation were stored at -70 degrees C with 30% DMSO in phosphate-buffered saline (PBS) and others were lysed with 0.9% NH4Cl and stored at -20 degrees C. Typical anaplasmal structures were seen by electron microscopy in the antigenic preparations containing the erythrocytes that had been stored with DMSO. The performance of the ELISA test was evaluated by testing 298 positive serum samples collected from immunized cattle, 39 negative serum samples collected from cattle imported from areas free of A. marginale and 50 samples collected from cattle naturally infected in the field. The test gave a specificity of 94.87% and a sensitivity of 100%.