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PubMed1986

Detection of an Anaplasma marginale common surface protein present in all stages of infection.

The establishment and maintenance of anaplasmosis-free cattle herds is impaired due to the lack of a rapid, sensitive, and specific serologic test to detect persistently infected cattle which serve as carriers for the organism. To develop an improved diagnostic test for anaplasmosis we screened Anaplasma marginale initial body proteins to identify a protein common to antigenically different isolates that is recognized by the host immune system at all stages of infection. Seronegative cattle were infected with either the Florida, Virginia, or North Texas isolate of A. marginale and monitored for infection by daily examination of Wright-stained blood smears for parasitized erythrocytes. Sera from cattle at different stages of infection, from acute through persistent, were used to immunoprecipitate A. marginale proteins that were metabolically radiolabeled with [35S]methionine or surface radiolabeled with 125I. Multiple A. marginale proteins were recognized by using sera either undiluted or at 1:10; however, only four or five proteins were sufficiently antigenic to elicit antibody reactive with a 1:1,000 serum dilution. A single protein with an apparent molecular mass of 86 kilodaltons was consistently recognized at all stages of infection regardless of the isolate used to infect the cattle. This protein was demonstrated to be on the surface of the A. marginale initial body and to be water soluble. We propose use of this 86-kilodalton protein to develop an improved serologic test for diagnosis of bovine anaplasmosis.

Anaplasma↗
PubMed1988

Detection of Anaplasma-marginale-infected tick vectors by using a cloned DNA probe.

Anaplasmosis is the most widely distributed of several important tick-borne diseases that constrain cattle production throughout much of the world. Evaluation of the effectiveness of disease control strategies that integrate vaccination with tick control requires the ability to monitor tick and cattle infection rates. To detect Anaplasma marginale in ticks and bovine erythrocytes, a 2-kilobase DNA fragment from a cloned A. marginale gene coding for a surface protein having a Mr of 105,000 was prepared and evaluated as a probe. The probe was species specific and detected A. marginale DNA derived from infected bovine erythrocytes and adult Dermacentor ticks infected either as nymphs or adults. Tick infection was confirmed by microscopy and test feeding on a susceptible calf. The sensitivity of the probe is suitable for detecting infected ticks in experimental and field epizootiology studies.

Anaplasma↗
PubMed2003

Simultaneous variation of the immunodominant outer membrane proteins, MSP2 and MSP3, during anaplasma marginale persistence in vivo.

Vector-borne bacterial pathogens persist in the mammalian host by varying surface antigens to evade the existing immune response. To test whether the model of surface coat switching and immune evasion can be extended to a vector-borne bacterial pathogen with multiple immunodominant surface proteins, we examined Anaplasma marginale, a rickettsia with two highly immunogenic outer membrane proteins, major surface protein 2 (MSP2) and MSP3. The simultaneous clearance of variants of the two most immunodominant surface proteins of A. marginale followed by emergence of unique variants indicates that the switch rates and immune selection for MSP2 and MSP3 are sufficiently similar to explain the cyclic bacteremia observed during infection in the immunocompetent host.

Amino Acid Sequence↗
PubMed1991

Anaplasma marginale in Lepus nigricollis (Blanford) treated with oxytetracycline.

The objective of this study was to test the efficiency and acceptability of oxytetracycline hydrochloride (OTC) in anaplasmosis. Five mg OTC was the only drug given subcutaneous twice by a week to anaplasmotized Lepus nigricollis. The therapeutic results were judged in relation to the parasitized erythrocytes (PE) present, assessment of the clinical acceptability of OTC was perfect in anaplasmosis (Anaplasma marginale).

Journal Article↗
PubMed1992

Phylogenetic relationship of Cowdria ruminantium, agent of heartwater, to Anaplasma marginale and other members of the order Rickettsiales determined on the basis of 16S rRNA sequence.

The phylogenetic relationship between Cowdria ruminantium and representative members of the orders Rickettsiales and Chlamydiales has been examined on the basis of the sequence of the 16S rRNA. Phylogeny reconstruction by using both parsimony and distance methods supports the conclusion that C. ruminantium is closely related to the Rickettsiales and in particular to the family Anaplasmataceae. A signature of nine base substitutions delineated the linkage of Anaplasma marginale with C. ruminantium and differentiated these two species from the 45 other members of the alpha group of Proteobacteria examined, and five of these base substitutions were unique among all members of the class Proteobacteria examined to date.

Alphaproteobacteria↗
PubMed2001

Identification of antigenic differences that discriminate between cattle vaccinated with Anaplasma centrale and cattle naturally infected with Anaplasma marginale.

Monoclonal antibodies were raised against the vaccine strain of Anaplasma centrale used in Australia. A monoclonal antibody that reacted with an 80 kDa antigen was used to develop an A. centrale-specific fluorescent antibody test that will be useful for confirming species identity in patent infections. Another monoclonal antibody that reacted with a 116 kDa antigen was used to develop an A. centrale-specific competitive inhibition enzyme-linked immunosorbent assay (ELISA) for the serological identification of vaccinated cattle. The sensitivity of the ELISA was 100% in cattle experimentally infected with A. centrale, 97.1% in a vaccinated beef herd and 98.3% in a vaccinated dairy herd. The specificity of the ELISA was 98.6% in non-vaccinated cattle outside the Anaplasma marginale-endemic area, 97.9% in non-vaccinated cattle within the A. marginale-endemic area and 100% in cattle experimentally infected with A. marginale. The ELISA detected antibodies to A. centrale in cattle up to 9 years after vaccination with no apparent decrease in sensitivity. The assay has proved extremely valuable in Australia for investigating reported failures of multivalent live vaccines used to protect cattle against anaplasmosis and babesiosis, and should be similarly useful elsewhere in the world where these types of vaccines are used, e.g. Israel and South America.

Anaplasma↗
PubMed1990

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.

Anaplasma↗
PubMed1978

Bovine abortion associated with Anaplasma marginale.

During the period from June 1974 to June 1975, five bovine fetuses between seven to nine months old were received for necropsy from four different counties of the S. Paulo State, Brazil. All of them were from brucellosis-free herds. Necropsy revealed slight liver enlargement generally accompanied by capsular petechial hemorrhages. Enlargement and congestion of the spleen, epicardial and endocardial petechiae were present in three fetuses and one of them had lungs with some hemorrhagic lobules. Cardiac blood films of all the fetuses stained by the Pappenheim's panoptic method showed Anaplasma marginale in two to 20% of red corpuscles. When stained with acridine orange and immunofluorescent methods blood films of the first fetus specifically showed A. marginale.

Abortion, Spontaneous↗
PubMed2002

Simultaneous detection of Anaplasma marginale and a new Ehrlichia species closely related to Ehrlichia chaffeensis by sequence analyses of 16S ribosomal DNA in Boophilus microplus ticks from Tibet.

To identify ehrlichial agents in Boophilus microplus ticks, DNA samples of B. microplus collected from the Tibet Autonomous Region and Sichuan Province of China were screened by a nested PCR. Sixteen of 43 (37%) DNA samples of B. microplus from Tibet were positive in nested PCR analysis. All 27 samples from Sichuan were negative. The screen identified two ehrlichial agents based on different 16S rRNA genes that were found after amplifying and sequencing the 5'-end fragments of the 16S rRNA genes. One sequence was identical to that of the gene of Anaplasma marginale, an etiological agent of animal anaplasmosis. The other sequence was most similar to that of the gene of Ehrlichia chaffeensis, an etiological agent of human monocytic ehrlichiosis. The sequence of 1,501 bases from the novel ehrlichial agent was obtained and showed the greatest levels of sequence similarity (97 to 98%) to 16S rRNA gene sequences of the members of the E. canis group of the genus EHRLICHIA: Sequence comparison of the 16S rRNA gene with the members of the genus Ehrlichia reveals that the novel ehrlichial agent detected in B. microplus ticks is a new species of the genus Ehrlichia and is most closely related to E. chaffeensis.

Anaplasma↗
PubMed1977

Survival of Aegyptianella pullorum, Anaplasma marginale and various parasitic protozoa following prolonged storage in liquid nitrogen.

Various protozoa species were examined using in vivo and in vitro methods to determine their ability to survive prolonged periods of storage in liquid nitrogen. The following protozoan species were successfully recovered after they had been cryopreserved for a period over 10 years: Trypanosoma lewisi, T. cruzi, T. congolense, T. brucei, T. rhodesiense, T. gambiense, T. evansi, T. equinum, T. equiperdum, Leishmania donovani, Plasmodium berghei, P. praecox (relictum), Babesia rodhaini and B. canis. The longest survival period that has been successfully tested so far is between 5 and 7 years for Eimeria species, for monoxenically (A--C) and axenically grown E. histolytica cultures between 2 and 7 years and trichomonades, including Crithidia sp., between 6 and 8 years. Aegyptianella pullorum was successfully recovered after 6.97 years and Anaplasma marginale after 1.92 years. The freezing techniques are briefly described.

Animals↗
PubMed1986

Anaplasma marginale, Eperythrozoon wenyoni: lectin reactions with bovine erythrocytes.

Normal bovine erythrocytes were agglutinated with four of five lectins specific for different oligosaccharides. The order of reactivity was wheat germ greater than ricin greater than soybean greater than peanut. Concanavalin A did not agglutinate normal bovine erythrocytes. After neuraminidase treatment of normal bovine erythrocytes, each lectin agglutinated the cells with decreased concentrations of lectin, verifying that partial removal of sialic acid exposes more of each lectin's binding sites or alters the binding site such that fewer molecules of lectin are required to initiate agglutination. A change in agglutination of erythrocytes using soybean agglutinin and peanut agglutinin occurred when cells were obtained from cattle infected with Eperythrozoon wenyoni. The results suggested that an alteration in erythrocyte membranes occurred as a result of this infection as manifested by the increased recognition of both the soybean agglutinin and peanut agglutinin receptor carbohydrates. A similar effect was indicated with erythrocytes obtained during an acute Anaplasma marginale infection; however, an ensuing reticulocytosis masked the effect, requiring the use of fluoresceinated lectins to verify that increased binding of each lectin occurred with infected cells when compared to normal cells.

Anaplasmataceae Infections↗
PubMed1975

Ultrastructure of Anaplasma marginale after freeze-fracture.

Stained thin sections and freeze-fractured replicas of Anaplasma marginale-infected bovine erythrocytes were examined by electron microscopy. Freeze-fracture replication not only verified basic Anaplasma ultrastructure, but also allowed visualization of structures not previously reported. Because of the partial 3-dimensional views obtained with freeze-fracture replication, a new structure that appears as a protruding tip was discernible. Also, the surface of Anaplasma's limiting membrane was less granular than the fractured surface of host erythrocyte. A corrugated surface with a periodicity of 10.5 nm was seen when the limiting membrane was fractured.

Anaplasma↗
PubMed1988

Nucleic acid levels in Anaplasma marginale during experimentally induced bovine anaplasmosis.

The DNA and RNA concentrations in uninfected and Anaplasma marginale-infected bovine erythrocytes were determined. Bovine anaplasmosis was experimentally induced and the nucleic acid levels were followed during the development of the A. marginale infection. Two distinct growth stages were found, the "multiplication" stage and the "transfer" stage. Based on this hypothetycal developmental cycle, the amount of DNA and RNA per Anaplasma initial body was estimated as 73.1 x 10(-3) pg and 45.7 x 10(-3) pg respectively.

Anaplasma↗
PubMed2005

Potential vertebrate reservoir hosts and invertebrate vectors of Anaplasma marginale and A. phagocytophilum in central Spain.

Organisms in the genus Anaplasma are obligate intracellular pathogens that multiply in both vertebrate and invertebrate hosts. The type species, A. marginale, causes bovine anaplasmosis and only infects ticks and ruminants. A. phagocytophilum causes human and animal granulocytic anaplasmosis, and genetically closely related strains show a wide host range, including ticks, ruminants, rodents, equids, canids, birds, and humans. Recent reports demonstrated that A. marginale and A. phagocytophilum co-exist in geographic areas and that concurrent infections occur in ruminants and ticks. In this study, we characterized A. marginale and A. phagocytophilum infections in wild and domestic animals, and ticks collected in central Spain by serology, PCR, and sequence of 16S rRNA genotypes. Species tested included humans, cattle, dogs, rodents, Iberian red deer, European wild boar, birds, and ticks. Species of hematophagous Diptera were analyzed as potential mechanical vectors of Anaplasma spp. A. marginale was detected in tabanids, ticks, cattle, and deer, while A. phagocytophilum was detected in ticks, deer, cattle, and birds. Concurrent infections of the two Anaplasma were found in cattle and deer. These results illustrate the complexity of the epizootiology of A. marginale and A. phagocytophilum in regions where both pathogens co-exist and share common reservoir hosts and vectors. The increasing contact between wildlife, domestic animals, and human populations increases the risk of outbreaks of human and bovine anaplasmosis, and the difficulty of implementing surveillance and control measures.

Anaplasma marginale↗