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.     

Preliminary studies of the development of Anaplasma marginale in salivary glands of adult, feeding Dermacentor andersoni ticks

On each day of feeding on susceptible calves, salivary glands obtained from groups of adult ticks that transmitted Anaplasma marginale were examined for A marginale colonies by use of light microscopy and transmission electron microscopy. On day 8 of feeding, salivary glands were examined, using fluorescein-labeled antibody and methyl green-pyronine stain. Use of fluorescein-labeled antibody consistently revealed small numbers of fluorescent foci in salivary gland acinar cells obtained from ticks that had fed for 8 days. Colonies of A marginale were seen by transmission electron microscopy only in salivary gland acini of male ticks; these colonies could not be identified, using light microscopy, in companion 1-micron plastic sections stained with Mallory stain. Methyl green-pyronine stain, used commonly to detect theilerial parasites in tick salivary glands, did not differentiate A marginale from cytoplasmic inclusions normally found in salivary gland acinar cells.     

Radioimmunoassay for Anaplasma marginale antibodies in cattle

A radioimmunoassay is described for use in the detection of Anaplasma marginale antibodies in cattle sera. Optimal sensitivity and specificity were obtained by using 2 antigens, an A marginale antigen and a RBC antigen (obtained before infection was established) from the same calf. In addition, sera were preabsorbed with RBC from healthy cattle and with sonicated Babesia bovis. Of 86 sera obtained from cattle with A marginale infection (as determined by blood smear examination or by results of subinoculation of blood from such infected cattle into splenectomized calves), 85 had positive results by use of this test. Of 100 sera obtained from cattle raised in an anaplasmosis-free area, 98 yielded negative results, and sera obtained from 35 cattle (97 sera) infected with B bigemina and from 18 cattle infected with Theileria orientalis yielded negative results. By use of this test, 99 of 100 sera obtained from cattle with B bovis infection were negative for A marginale. Anaplasma marginale antibodies were detected in 18 cattle that had been pastured in a Boophilus microplus-free area for 2 years after natural infection. After 3 years, 16 of these cattle were still seropositive for A marginale. Sixteen cattle pastured in a Bo microplus-infested area had detectable antibody against A marginale 27 months after initial infection with A marginale. Sensitivity and specificity of the test were assessed as 98.8% for each.     

Serologic and molecular characterization of Anaplasma species infection in farm animals and ticks from Sicily

Although Anaplasma marginale was known to be endemic in Italy, the diversity of Anaplasma spp. from this area have not been characterized. In this study, the prevalence of Anaplasma spp. antibodies in randomly selected farm animals collected on the island of Sicily was determined by use of a MSP5 cELISA for Anaplasma spp. and an immunofluorescence test specific for Anaplasma phagocytophilum. Genetic variation among strains of Anaplasma spp. from animals and ticks was characterized using the A. marginale msp1alpha and the Anaplasma spp. msp4 genes. Eight species of ticks were collected and tested by PCR. Seropositivity for Anaplasma spp. and A. phagocytophilum was detected in bovine and ovine samples. All the donkeys were seropositive for A. phagocytophilum but not for Anaplasma spp. Four A. marginale genotypes were identified by msp4 sequences from bovine and tick samples. Two new genotypes of Anaplasma ovis were characterized in sheep. The sequences of A. phagocytophilum from three donkeys proved to be identical to the sequence of the MRK equine isolate from California. Six A. marginale genotypes were found in cattle and one tick using the A. marginale msp1alpha sequences. All genotypes had four repeated sequences in the N-terminal portion of the MSP1a, except for one that had five repeats. The Italian strains of A. marginale contained three repeat sequences that were not reported previously. Definition of the diversity of Anaplasma spp. in Sicily reported, herein is fundamental to development of control strategies for A. marginale, A. ovis and A. phagocytophilum in Sicily.     

A msp1alpha polymerase chain reaction assay for specific detection and differentiation of Anaplasma marginale isolates

Anaplasma marginale is the causative agent of bovine anaplasmosis, a disease which can be protected by vaccination with the less pathogenic Anaplasma species, A. centrale. Currently, there is no polymerase chain reaction (PCR) assay available which differentiates between different species of Anaplasma or which can differentiate isolates of A. marginale within outbreaks and between different countries. A molecular test specific for A. marginale would be ideal for the identification of Anaplasma species in wild ruminants, as possible reservoirs of anaplasmosis, and to differentiate between A. marginale from A. centrale. A PCR assay was designed to amplify the major surface protein 1alpha gene of the rickettsial bovine pathogen, A. marginale both as an inter- and intra-specific test. The test did not amplify A. centrale or A. ovis, and discriminated A. marginale by amplifying repeat regions within the msp1alpha gene which vary in number between many isolates. The nested A. marginale amplicons varied in size from 630 to 1190bp representing one to eight internal repeats. All 22 Australian isolates tested amplified a 630bp product (one repeat) in contrast to all 19 non-Australian isolates tested. Eight sequences from Australian isolates from different geographical regions confirmed the conserved nature of the Australian A. marginale msp1alpha genes. The Australian 'repeat unit' MSP1a deduced amino acid sequence has been designated as Australian type 1. The msp1alpha PCR method developed here enabled the amplification and comparison of A. marginale isolates originating from North and South America, Africa, Israel and Australia. The method is sensitive and specific for A. marginale. Although additional msp1alpha products were amplified from at least two Australian isolates, the results suggest limited introduction of A. marginale into Australia.     

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.     

Survey of anaplasmosis in elk of the Clearwater National Forest (Idaho).

Samples of blood from 27 free-roaming elk (Cervus canadensis canadensis) from the Clearwater National Forest in north central Idaho were tested by the rapid card agglutination test and complement-fixation test for the presence of antibodies against Anaplasma marginale. The serum card test and complement-fixation test gave incomplete and false-positive reactions; the plasma card test did not give any reactions. Anaplasma bodies or other blood parasites were not observed in stained smears of elk blood. Blood from 11 elk, including 2 that were serum card test-positive, did not produce clinical, hematologic, or serologic evidence of infection in 3 anaplasmosis-susceptible bovine calves.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Anaplasma centrale and Anaplasma marginale

An enzyme-linked immunoassay (ELISA) was applied to detect antibodies to A. centrale and A. marginale using homologous and heterologous antigens. The assay was compared with the indirect fluorescent antibody (IFA) test, and although a similar degree of sensitivity was obtained, the ELISA test had several advantages. Partially purified Anaplasma initial bodies used for antigen preparations contained negligible amounts of residual erythrocytic material, and did not interfere with the specificity of the ELISA. The antigenic similarity between A. marginale and A. centrale was further substantiated by cross-reactivity obtained with heterologous antigens in both ELISA and IFA tests, and antibodies produced during natural infection with A. marginale were indistinguishable in both tests from those produced following vaccination with A. centrale.     

Development of an indirect fluorescent antibody test, using microfluorometry as a diagnostic test for bovine anaplasmosis

The indirect fluorescent antibody test for the diagnosis of Anaplasma marginale infection in cattle was modified for use with microfluorometry. The test was standardized by use of a fluorometer that measures intensity of fluorescence. Standardization included A marginale-infected blood smears on microscope slides as antigen, serum from an inoculated calf as a positive control containing specific antibody, and an affinity-purified fluorescein-conjugated anti-bovine immunoglobulin as 2nd antibody. The modified test and microfluorometry allowed for titration of sera from A marginale (Florida isolate)-inoculated cattle with a degree of accuracy exceeding visual determinations. In addition, the fluorometric test was more sensitive than the complement fixation or card agglutination tests in identifying cattle that had previous Anaplasma infections.     

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.     

Single-step technique for staining Anaplasma marginale in bovine blood smears.

Three available differential stains, Camco-Quik, Diff-Quik, and Wright-Giesma were compared for detection of intraerythrocytic Anaplasma marginale in bovine blood smears. In samples where < 1% to more than 51% of the RBC were infected, statistical analysis of the data indicated no significant difference in the detection of A marginale with Camco-Quik or Diff-Quik stains. However, a significantly lower percentage of infected RBC were detected when blood smears were stained with the Wright-Giemsa stain, compared with the other 2 methods.     

In vivo endothelial cell infection by Anaplasma marginale

Anaplasma marginale has recently been shown to infect endothelial cells in vitro, but it remains unknown as to whether endothelial infection also occurs in vivo. In this report, we demonstrate through dual fluorescence microscopy that A marginale, detected by the monoclonal antibody ANAF16C1, co-localizes with the endothelial cell marker, von Willebrand factor, in tissue sections from an experimentally inoculated calf. The results indicate that A marginale infection includes endothelial cells and has implications for both pathogenesis and immune mechanisms.     

Heterologous antibody responses of calves to Anaplasma centrale and A. marginale

Antigens of Anaplasma centrale, Onderstepoort isolate, and A. marginale, Wacol isolate, were analysed by a Western blotting technique. Sera from A. centrale-infected calves reacted to 41- and 38-kDa antigens in A. centrale and a 41-kDa antigen in A. marginale. Serum collected during the primary reaction from an A. marginale-infected calf reacted only to the 41-kDa antigen of A. marginale; heterologous antibody response to the 41-kDa antigen of A. centrale did occur later during the infection, but remained markedly weaker than the homologous response. The serologic cross-reactivity to this 41-kDa Anaplasma antigen confirms that it is common to the genus and also that it is a heterogeneous complex.     

Spherocytosis associated with anaplasmosis in two cows

Spherocytes were detected on blood smears of 2 Angus cows. The RBC from both cows had increased fragility in hypotonic saline solution, supporting the presence of spherocytes. Other laboratory tests revealed a macrocytic, regenerative anemia with anisocytosis, polychromasia, basophilic stippling, Howell-Jolly bodies, nucleated RBC, and Anaplasma marginale organisms. A complement-fixation test was positive for anaplasmosis in cow 1 and a direct Coombs' test was positive for immunoglobulin G in both cows.     

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 congenital anaplasmosis associated with bovine viral diarrhoea virus (BVDV) infection in a crossbred calf

Clinical disease resulting from the vertical transmission of Anaplasma marginale has only been reported on 5 occasions despite studies demonstrating successful in utero transmission. During the reported experimental induction of congenital anaplasmosis in calves, the outcome was variable but mostly led to inapparent or mild infection. There are previous case reports of fatal congenital anaplasmosis following natural infection. The clinical findings in a 2-day-old calf presented to the Onderstepoort Veterinary Academic Hospital with clinical signs of congenital anaplasmosis, which was unresponsive to treatment, are described. Subsequent post mortem diagnostic tests revealed that this calf was co-infected with bovine viral diarrhoea virus (BVDV). It is postulated that immunosuppression resulting from BVDV infection predisposed to severe, fatal anaplasmosis in this calf.     

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%.     

Infectivity and antigenicity of Anaplasma marginale from tick cell culture

The infectivity and immunogenicity of Anaplasma marginale grown in a tick cell culture from embryonic Dermacentor variabilis ticks were assessed in splenectomized and intact calves, respectively. Culture 1 consisted of the cell line inoculated with midguts of adult ticks infected with the Mississippi isolate of A marginale and dissected 5 to 10 days after repletion and detachment from an experimentally infected calf. Cultures 2 and 3 consisted of the cell line inoculated with midguts of ticks infected with the Virginia isolate of the organism. Inoculum for culture 2 was derived from nymphal ticks dissected 5 to 10 days after repletion and detachment from the infected calf; inoculum for culture 3 was midguts from adult ticks that were fed as nymphs, allowed to molt in the laboratory and dissected 21 to 24 days after molting. In trial 1, cultures 1, 2, and 3 were maintained at pH 6.9 and incubated at 28 C; in trial 2, cultures 1 and 3 were maintained at pH 7.4 and incubated at either 28 C or 37 C. Cultures 1, 2, and 3 failed to induce infection when injected IV and SC into 6 calves in 2 separate trials. Pre-challenge sera from these calves reacted with 2 purified Anaplasma antigens in the ELISA, but failed to react in the complement-fixation test. Results of a trial to use cultures 1 and 3 in combination with an oil-in-water adjuvant to immunize intact calves against A marginale were inconclusive. However, pre-challenge sera from immunized calves reacted with the 2 purified Anaplasma initial body antigens in the ELISA but failed to react in the complement-fixation text.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assessment of a low virulence Australian isolate of Anaplasma marginale for pathogenicity, immunogenicity and transmissibility by Boophilus microplus

A 14-year-old cow (Dawn) born and kept in a Boophilus microplus-free region gave birth to a calf, which showed the presence of an Anaplasma marginale infection after splenectomy. The calf's grand dam was from a B. microplus infected area and we assume the infection originated via the transplacental route over two generations. An isolate, prepared from the calf, had similar or lower pathogenicity as Anaplasma centrale, and previously exposed steers were resistant to challenge by four A. marginale field isolates. Two attempts to transmit the isolate using B. microplus were unsuccessful. Our results indicate that Dawn A. marginale may be a useful vaccine in Australia and warrants larger scale validation of its safety and potency locally as well as of the protection it affords against African and New World isolates.     

Susceptibility of Bos indicus and Bos taurus to Anaplasma marginale and Babesia bigemina infections

The reaction of Bos taurus and pure-bred Bos indicus heifers to infection with the intraerythrocytic parasites Anaplasma marginale and Babesia bigemina was studied. B. bigemina infection at 18 months and A. marginale infection at 13 or 24 months resulted in slightly less severe reactions in pure-bred Bos indicus cattle than in Bos taurus. In both breeds, the reaction to A. marginale infection was more severe in older cattle. The severity of B. bigemina infection was not affected by a previous infection with A. marginale.