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PubMed1992

Prevalence of Babesia bovis and Anaplasma marginale at selected localities in Sri Lanka.

Sera were collected from a minimum of 20 cattle aged nine to 36 months at each of 14 localities in five climatic zones of Sri Lanka. Sera were tested for antibodies to Babesia bovis and Anaplasma by an indirect fluorescent antibody test and a card agglutination test, respectively. Antibodies to B. bovis and Anaplasma were detected in all samples tested from each of 14 and 12 localities respectively. In general, prevalences were consistently high among localities below 1,200 m and lower and more variable in the hill country. Management practice rather than climate was considered to be the main factor influencing prevalences. Management is discussed in relation to the risk of disease outbreak.

Agglutination Tests↗
PubMed1985

Chemoprophylaxis of Anaplasma ovis infection in sheep with a long-acting oxytetracycline.

The chemoprophylactic efficacy of long-acting oxytetracycline was determined in 13 susceptible ewes infected with Anaplasma ovis. The drug was administered intramuscularly at a dose rate of 20 mg kg-1 body weight during the prepatent period. When the ewes were exposed to an equivalent homologous challenge on Day 45 post-infection, each showed a mild or inapparent reaction. The host reactions, i.e., body temperature, parasitaemia, packed-cell volume (PCV), haemoglobin, humoral and cell mediated immunity (CMI) were also studied. The results indicate that the use of oxytetracycline during the incubation period would minimise clinical signs of an Anaplasma infection; this may be due to an increasing CMI response.

Anaplasma↗
PubMed1989

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.

Anaplasma↗
PubMed1994

Antigenic profile of a pure isolate of Anaplasma marginale of Brazilian origin, using a western blot technique.

Anaplasma marginale initial bodies of the Brazilian isolate AUFV1 were purified from infected erythrocytes using a combination of lysis, ultrasonic disruption and differential centrifugation. Initial bodies were solubilised with a buffer containing protease inhibitors and non-ionic detergents. Immunochemical analysis by the Western blot technique revealed at least five proteins with apparent molecular weights (MW) of 105, 100, 97, 87 and 38 kDa when homologous sera were used as primary antibodies. Sera from cattle from Mato Grosso do Sul State in Brazil revealed five proteins of 105, 100, 87, 38 and 25 kDa; other heterologous sera obtained from Illinois, USA, bound to four antigens with MW of 105, 100, 87 and 38 kDa, the latter being stronger and broader than the others. No bands were observed in the non-infected erythrocyte control when the different A. marginale sera or antibodies against Babesia bovis and Babesia bigemina were used. Antibodies from cattle infected with the A. marginale isolates bound to proteins of 105, 100, 87 and 38 kDa, indicating that there are at least four peptides common to the isolates. The major surface proteins, designated MSP-1, MSP-2 and MSP-3, are also present in the Brazilian isolate AUFV1 and the sample from the National Research Centre of Beef Cattle in Mato Grosso do Sul State. One practical consideration of this study is the possibility of cross-protection between different Anaplasma isolates including some from Brazil.

Anaplasma↗
PubMed1984

Serologic comparisons of 4 Anaplasma isolates as measured by the complement-fixation test.

Anaplasma marginale isolated from Virginia ( VAM ), from North Texas ( NTAM ), from Florida (FAM), and Anaplasma ovis from Idaho were used in these trials. Complement-fixation antigens from each of the 4 isolates were used to compare complement-fixing antibody titers of 10 cattle infected with VAM , 17 with FAM, and 6 with NTAM . Strong cross-reactions occurred with all antigens and sera. The homologous system generally showed higher average antibody titers. The serum antibody titers occurring with the A. ovis antigen were significantly lower than those seen with A. marginale antigens. Serum antibody titer differences as measured by FAM and NTAM antigens on sera from VAM , NTAM and FAM failed to reach significance. Serum antibody titer comparisons using VAM antigen were significantly different from those occurring with FAM and NTAM in most instances.

Anaplasma↗
PubMed1993

Antigen profiles of Anaplasma ovis and A. mesaeterum and cross infection trials with them and A. marginale.

Antigen profiles of Anaplasma ovis and A. mesaeterum were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, and cross infection trials were performed by A. ovis challenge to A. mesaeterum- or A. marginale-inoculated goats and A. marginale challenge to an A. ovis-inoculated calf. Antigen analysis showed unique and common proteins of A. ovis and A. mesaeterum and identified a major 38 kDa protein to be a dominant immunogen bearing two epitopes common to Anaplasma species. An epitope specific to A. ovis and A. marginale, absent from A. mesaeterum, was recognised on the major protein of A. ovis. Pre-inoculation of goats with A. mesaeterum induced continuous antibody response with low parasitemia and partial protection against A. ovis challenge, as demonstrated by lower peak parasitemia and normal body temperature compared with values for goats inoculated with A. ovis alone. Pre-inoculation of goats with A. marginale and a calf with A. ovis induced weak or no antibody response without parasitemia, and was of no effect for preventing anaplasmosis by heterologous challenge. These results suggest that the development of parasitemia is essential for inducing continuous antibody by which cross protection would be possible.

Anaplasma↗
PubMed2004

A novel antigen from Anaplasma marginale: characterization, expression and preliminary evaluation of the recombinant protein.

Through a process of protein fractionation and vaccination we previously identified four native antigens that confer a degree of protection against challenge with Anaplasma marginale. One of these, Ana 29 has been successfully cloned and sequenced using degenerate primers designed to N-terminal and internal peptide sequences. The full-length gene codes for a protein with a theoretical molecular weight of 27 kDa and pI 8.6. The sequence is highly conserved, showing 99% identity between two Australian and an American isolate of A. marginale. The gene sequences from these isolates also share 99% identity with the strain of Anaplasma centrale used in the commercial Australian vaccine. Protein prediction algorithms suggest the native protein is an integral membrane protein. This protein has been over-expressed and purified from Escherichia coli and used in vaccination trials in cattle using two adjuvants. The initial results from the trial show a significant level of protection was obtained with one adjuvant; in comparison, the second adjuvant slightly aggravated the disease. Preliminary data suggests a good correlation between the induction of an IgG2 response and protection.

Amino Acid Sequence↗
PubMed2004

Adhesion of outer membrane proteins containing tandem repeats of Anaplasma and Ehrlichia species (Rickettsiales: Anaplasmataceae) to tick cells.

Infection of cells by tick-borne rickettsiae appears to be mediated by outer membrane proteins that allow pathogens to adhere to host cells. Major surface protein (MSP) 1a of Anaplasma marginale, the type species for the genus Anaplasma, was shown previously to be an adhesin for tick cells. The A. marginale MSP1a has a variable number of tandem 28 or 29 amino acid repeats located in the amino terminal region of the protein that contains an adhesion domain that is necessary and sufficient for infection of tick cells. The MSP1a studies demonstrated the importance of combining structural and functional characteristics for identification of adhesive proteins. In the present study other outer membrane proteins containing tandem repeats were selected from organisms of the family Anaplasmataceae and studied for their adhesive properties to tick cells. The adhesive properties and protein characteristics were then analyzed in order to provide a predictor of the adhesion function of proteins identified from genome sequences. Proteins selected included the A. marginale MSP1a, A. phagocytophilum 100 and 130 kDa, Ehrlichia chaffeensis 120 kDa, E. canis 140 kDa and E. ruminantium "mucin", which were all cloned and expressed in Escherichia coli and then tested as adhesins for cultured IDE8 cells. Of the proteins studied, the A. marginale MSP1a and the E. ruminantium "mucin" were found to be adhesins for tick cells. Although all of these recombinant outer membrane proteins were glycosylated, the A. marginale MSP1a and E. ruminantium "mucin" adhesins shared a common feature of having a high Ser/Thr content in the tandem repeats. The results reported herein provide new information on the role of E. ruminantium "mucin" as an adhesin for tick cells and also suggest a role of glycans in adhesin molecules.

Anaplasma↗
PubMed2005

Anaplasma marginale: lack of cross-protection between strains that share MSP1a variable region and MSP4.

In Mexico, there are no commercial alternatives for the immunoprophylaxis of bovine Anaplasmosis, a disease responsible for great economic losses. Blood derived Anaplasma marginale used for immunizing susceptible cattle has shown promising results for homologous protection and controversial results against unrelated strains. The present study examined, under controlled conditions, the cross-protective potential of an immunogen composed of blood derived A. marginale of three strains against challenge with strains not included in the immunogens. Groups 1 and 2 were immunized with blood derived Anaplasma from strains Mexico, Morelos and Yucatan, group 4 with strains Morelos, Veracruz and Yucatan, two more groups (2 and 5) of equal conditions were inoculated with an adjuvant alone. Groups 1, 4 and 5 were challenged with Mexico strain; groups 2 and 3 were challenge-inoculated with strain Veracruz; groups 3 and 5 with strains Veracruz and Mexico as controls. Only animals in group 1, immunized and challenged with strain Mexico showed adequate protection. Both groups challenged with strains not included in the immunogens developed poor protection, while all the controls had to be treated to prevent death.

Anaplasma marginale↗
PubMed2003

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.

Anaplasma marginale↗
PubMed2005

Serological evidence of infection of Anaplasma and Ehrlichia in domestic animals in Xinjiang Uygur Autonomous Region area, China.

Serological methods were utilized to detect Anaplasma and Ehrlichia infection in domestic animals in Xinjiang Uygur Autonomous Region, China. By using an indirect immunofluorescence assay (IFA), antibodies that reacted with Anaplasmaphagocytophilum and Ehrlichiachaffeensis were detected mainly in ruminants kept on pastureland in Altai, Ili and Kashgar area. Antibody titers up to 1:320 were recorded. These results indicate that ruminants kept in these areas may be infected with some species of Anaplasma and Ehrlichia.

Anaplasma phagocytophilum↗
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↗
PubMed1997

Ultrastructure of Anaplasma marginale with an inclusion appendage, isolated in Minas Gerais State, Brazil.

This is the first report on the occurrence and isolation of a strain of Anaplasma marginale with an inclusion appendage in Brazil. The inclusion appendage presented longitudinal electron-dense striations and did not originate directly from the body of the rickettsia but from an electron-dense complex located at the junction of the inclusion membrane and inclusion appendage. The inclusion appendage remained in the host cell after the Anaplasma inclusion appeared to be leaving the red blood cell. Other ultrastructures of this rickettsia are described and its epidemiological importance is discussed.

Anaplasma↗
PubMed2006

An Ixodes scapularis protein required for survival of Anaplasma phagocytophilum in tick salivary glands.

Anaplasma phagocytophilum is the agent of human anaplasmosis, the second most common tick-borne illness in the United States. This pathogen, which is closely related to obligate intracellular organisms in the genera Rickettsia, Ehrlichia, and Anaplasma, persists in ticks and mammalian hosts; however, the mechanisms for survival in the arthropod are not known. We now show that A. phagocytophilum induces expression of the Ixodes scapularis salp16 gene in the arthropod salivary glands during vector engorgement. RNA interference-mediated silencing of salp16 gene expression interfered with the survival of A. phagocytophilum that entered ticks fed on A. phagocytophilum-infected mice. A. phagocytophilum migrated normally from A. phagocytophilum-infected mice to the gut of engorging salp16-deficient ticks, but up to 90% of the bacteria that entered the ticks were not able to successfully infect I. scapularis salivary glands. These data demonstrate the specific requirement of a pathogen for a tick salivary protein to persist within the arthropod and provide a paradigm for understanding how Rickettsia-like pathogens are maintained within vectors.

Anaplasma phagocytophilum↗
PubMed2004

Acquisition of different isolates of Anaplasma phagocytophilum by Ixodes scapularis from a model animal.

The prevalence of etiologic agents in ticks reflects the intensity of their transmission in natural cycles and is an important measure of their potential to cause human disease. The distribution of Anaplasma phagocytophilum within the range of its primary vectors is patchy. Even nearby sites differ dramatically in the prevalence of Anaplasma in questing ticks. We hypothesized that this irregular distribution may be due in part to variations in acquisition rates of different isolates of A. phagocytophilum by I. scapularis ticks from infected animals. BALB/c mice were infected with seven isolates of A. phagocytophilum from different geographical regions: four isolates from the Northeastern United States (Bridgeport, Dawson, Gaillard, NY-8), two from the Midwest (Webster, Sp-Is), and one from California (MRK). Larval ticks were placed on infected mice for 16 consecutive weeks and allowed to feed to repletion. The prevalence of infection in the freshly molted nymphs was then determined by PCR. The proportion of ticks that became infected with either isolate fluctuated over the duration of infection. Mice harboring the isolate Sp-Is were most infectious for ticks at 3 weeks postinfection. Mice infected with the other six isolates exhibited several peaks of infectivity. Timing and relative heights of these peaks differed between isolates. Geographical proximity of the studied isolates did not predetermine their similarity, and isolates originating from the same region differed more in their ability to infect ticks than isolates from different regions. However, it appears unlikely that described differences in agent acquisition by ticks alone are sufficient to account for the irregular distribution of A. phagocytophilum in vector populations.

Anaplasma phagocytophilum↗
PubMed1992

Persistence of Anaplasma marginale (Rickettsiales: Anaplasmataceae) in male Dermacentor andersoni (Acari: Ixodidae) transferred successively from infected to susceptible calves.

The persistence of Anaplasma marginale Theiler in male Dermacentor andersoni Stiles ticks exposed to the organism as adults was studied as the ticks were successively transferred to five susceptible calves. All calves fed upon by these ticks rapidly developed clinical anaplasmosis; incubation periods of infection ranged from 19 to 26 d and did not change significantly with successive feedings. Development of A. marginale in tick midgut and salivary glands was followed daily during tick feeding (total, 35 d) with light microscopy and DNA hybridization. With microscopy, A. marginale colonies persisted in midgut cells throughout the experiment. Large colonies were observed in gut muscle cells on days 8 through 35 and were the predominant infected cell type during this part of feeding. Colonies were seen in salivary gland acini from day 2 throughout the 35-d experiment. The DNA probe confirmed the presence of Anaplasma DNA in midgut and salivary glands throughout the experiment. Quantitative estimates of infection intensity in tissues of individual ticks approximated 10(7) initial body equivalents, confirming heavy infections. A marginale in midgut tissues decreased with feeding time, whereas the estimated number of organisms in salivary glands remained constant. These data demonstrate that D. andersoni males are efficient vectors of A. marginale and may be potential reservoirs of infection for ruminants for extended periods.

Anaplasma↗
PubMed1993

Detection of Anaplasma marginale (Rickettsiales: Anaplasmataceae) in hemolymph of Dermacentor andersoni (Acari: Ixodidae) with the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to detect Anaplasma marginale in hemolymph collected from live Dermacentor andersoni Stiles ticks. Hemolymph was collected from severed legs of male and female ticks exposed to A. marginale as either nymphs or adults. Heat treatment was found to be the optimum method of hemolymph preparation for PCR. Hemolymph samples were collected and pooled from adult ticks exposed as nymphs on days 0-10 of feeding on a susceptible calf. For male and female ticks exposed as adults, samples were collected as ticks fed 7 d on an infected calf, while being held 9 d between feedings, and during a second feeding of 10 d (or to repletion) when they transmitted the parasite. Hemolymph samples were collected from uninfected ticks at the same times to serve as controls. Anaplasma marginale DNA was amplified with primers BAP-2 (5'-GTATGGCACGTAGTCTTGGGATCA-3') and AL34S (5'-CAGCAGCAGCAAGACCTTCA-3'), which flank a 409-bp fragment of the A. marginale Florida isolate msp1 beta gene. Infected tick hemolymph was PCR-positive for A. marginale at all collection times, including unfed adults infected as nymphs and previously unexposed adults that fed on infected calves for only 1 d. The PCR-based assay of tick hemolymph proved to be a sensitive method for identification of infected ticks, potentially without killing them; it would be well suited for identification of laboratory- or field-infected ticks that could then be used for further studies. The primers used in this assay were also found specific when tested with species of 18 different genera, and universal for 7 A. marginale isolates from diverse geographical areas of the United States.

Anaplasma↗
PubMed1996

Establishment of the tick (Acari:Ixodidae)-borne cattle pathogen Anaplasma marginale (Rickettsiales:Anaplasmataceae) in tick cell culture.

Anaplasma marginale is a tick-borne rickettsia that causes bovine anaplasmosis worldwide. Despite its importance, A. marginale has thus far not been established in a continuous culture system. We have propagated A. marginale continuously for the 1st time in a tick cell line derived from the black-legged tick, Ixodes scapularis Say, using infected bovine blood as the inoculum. Erythrocytic stages invaded the tick cells and multiplied in membrane-lined vacuoles to form colonies typical of those observed in naturally infected ticks as demonstrated by light and electron microscopy. The rickettsiae have been passaged serially for 3 yr and have been cryopreserved in liquid nitrogen. Antigens present in A. marginale from tick cell culture were recognized by bovine immune serum against the blood stages of A. marginale. A. marginale grown in this tick cell line was infective for calves, and male ticks fed on the calves transmitted A. marginale to a susceptible calf. The ability to culture A. marginale removes a major impediment to the study of Anaplasma biology in vitro, and will enhance development of vaccines and diagnostic tests.

Anaplasma↗