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PubMed1980

Susceptibility of Bos indicus crossbred and Bos taurus cattle to Anaplasma marginale infection.

The effects of infection with Anaplasma marginale in groups of Bos indicus-Brahman crossbred and Bos taurus cattle aged 27 and 48 months were studied using the parameters of body weight change, packed cell volume, parasitaemia and humoral antibody. Twenty-six animals (8 Bos taurus and 18 Bos indicus crosses) of each age group were infected. No significant differences between any of the groups were observed.

Anaplasma↗
PubMed1991

Crossprotective immunity between the Florida and a Zimbabwe stock of Anaplasma marginale.

Cattle immunised by infection with the Florida stock of Anaplasma marginale were protected against subsequent homologous challenge, as demonstrated by complete prevention of microscopically detectable parasitaemia. Identically immunised cattle were partially protected against challenge with the Norton, Zimbabwe stock of A. marginale as determined by the significant prolongation of the prepatent period, a significantly lower peak parasitaemia, and a significantly lower percentage drop in haemoglobin concentration when compared to non-immunised calves challenged identically. The partial protection induced by live Florida stock immunisation demonstrates that while protection-inducing epitopes are shared between the two stocks, induction of complete immunity against a Zimbabwe stock may require alternative presentation of Florida stock epitopes or inclusion of additional Zimbabwe stock epitopes in the immunogen.

Anaplasma↗
PubMed2006

Quantitation of Anaplasma marginale major surface protein (MSP)1a and MSP2 epitope-specific CD4+ T lymphocytes using bovine DRB3*1101 and DRB3*1201 tetramers.

Antigen-specific CD4+ T cells play a critical role in protective immunity to many infectious pathogens. Although the antigen-specific CD4+ T cells can be measured by functional assays such as proliferation or cytokine enzyme-linked immunospot, such assays are limited to a specific function and cannot quantify anergic or suppressed T cells. In contrast, major histocompatiblity complex (MHC) class II tetramers can enumerate epitope-specific CD4+ T cells independent of function. In this paper, we report the construction of bovine leukocyte antigen MHC class II tetramers using a novel mammalian cell system to express soluble class II DRA/DRB3 molecules and defined immunodominant peptide epitopes of Anaplasma marginale major surface proteins (MSPs). Phycoerythrin-labeled tetramers were either loaded with exogenous peptide or constructed with the peptide epitope linked to the N terminus of the DRB3 chain. A DRB3*1101 tetramer loaded with MSP1a peptide F2-5B (ARSVLETLAGHVDALG) and DRB3*1201 tetramers loaded with MSP1a peptide F2-1-1b (GEGYATYLAQAFA) or MSP2 peptide P16-7 (NFAYFGGELGVRFAF) specifically stained antigen-specific CD4+ T cell lines and clones. Tetramers constructed with the T-cell epitope linked to the DRB3 chain were slightly better at labeling CD4+ T cells. In one cell line, the number of tetramer-positive T cells increased to approximately 94% of the CD4+ T cells after culture for 21 weeks with specific antigen. This novel technology should be useful to track the fate of antigen-specific CD4+ T-cell responses in cattle after immunization or infection with persistent pathogens, such as A. marginale, that modulate the host immune response.

Amino Acid Sequence↗
PubMed2006

Anaplasma phagocytophilum in central and western Wisconsin: a molecular survey.

Anaplasma phagocytophilum is an obligate intracellular bacterium that is transmitted to humans through the bite of Ixodes spp. ticks, and causes a febrile disease known as human granulocytic anaplasmosis. The presence of A. phagocytophilum in Wisconsin white-tailed deer blood and in deer ticks was assessed using PCR and DNA sequencing. Sampling sites in the western part of the state (Buffalo County) and central region (Waushara, Waupaca, and Green Lake counties) were used. In Buffalo County, 5.6% of deer and 8.9% of ticks were infected. At Hartman Creek State Park (Waupaca County), 11.5% of ticks were infected, while the observed prevalence in deer from counties to the south of the park (Waushara and Green Lake) reached 19-26%. Based on 16S rRNA sequences, A. phagocytophilum strains associated and not associated with human infections were identified. Furthermore, two novel A. phagocytophilum variants were found in deer blood samples. Transmission of Lyme disease has been documented in both the Western and Central regions we sampled, and the presence of A. phagocytophilum in naturally occurring tick populations could present an additional risk of disease to humans that enter tick habitats.

Anaplasma phagocytophilum↗
PubMed1996

Peripheral blood lymphocyte proliferative responses in cattle infected with or vaccinated against Anaplasma marginale.

An assay was developed for measurement of the peripheral blood lymphocyte proliferative response (PBLPR) in cattle infected with or immunised against Anaplasma marginale. PBLPR was not evident in all cattle that had recovered from A. marginale infection. However, A. marginale-sensitised lymphocytes were detected in the spleens of all immune cattle tested in the absence of detectable PBLPR. During the course of initial infection, cattle exhibited detectable PBLPR for a period corresponding with and up to 2 weeks after patent parasitaemia, followed by a second, usually larger peak in PBLPR corresponding to the time of sub-clinical relapse of cattle. Analysis of the PBLPR of A. marginale chronically infected cattle demonstrated highly variable PBLPR between individuals and over time. A positive PBLPR was induced in cattle by vaccination using a crude A. marginale antigen preparation. The PBLPR of vaccinated cattle subsequently infected with A. marginale was markedly different from that of naive cattle, with reduced PBLPR being associated with the onset of parasitaemia. The antigen used in the PBLPR assay was inactivated by proteolysis. Proteolysis also abolished immunity that had been induced in cattle vaccinated using the antigen preparation. A marginale-sensitised PBL did not proliferate in response to antigen from the heterologous species A. centrale. A. centrale-sensitised PBL, however, responded to A. marginale antigen. Interferon-gamma (IFN-gamma) was detected in PBLPR-assay supernatants and was associated with a strong PBLPR.

Anaplasma↗
PubMed2006

Differences in the transmissibility of two Anaplasma phagocytophilum strains by the North American tick vector species, Ixodes pacificus and Ixodes scapularis (Acari: Ixodidae).

The etiologic agent of granulocytic anaplasmosis, Anaplasma phagocytophilum, has a circum-global distribution within the northern hemisphere and shows a host species predilection that varies by the geographic region in which the disease is found. Adaptation by the bacterium to a host species potentially contributes to the variation found worldwide but this is confounded by the bacterium's relationship with its tick vectors, all of which belong to the Ixodes ricinus group. We tested the hypothesis that tick vector species collected from geographic regions sympatric with particular A. phagocytophilum strains will show evidence of a higher degree of vector competence than will tick species and allopatric A. phagocytophilum strains. A reciprocal cross-transmission experiment was performed using an eastern and a western North American strain of A. phagocytophilum (Webster and MRK, respectively) and the two tick species, I. scapularis and I. pacificus, most commonly associated with human and animal transmission of the bacteria in the United States. The western tick, I. pacificus, showed a significantly higher vector competence for A. phagocytophilum than I. scapularis and the eastern isolate, Webster, was more transmissible than its western counterpart, MRK. These results indicate that geographic variation in host susceptibility to A. phagocytophilum strains may play a more important role in the epidemiology of granulocytic anaplasmosis than does the competence of its tick vectors to transmit the pathogen.

Anaplasma phagocytophilum↗
PubMed1992

Sequence of a putative glutathione synthetase II gene and flanking regions from Anaplasma centrale.

The complete nucleotide sequence of a putative glutathione synthetase gene (gsh II) has been determined from Anaplasma centrale. The predicted 308 amino acid protein has a molecular weight of 34,222 and is 32% identical to the enzyme, glutathione synthetase (EC 6.3.2.3), encoded by Escherichia coli gsh II. The previously proposed ATP-binding site is not highly conserved. The putative glutathione synthetase gene (gsh II) is preceded by an unassigned open reading frame. Downstream of gsh II is the 5' region of an open reading frame encoding a protein with significant similarity to bacterial D-alanine:D-alanine ligases (ADP forming) (EC 6.3.2.4). The predicted partial amino acid sequence is 33% identical to the amino acid sequence of the protein encoded by the E. coli ddl gene.

Adenosine Triphosphate↗
PubMed1983

Acquired cellular responsiveness in cattle cleared of Anaplasma marginale 28 months earlier.

The leukocyte migration inhibition test (LMIT) was used to assess cell-mediated immune responses of cattle against Anaplasma marginale. Leukocytes from 7 of 11 cows that had been cleared of the carrier state by antibiotic therapy 28 months earlier were inhibited from their normal migration by A. marginale antigens (P less than 0.001). After reexposure to virulent organisms, all animals were positive to the LMIT. There was not a significant relationship (r = 0.49) between LMIT values before and after reexposure. Results suggest that animals free of anaplasmosis for more than 2 years are capable of cell-mediated immune responses, despite decreased in vitro responsiveness at the time of reexposure. Acquired cellular responses did not prevent reinfection as all animals became A. marginale carriers.

Anaplasma↗
PubMed1993

Opsonization of Anaplasma marginale mediated by bovine antibody against surface protein MSP-1.

Antibody from cattle immunized with purified major surface protein-1 (MSP-1) was demonstrated to significantly enhance phagocytosis of Florida strain Anaplasma marginale by bovine macrophages in vitro. Serum immunoglobulin from individual MSP-1 immunized, protected cattle varied in ability to promote phagocytosis, however all sera were significantly opsonic as compared with sera from sham immunized control cattle.

Anaplasma↗
PubMed1994

Heterologous strain challenge of cattle immunized with Anaplasma marginale outer membranes.

In this study, we tested the hypothesis that outer membrane immunization would induce protection against an antigenically variant strain. Outer membranes were isolated from the Virginia strain of Anaplasma marginale using density gradient centrifugation, combined with saponin adjuvant, and used to immunize Friesian cattle in Zimbabwe. Immunized cattle developed high antibody titers (80,000-160,000) against outer membrane polypeptides including MSP-2 and MSP-5 in both the homologous Virginia and heterologous Zimbabwe strains. Outer membrane immunized cattle were protected significantly following challenge with 10(4) Zimbabwe strain parasitized erythrocytes, demonstrated by significant differences in prepatent period and peak rickettsemia compared with adjuvant immunized control cattle. One outer membrane immunized animal was completely protected against infection. However, there were no overall significant differences in severity of anemia between cattle immunized with outer membrane and the control group, indicating that a significant reduction in rickettsemia does not necessarily result in less severe anemia.

Anaplasma↗
PubMed1994

Recombinant vaccinia virus expression of Anaplasma marginale surface protein MSP-1a: effect of promoters, leader sequences and GPI anchor sequence on antibody response.

Anaplasma marginale surface protein MSP-1a was expressed by recombinant vaccinia viruses with different promoters and as hybrid proteins. Transcription of msp1 alpha with P11 late promoter resulted in more MSP-1a than with P7.5 early-late promoter; however, mice immunized with the recombinants had similar antibody titres. Recombinants expressing hybrid MSP-1a with either a murine leukaemia virus or a trypanosomal glycoprotein signal sequence did not enhance antibody responses and resulted in a diffuse intracellular distribution of MSP-1a which did not accumulate in the Golgi apparatus as was noted in the absence of these signal sequences. In contrast, antibody titres to MSP-1a in mice immunized with a recombinant virus expressing hybrid MSP-1a with a trypanosomal GPI anchor signal sequence were significantly increased over all other constructs.

Anaplasma↗
PubMed1983

The use of tick transmission by Boophilus microplus to isolate pure strains of Babesia bovis, Babesia bigemina and Anaplasma marginale from cattle with mixed infections.

Pure strains of Babesia bovis, Babesia bigemina and Anaplasma marginale were isolated from cattle infected with all 3 species as well as Theileria sp. and Eperythrozoon teganodes, using only transmission by the tick, Boophilus microplus. Unengorged adult ticks transferred to susceptible cattle transmitted A. marginale, but not Babesia. Engorged adults gave rise to progeny that transmitted Babesia, B. bovis by larvae and B. bigemina by male ticks. The Theileria and E. teganodes were not transmitted by the ticks and thus did not appear in calves used for isolating the pure strains of Babesia and A. marginale.

Anaplasma↗
PubMed1985

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.

Age Factors↗
PubMed1986

Prevalence of Anaplasma marginale, Babesia bigemina and B. bovis in Nigerian cattle using serological methods.

Serum samples collected randomly from 500 cattle from the 10 northern states of Nigeria were tested for antibodies against Anaplasma marginale by indirect fluorescent antibody (IFA), card agglutination (CT) and capillary tube-agglutination (CA) tests. The serum samples were also examined for antibodies to Babesia bigemina and B. bovis by the IFA test only. Of the serum samples tested, 79.4% had antibodies against A. marginale by the IFA test, 40 and 25% in the CT and CA tests, respectively. The IFA test results for B. bigemina and B. bovis were 29.4 and 14.1%, respectively.

Agglutination Tests↗
PubMed1988

A sensitive ELISA technique for the diagnosis of Anaplasma marginale infections.

A sensitive enzyme-linked immunosorbent assay (ELISA) technique using a horse radish peroxidase conjugate is described for measuring Anaplasma marginale antibodies in bovine serum. This technique utilizes two antigen preparations, a 'negative' antigen derived from an animal prior to infection and a 'positive' antigen derived from A. marginale-infected red cells from the same animal following infection. This markedly reduces cross-reactions which are a result of isoantigens. Absorbance values obtained using the 'negative' antigen are subtracted from those obtained using the 'positive' antigen to give a net figure. Of 100 A. marginale-positive sera tested no false negative results were obtained. All 11 animals maintained tick-free after initial diagnosis of naturally transmitted anaplasmosis were positive 3 years later, 15 A. marginale-infected animals maintained with ticks were positive 27 months after initial infection and a further 26 animals infected with A. marginale by blood inoculation were positive 3 months later. Three per cent of negative sera, 2% of B. bovis and 4% of B. bigemina-positive sera gave positive reactions.

Anaplasma↗
PubMed1989

Infectivity of cryopreserved Babesia bovis, Babesia bigemina and Anaplasma centrale for cattle after thawing, dilution and incubation at 30 degrees C.

Blood containing either Babesia bovis, Babesia bigemina or Anaplasma centrale was mixed with an equal volume of 3 M glycerol in phosphate-buffered saline with or without glucose and then stored in liquid nitrogen for 2-30 days. After being thawed, the parasitized blood was subjected to various procedures, including dilution up to 1000-fold followed by incubation at 30 or 4 degrees C for 8 h, before infectivity of the parasites was tested in a total of 70 cattle. The results showed that the blood cryopreserved with glycerol remained highly infective after thawing, despite dilution and incubation for 8 h at 30 degrees C. The results have practical application in the use of frozen, live vaccines against bovine babesiosis and anaplasmosis.

Anaplasma↗
PubMed1993

Multiplex polymerase chain reaction based assay for the detection of Babesia bigemina, Babesia bovis and Anaplasma marginale DNA in bovine blood.

A highly sensitive polymerase chain reaction (PCR) based method was developed to detect, in the same blood sample, DNA of hemoparasites frequently found together infecting cattle in tropical and subtropical areas. Bovine blood containing equal parasitemias of Babesia bigemina, B. bovis and Anaplasma marginale infected erythrocytes was mixed to standardize the test. Twenty microliters of 10-fold dilutions from the pooled blood sample were resuspended in PCR mixture buffer containing each of the species-specific sets of primers. Group I primers (BiIA/IB, BoF/R and Am9/10) which specifically bind B. bigemina, B. bovis and A. marginale DNA were used to amplify a fragment of DNA from genomic parasite DNA. Group II nested primers (BiIAN/IBN, BoFN/RN and Am11/12) were used to prepare, via incorporation of digoxigenin-11-dUTP by PCR, nonradioactive probes specific for internal sequences present in DNA amplified with Group I primers. Agarose gel electrophoresis and Southern blot hybridization studies showed that by using Group I primers, DNA fragments of 278 bp, 350 bp and 200 bp were specifically amplified in samples containing B. bigemina, B. bovis and A. marginale DNA, respectively. The analytical sensitivity of the multiple PCR test, as evaluated by nucleic acid hybridization with the nonradioactive probe, was 0.00001%, 0.00001% and 0.0001% infected erythrocytes for B. bigemina, B. bovis and A. marginale, respectively. Blood collected from cattle previously inoculated with B. bovis (4 years), A. marginale (2 years) and B. bigemina (1 year) was demonstrated to be latently infected by using the Multiplex PCR test.

Anaplasma↗
PubMed1996

Morphology and development of Anaplasma marginale in midgut of engorged female ticks of Boophilus microplus.

Morphology and development of Anaplasma marginale were studied in midgut epithelial cells of adult Boophilus microplus females using light and transmission electron microscopy. Hemoparasite-free B. microplus larvae were used to infest Holstein calves experimentally inoculated with A. marginale. When A. marginale parasitemia varied from 0.4% to 4.3%, 140 engorged female ticks were collected and kept individually in steel wire tubes and incubated outdoors on Brachiaria decumbens grass pasture. Female ticks in groups of 20 were dissected 14, 19, 26, 33, 40, 47 and 50 days after detachment. Engorged female ticks collected from an A. marginale-free calf were kept under the same conditions as control ticks. Gut sections of infected and control ticks were processed using light and electron microscopy. Infected gut sections were processed by the immunocytochemical peroxidase-antiperoxidase technique. Under light microscopy A. marginale colonies were found in midgut epithelial cells of engorged B. microplus females from 19 days on after detachment from donor calves. Colonies were 6.1-10.3 microns in diameter, ovoid or irregular and contained round or shaped organisms. Two distinct morphological types of colonies (BM1 and BM2) were found. BM1 colonies had a dense matrix strongly stained in which organisms were barely visible; BM2 had large separated organisms densely stained inside a matrix which was lightly stained. A. marginale colonies immunocytochemically stained were found in gut tissue sections using light microscopy. Using transmission electron microscopy organisms of different morphologies were found in midgut epithelial cells of engorged females, suggesting sequential phases of development of A. marginale in the vector. Modifications occurred in colonies and in the organisms within them. No symbiotic rickettsiae were found in intestinal cells of control ticks.

Anaplasma↗