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PubMed2006

A paretic condition in an Anaplasma phagocytophilum infected roe deer calf.

This paper describes a case of Anaplasma phagocytophilum infection in a roe deer (Capreolus capreolus) calf in Norway. The calf was found deserted, paretic, and heavily infested with Ixodes ricinus ticks. It was euthanized and investigated postmortem. Anaplasma phagocytophilum was detected in several tissues by polymerase chain reaction (PCR) and 16S rRNA sequence analyses. Analyses for Borrelia burgdorferi sensu lato and tick-borne encephalitis (TBE) virus infections were negative. This is the first report of a possible paretic condition in A. phagocytophilum infected roe deer.

Anaplasma phagocytophilum↗
PubMed2002

Infection with Anaplasma phagocytophila in cervids from Slovenia: evidence of two genotypic lineages.

OBJECTIVE: Human granulocytic ehrlichiosis (HGE) was recently recognized as an emerging tick-borne infection in Europe. The disease is caused by Anaplasma (previously Ehrlichia) phagocytophila. The first confirmed acute human disease caused by A. phagocytophila was reported from Slovenia in 1998. The tick Ixodes ricinus was identified as the likely vector for this pathogen of humans and animals in Europe. In order to assess the possibility that roe and red deer in Slovenia serve as potential reservoir hosts for A. phagocytophila, materials from both species were examined. METHODS: Samples were obtained from 32 red deer (Cervus elaphus) and 56 roe deer (Capreolus capreolus). Polyvalent antibodies to the USG3 isolate of Anaplasma phagocytophila were detected by indirect immunofluorescence assay (IFA). DNA was extracted from spleen tissue. The 16S rRNA gene and a portion of the groESL heat shock operon were used for PCR detection and subsequent direct sequencing of amplified products. RESULTS: Serological and PCR results indicated that high proportions of roe and red deer were infected with A. phagocytophila. Infection was confirmed in 74% of the animals by IFA and in 86% of animals by PCR. While similar prevalences by PCR were seen in the two species (approximately 86%), the prevalence of antibodies was much higher in roe deer (94% vs. 35% in red deer). Sequence analysis of a 1256-bp fragment of the groESL operon revealed genetic diversity among collected samples. Identity of sequences ranging from 98% to 100%. None of the A. phagocytophila groESL and 16S rRNA gene sequences from red or roe deer were identical to the sequences previously obtained from human patients with ehrlichiosis from Slovenia or elsewhere in the world. All red deer sequences clustered with those obtained from humans, whereas all but two sequences from roe deer clustered separately. CONCLUSIONS: The results of our study indicate that a high percentage of red deer and roe deer in Slovenia are infected with A. phagocytophila. Analysis of groESL and 16S rRNA gene sequences revealed two distinct genetic lineages. Among deer, one variant was primarily associated with roe deer. Although none of the sequences from red deer was identical to those found in humans, they were more closely related.

Anaplasma phagocytophilum↗
PubMed2006

High prevalence of Anaplasma phagocytophilum infection in ticks removed from human skin in north-eastern Poland.

In order to examine the risk of Anaplasma phagocytophilum infection in humans, 76 Ixodes ricinus ticks and 3 Dermacentor reticulatus removed from humans were examined for the presence of Anaplasma phagocytophilum by PCR targeting the16S rRNA gene. The average infection rate was 23.7% (18/76) in Ixodes ricinus ticks. A very high infection rate reaching 36.8% (14/38) was detected in female Ixodes ricinus, followed by males--16.6% and nymphs--10.5%. No infection was noted in larvae. All Dermacentor reticulatus also tested negative. The positive PCR products were sequenced and deposited in the Gen Bank (Accession No. DQ006828).

Anaplasma phagocytophilum↗
PubMed1990

Use of the dot enzyme-linked immunosorbent assay with isolated Anaplasma marginale initial bodies for serodiagnosis of anaplasmosis in cattle.

Isolated Anaplasma marginale initial bodies were successfully used in a dot ELISA for rapid detection of antibodies to Anaplasma organisms. The enzyme immunoassay used only 25 ng of antigen dotted onto nitrocellulose disks. Antigen-antibody complexes were detected by use of alkaline phosphatase-conjugated protein A, and reactions were read visually after addition of a precipitable, chromogenic substrate. The test allowed the processing of multiple sera, either for screening or for titer determination, in less than 3 hours and was found to be as sensitive as the indirect fluorescent antibody test. The overall performance of the dot ELISA, using isolated A marginale initial bodies, for 580 bovine serum samples was as follows: sensitivity, 93%; specificity, 96%; and predictive value, 95%. Cross-reactivity was not observed with sera positive to Babesia bovis and B bigemina, Trypanosoma vivax, or common bacteria or viruses infecting cattle. The antigen dotted onto nitrocellulose disks was stable when stored at -20, 4, or 25 C. Compared with the indirect fluorescent antibody test, the dot ELISA allowed easier, faster, and more objective interpretation of results. Its simplicity and low cost combined with high sensitivity and specificity indicate that this assay could effectively replace serologic assays currently used for diagnosis of anaplasmosis in cattle.

Anaplasma↗
PubMed1988

Differential extraction of antigens of Anaplasma marginale.

Strain-, group-, and genus-specific antigens of the Florida isolate of Anaplasma marginale had different solubilities in zwitterionic, nonionic, and anionic detergents. On the basis of their solubility in nonionic detergent, antigens were grouped into 3 classes: (i) a 108-kilodalton (kD) group-specific antigen and a series of poorly defined antigens (70 to 100 kD) were completely soluble in nonionic detergent; (ii) 96 kD and 75 kD group-specific antigens, and a 91-kD strain-specific antigen were completely insoluble in nonionic detergent; and (iii) a 47-kD group-specific antigen and a 41-kD genus-specific antigen were partially soluble in nonionic detergent. A mercaptoethanol-sensitive antigen of 200 kD also was present in the insoluble fraction. Differences in solubility of taxonomic antigens of Anaplasma may reflect differences in function. Antigens totally insoluble in nonionic detergent were possibly integral membrane proteins, and those completely soluble in nonionic detergent were more likely peripheral membrane proteins that were less tightly bound to hydrophobic components of the membrane. Seemingly, all antigens were associated with membranes. Bovine preimmune and immune sera used did not react with any proteins of noninfected RBC.

Anaplasma↗
PubMed1988

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.

Anaplasma↗
PubMed1987

The genome of anaplasma: DNA base composition and DNA/DNA hybridization.

The Tm value of DNA from Anaplasma centrale and Anaplasma marginale was found to be 87.1 degrees C and 89.3 degrees C, respectively. The G + C content, calculated from the Tm, was 45.1% for A. centrale and 48.5% for A. marginale. Identical hybridization patterns were obtained when the DNA from one species was hybridized to restriction endonuclease-digested DNA from the other species.

Anaplasma↗
PubMed1985

High-yield preparation of purified Anaplasma marginale from infected bovine red blood cells.

Purification of Anaplasma marginale from infected bovine RBC was achieved through enzyme treatment and density-gradient centrifugation. A relative yield of 41.6% was obtained by dividing the number of organisms in the final purified preparation by the number of A marginale-infected RBC. Purified parasites were verified as A marginale by light microscopy, electron microscopy, and immunologic tests. The purified parasites reacted positively with calf and rabbit anti-A marginale sera in interfacial and slide agglutination tests. Anti-bovine RBC serum did not agglutinate purified A marginale, indicating absence of any contaminating RBC stroma. Anaplasma marginale was antigenic, but did not cause infection when the preparation was inoculated into a susceptible calf. The density of A marginale was determined to be 1.19 g/ml and cell diameters ranged from 0.25 to 0.63 micron. This method provided procedures for obtaining A marginale free of bovine RBC antigens for accurate biochemical assays and vaccine production.

Agglutination Tests↗
PubMed1985

Anaplasma marginale infections in American bison: experimental infection and serologic study.

Anaplasma marginale was experimentally transmitted from cattle to bison and back to cattle. Of the 2 splenectomized and 1 intact American bison calves (Bison bison) inoculated with a North Texas A marginale stabilate, 1 splenectomized and 1 intact bison exhibited clinical signs of anaplasmosis. Active parasitemias in these bison were observed along with positive reactions in the rapid card agglutination and complement fixation tests. Blood from the infected bison produced disease in splenectomized bovine calves. Screening tests for anti-Anaplasma antibodies in 178 blood samples collected from adult bison from the National Bison Range, Montana, revealed 1 rapid card agglutination test-positive sample, and 110 negative, 40 suspect, and 28 positive (15.7%) complement fixation test samples.

Agglutination Tests↗
PubMed1979

Blood parasites of sheep in the Netherlands. I. Anaplasma mesaeterum sp.n. (Rickettsiales, Anaplasmataceae).

On two occasions an anaplasm was isolated from sheep on the Dutch island of Ameland. The organism proved to be highly pathogenic for splenectomised sheep; a non-splenectomised animal recovered spontaneously after the packed cell volume had decreased by 40%. Treatment with oxytetracycline was effective. Its pathogenicity for goats appeared to be low, and the organism was apparently not infective to splenectomised cattle. This anaplasm differs from Anaplasma ovis in that less than 30% of the organisms are marginally situated in the red cell, as against over 70% in A. ovis; cross-immunity with A. ovis was incomplete and the latter appeared to be far more pathogenic to goats than the Dutch anaplasm, for which the name Anaplasma mesaeterum sp.n. is proposed. Its ultrastructure is similar to that of A. marginale and A. ovis. The vector is either Ixodes ricinus or Haemaphysalis punctata. Its practical importance remains to be ascertained.

Anaplasma↗
PubMed1984

Serologic and clinical responses of premunized, vaccinated, and previously infected cattle to challenge exposure by two different Anaplasma marginale isolates.

Two Anaplasma marginale isolates, one originating in Florida (FAM) and the other from Virginia (VAM), were compared immunologically by cross-challenge exposure of 14 Anaplasma carrier cattle, 8 previously infected cattle, and 6 splenectomized carrier calves. In addition, 28 cattle vaccinated with a commercially available adjuvant killed vaccine and 22 nonvaccinated cattle were challenge exposed with either FAM or VAM. A detectable clinical response was not produced by either FAM or VAM challenge exposure in carrier and previously infected cattle; however, evidence of A marginale growth as characterized by low percentages of parasitemia and increased serum complement-fixation titers was seen in carrier cattle given a heterologous challenge organism and in previously infected cattle inoculated with either homologous or heterologous organisms. Among splenectomized calves, there was virtually no cross protection to the heterologous challenge exposure, whereas a homologous challenge failed to elicit any detectable response. Vaccinated cattle were resistant to VAM exposure, but the clinical response to FAM exposure was severe with a 47% mortality. Most of these cattle displayed typical acute anaplasmosis that was only marginally less severe than that encountered in nonvaccinated cattle.

Anaplasma↗
PubMed1983

Infectivity virulence and immunogenicity of Anaplasma centrale live blood vaccine.

Cross-bred Bos taurus calves, aged between 6 and 8 months, were inoculated with the Onderstepoort Anaplasma centrale live blood vaccine. One group of 15 calves were inoculated once only, while a 2nd group of 15 were revaccinated 6 months later. All the animals were challenged with approximately 1 X 10(10) Anaplasma marginale parasites of a known virulent strain 8 months after the first vaccination. The results of blood smear examination and the card agglutination test indicated that only 20 out of 30 animals vaccinated contracted A. centrale infections after the first attempt, and 3 out of 5 after the second. The vaccine conferred only partial immunity to challenge with a virulent A. marginale strain.

Anaplasma↗
PubMed1984

Antigen beads: a novel approach for the delivery of a soluble Anaplasma immunogen.

An important consideration in the development of effective immunization regimens against bovine anaplasmosis is the optimization of vaccine delivery systems. This report describes the formulation of proteinaceous antigen-containing spheres derived from supernatant fluids of Analplasma marginale cultures. The antigen beads were designed for use as potential dispersal agents for a soluble Anaplasma immunogen. Immunospecificity assays demonstrated that A marginale antigen was located on the surfaces of individual antigen beads. Use of Anaplasma antigen beads with a potent adjuvant, such as saponin, was proposed.

Adjuvants, Immunologic↗
PubMed1984

Response of cattle upon reexposure to Anaplasma marginale after elimination of chronic carrier infections.

Sixteen cattle serotest-negative for anaplasmosis with either no previous exposure (2 animals) or cleared 8 months earlier of their carrier state by chemotherapy (14 animals) were each exposed to Anaplasma marginale. Anaplasma serotest titers were determined by complement-fixation and rapid card agglutination tests conducted during a 63-day trial period. Serologic reactions indicated that all cattle (both groups) were converted to seropositive by the 21st day after exposure. Fluctuations in PCV were seen in the 2 groups between days 21 and 35. However, parasitemia levels were detectable only in the 2 previously unexposed control cattle. Three splenectomized calves, given 10 ml of blood from 3 of the former carrier cattle 14 days after the latter were reexposed, developed severe clinical and hematologic signs of anaplasmosis and seroconverted from negative to positive on both serologic tests. The need to acquire a better understanding of immunity in anaplasmosis is discussed.

Anaplasma↗
PubMed1984

Immune serum against Anaplasma marginale initial bodies neutralizes infectivity for cattle.

The first demonstration of immune serum-mediated neutralization of Anaplasma marginale and identification of the involved surface antigens is presented. Anaplasma marginale initial bodies were purified from parasitized erythrocytes by using ultrasonic disruption and differential centrifugation. The initial bodies were morphologically intact, as determined by electron microscopy, were not agglutinated by anti-bovine erythrocyte sera, and were infective. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated minimal erythrocytic contamination in the purified initial bodies. Rabbit antisera against initial bodies completely neutralized the infectivity of purified initial bodies for splenectomized calves, whereas normal rabbit serum did not alter infectivity. Nine initial body proteins were surface radioiodinated by using lactoperoxidase, and five of these were precipitated by the neutralizing antibody (105,000, 86,000, 61,000, 36,000, and 31,000 apparent m.w.). The results demonstrate that initial body determinants capable of inducing neutralizing antibody are present. The identification of surface radiolabeled proteins immunoprecipitated by the neutralizing antibody provides candidates to be tested as protective immunogens in cattle.

Anaplasma↗
PubMed1982

Babesia, Theileria and Anaplasma spp. infecting sable antelope, Hippotragus niger (Harris, 1838), in southern Africa.

Some observations are recorded on blood parasites of sable antelopes. Blood smears of 124 of these antelopes from South Africa and Zimbabwe were examined and 7 were found to be positive for a Babesia sp., identified as Babesia irvinesmithi Martinaglia 1936. A total of 70 of the smears were positive for theilerial piroplasms, while 1 smear had macroschizonts (with cytomeres) and microschizonts of a Theileria (= Cytauxzoon) sp. One blood smear was positive for an Anaplasma sp. Attempts to isolate the Babesia sp. by subinoculating blood from sable to splenectomized and intact sable and splenectomized cattle were unsuccessful. Attempts to infect sable with Babesia bovis and Babesia bigemina were likewise unsuccessful. Theilerial piroplasms reached high levels in a splenectomized sable but could not be transmitted with blood to cattle. The Anaplasma sp. was found to be infective for sheep but not for cattle.

Anaplasma↗
PubMed1980

Anaplasma marginale in bovine erythrocyte cultures.

Bovine erythrocytes infected with Anaplasma marginale were standardized with normal bovine erythrocytes at 1.4% and 3.5% parasitemia and cultured under low oxygen pressure at 37 C. The number of erythrocytes infected by A marginale appeared to increase 3.3-fold by day 11 and 4.5-fold by day 3, as determined by microscopic examination of Giemsa-stained thin films of the culture suspension. Anaplasma parasites in experimental cultures were proven to be viable and pathogenic by calf inoculation for up to 16 days.

Anaplasma↗