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PubMed2006

Isolation of Anaplasma phagocytophilum strain Ap-variant 1 in a tick-derived cell line.

Ten isolates of the Ap-Variant 1 strain of Anaplasma phagocytophilum were made in the Ixodes scapularis (I. scapularis)-derived cell line, ISE6. Two isolates were obtained from laboratory-infected goats and eight isolates were obtained from field-collected I. scapularis ticks. Each isolate showed 16S rRNA sequences identical to those as previously described for the Ap-Variant 1 strain. These are the first tissue culture isolates of the Ap-Variant 1 strain and will allow for further characterization of the biological and antigenic properties of this strain.

Anaplasma phagocytophilum↗
PubMed2003

Ehrlichia platys (Anaplasma platys) in dogs from Maracaibo, Venezuela: an ultrastructural study of experimental and natural infections.

Since 1982 Ehrlichia platys, now emended as Anaplasma platys, has been diagnosed in dogs from Maracaibo, Venezuela, using buffy coat smears stained with Dip Quick. Three dogs were inoculated with an A. platys strain. When parasitemia reached 60-97%, blood samples obtained from the inoculated dogs and from two naturally infected dogs were centrifuged to obtain platelet-rich plasma, which was mixed with 0.1% glutaraldehyde at 37 C for 10 minutes. Platelet pellets were fixed in 3% glutaraldehyde for 72 hours and processed for conventional transmission electron microscopy. Platelets contained pleomorphic organisms with a distinct double membrane that was not observed when the bodies were in a determinate developmental stage. There were 1-15 individual bodies included in a host cell vacuole. The organisms had an electron-lucent inner area, whereas the internal surface of their inner plasma membranes exhibited an electron-dense rough substance. In naturally infected dogs, organisms with different ultrastructural features were found inside the same platelet. Some organisms contained central dense material surrounded by a pale zone, which was in turn surrounded by a moderately dense peripheral area. Other organisms contained an eccentrically electron-dense material. The intravacuolar space appeared fully electron-lucent. Each organism usually exhibited inner fine strands. Empty structures displaying junctions with the vacuolar membrane were observed. Our results indicate that distinct ultrastructural characteristics are associated with different stages of A. platys development and may differ among A. platys strains.

Anaplasma↗
PubMed2007

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.

Anaplasma marginale↗
PubMed2007

Experimental infection of C3H/HeJ mice with the NY18 isolate of Anaplasma phagocytophilum.

Human granulocytic anaplasmosis (HGA), an emerging disease of public health concern in many areas of the world, is caused by Anaplasma phagocytophilum. Small animal models of A phagocytophilum in laboratory mice have been developed and used to study the pathogenesis of HGA. In this study, we characterized the pathologic changes in acute infection of C3H/HeJ mice experimentally infected with the NY18 isolate of A phagocytophilum. Although no clinical signs were noted, acute infection was associated with gross splenomegaly, microscopic inflammatory lesions in the lung and liver, hyperplastic lesions on the spleen, and clinical pathology abnormalities including neutropenia and monocytosis. This study emphasizes the use of well-defined animal models as a valuable tool for the study of A phagocytophilum infections.

Anaplasma phagocytophilum↗
PubMed2006

Genomic and proteomic approaches to vaccine candidate identification for Anaplasma marginale.

Outer membrane protein preparations have been demonstrated to elicit protective immunity for Anaplasma marginale. Attempts to recreate this protective immunity using known surface proteins have been unsuccessful; therefore, novel outer membrane proteins have been searched for using a proteomic/genomic approach. Annotation of the whole genome sequence identified 62 outer membrane protein candidates based on sequence similarity to known surface proteins. In a proteomics approach for the identification of immunostimulatory outer membrane proteins, outer membrane preparations that were separated on 2D gels were used to immunize calves . Antisera from the calves were used to detect immunoreactive proteins, which were then selected and subjected to mass spectrometric analyses. These data were mapped back to the annotated A. marginale genome and have identified several new outer membrane proteins that are vaccine candidates.

Anaplasma marginale↗
PubMed2003

Antigenic characterization of Brazilian isolates of Anaplasma marginale.

Antigenic characterization of Anaplasma marginale isolates, by identifying conserved and variable epitopes of major surface proteins (MSP), is an important tool for vaccine development against this rickettsia. The B cell epitopes of A. marginale isolates from three microregions of the State of Pernambuco and one from the State of Mato Grosso do Sul, Brazil, were characterized by indirect fluorescent antibody technique (IFAT) and Western blot (WB) with 15 monoclonal antibodies (MAbs). The epitope recognized by MAb ANA22B1 (MSP-1a) was conserved by IFAT and WB (73-81 kDa). MSP-2 epitopes recognized by MAbs ANAO58A2 and ANAO70A2 were conserved by IFAT, while ANAO50A2 and ANA66A2 epitopes were polymorphic; in the WB, the MAbs ANAO50A2 and ANAO70A2 identified bands of 45 kDa only in the Pernambuco-Mata isolate. None of the isolates reacted with MAb ANAR75C2 (MSP-3). The MSP-4 epitope recognized by MAb ANAR76A1 was conserved by IFAT, as well as the MSP-5 epitope recognized by MAb ANAF16C1 by IFAT and WB (16 kDa). The MAbs ANAR17A6, ANAR83B3, ANAR94C1, ANAO24D5 and ANAR19A6 identified conserved epitopes by IFAT. MSP-1, MSP-2 and MSP-4, which previously showed partial protection in experimental trials, are also potential immunogens to be employed in Brazil, due to the B cell epitope conservation.

Anaplasma↗
PubMed2005

Serological survey of Babesia bovis, Babesia bigemina, and Anaplasma marginale antibodies in cattle from the semi-arid region of the state of Bahia, Brazil, by enzyme-linked immunosorbent assays.

The objectives of this work were to determine the prevalence of Babesia bovis, Babesia bigemina, and Anaplasma marginale detecting antibodies in cattle raised in the semi-arid region of the state of Bahia, Brazil, through indirect enzyme linked immunosorbent assays (ELISA) and to compare the performances of indirect enzyme-linked immunosorbent assays with crude (I-ELISA-CrAnaAg) and recombinant major surface protein-5 (I-ELISA-MSP-5Ag), as antigens to detect antibodies against A. marginale. An stable enzootic area was found in Senhor do Bonfim and Euclides da Cunha for B. bovis that showed 86 and 95.5% of prevalence, respectively, and for B. bigemina with 90.8 and 91.3%. On the other hand, Uauá and Juazeiro, were characterized as enzootically unstable areas, since prevalences were: B. bovis--63.7 and 56.4% and B. bigemina--53 and 54.8%, respectively. The prevalence of A. marginale in the four municipalities was above 97% with I-ELISA-CrAnaAg and 94.8% with I-ELISA-MSP-5Ag which is an indication of stable enzootic condition for the rickettsia. The I-ELISA-CrAnaAg and I-ELISA-MSP-5Ag showed a highly significant association (r = 0.977), which means that both ELISA tests are suitable for epidemiological studies of A. marginale.

Anaplasma marginale↗
PubMed2006

Development of enzyme-linked immunosorbent assays based on recombinant MSP1a and MSP2 of Anaplasma marginale.

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant MSP1a and MSP2 from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. The high sensitivities (99% for both tests) and specificities (100% for both tests) were confirmed with sera from cattle positive or negative for A. marginale antibodies, respectively, by immunofluorescent antibody test. By the analysis of 583 sera from cattle of three regions of the state of Pernambuco, Brazil, the agreement between both tests was high, with a kappa index of 0.89. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

Anaplasma marginale↗
PubMed2006

Comparison between indirect enzyme-linked immunosorbent assays for Anaplasma marginale antibodies with recombinant major surface protein 5 and initial body antigens.

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant major surface protein 5 (rMSP5) and initial body (IB) antigens from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. Both tests showed the same sensitivity (98.2%) and specificities (100% for rMSP5 and 93.8% for IB ELISA) which did not differ statistically. No cross-reactions were detected with Babesia bigemina antibodies, but 5 (rMSP5 ELISA) to 15% (IB ELISA) of cross-reactions were detected with B. bovis antibodies. However, such difference was not statistically significant. Prevalences of seropositive crossbred beef cattle raised extensively in Miranda county, state of Mato Grosso do Sul, Brazil, were 78.1% by rMSP5 ELISA and 79.7% by IB ELISA. In the analysis of sera from dairy calves naturally-infected with A. marginale, the dynamics of antibody production was very similar between both tests, with maternal antibodies reaching the lowest levels at 15-30 days, followed by an increase in the mean optical densities in both ELISAs, suggesting the development of active immunity against A. marginale. Results showed that all calves were seropositive by one-year old, characterizing a situation of enzootic stability. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

Anaplasma marginale↗
PubMed2005

Variation among geographically separated populations of Dermacentor andersoni (Acari: Ixodidae) in midgut susceptibility to Anaplasma marginale (Rickettsiales: Anaplasmataceae).

Anaplasma marginale is a tick-borne rickettsial pathogen of cattle that is endemic throughout large areas of the United States. Cattle that survive acute infection become life-long persistently infected carriers. In the intermountain west the Rocky Mountain wood tick, Dermacentor andersoni Stiles, is the most common vector of A. marginale. Male D. andersoni acquire A. marginale when feeding on persistently infected cattle and biologically transmit it when they transfer from infected to susceptible hosts. Host-seeking adult D. andersoni were collected from four widely separated natural populations and tested for susceptibility to midgut colonization with A. marginale. Male ticks were fed on calves persistently infected with a strain of A. marginale naturally transmitted by D. andersoni. Gut infection rates ranged from 12.5% of ticks collected from a mountain site near Hamilton, MT, to 62.5% of ticks from a rangeland site near Riley, OR. Sites near Miles City, MT, and Kamloops, British Columbia, Canada, had intermediate levels of susceptibility. The infection rates differed significantly among populations, and the same populations sampled in two consecutive years were not significantly different from one year to the next. Although there was variation among the populations in the size of ticks, size was unrelated to acquisition of gut infection. Quantitative polymerase chain reaction (PCR) demonstrated that there was no significant difference between populations in the mean number of genome copies in the guts of infected ticks. A. marginale from infected ticks was genotyped to confirm that they were all infected with the laboratory strain, and a sample of 682 field-collected D. andersoni was surveyed for A. marginale by nested PCR; none were found to be naturally infected. Infection of the gut is an essential constituent of vector competence for A. marginale; in this study, we have demonstrated significant variation among populations in this key component of vector competence.

Anaplasma marginale↗
PubMed2005

Relative efficiency of biological transmission of Anaplasma marginale (Rickettsiales: Anaplasmataceae) by Dermacentor andersoni (Acari: Ixodidae) compared with mechanical transmission by Stomoxys calcitrans (Diptera: Muscidae).

Anaplasma marginale Theiler is a tick-borne intraerythrocytic rickettsial pathogen of cattle that also can be mechanically transmitted by biting flies. Rickettsemia during the acute phase of infection may reach as high as 10(9) infected erythrocytes (IEs) per milliliter of blood. Animals that survive acute infection develop a life-long persistent infection that cycles between 10(2.5) and 10(7) IE/ ml of blood. We compared stable fly Stomoxys calcitrans (L.) -borne mechanical transmission during acute infection with Rocky Mountain wood tick, Dermacentor andersoni Stiles-borne biological transmission in the persistent phase of infection to demonstrate quantitatively that biological transmission by ticks is considerably more efficient than mechanical transmission by biting flies. Stable flies that partially fed on an acutely infected calf and were immediately transferred to susceptible calves to complete their bloodmeals failed to transmit A. marginale. Ticks that fed on the original acquisition host after it reached the persistent phase of infection (>300-fold lower rickettsemia) successfully transmitted A. marginale after transfer to the same calves that failed to acquire infection after fly feeding. Failure of fly-borne mechanical transmission at a rickettsemia >300-fold higher than that from which ticks transmit with 100% efficiency demonstrates that tick-borne biological transmission is at least 2 orders of magnitude more efficient than direct stable fly-borne mechanical transmission.

Anaplasma marginale↗
PubMed2006

Detection of Anaplasma phagocytophilum and Babesia odocoilei DNA in Ixodes scapularis (Acari: Ixodidae) collected in Indiana.

The blacklegged tick, Ixodes scapularis Say, first reported in Indiana in 1987, has now been detected in more than half of Indiana's counties. The first case of human granulocytic ehrlichiosis (human anaplasmosis) in Indiana was reported in 2002. We now report the detection of Anaplasma phagocytophilum and Babesia odocoilei (Emerson and Wright 1968) in I. scapularis ticks collected in northern Indiana. Using polymerase chain reaction analysis, 41 of 193 adult ticks (21.2%) collected from deer were positive for A. phagocytophylum, and 22 (11.4%) were positive for Babesia sp. Restriction fragment analysis of 12, and sequencing of another five of the amplified products identified these parasites as B. odocoilei. Five ticks (2.6%) were coinfected. Eight of 68 questing adult ticks (11.8%) were positive for A. phagocytophilum; seven (10.3%) were positive for Babesia sp. Six of the latter seven positive samples were determined to be B. odocoilei by restriction fragment analysis and sequencing of two samples. None of 39 pools of nymphs was positive for Babesia sp. Three of 15 ticks (20%) collected from a dog were positive for A. phagocytophilum and three ticks (20%) were positive for Babesia sp. One was confirmed as B. odocoilei. One tick was coinfected. This is the first report of the presence of these two agents in ticks in Indiana.

Anaplasma phagocytophilum↗
PubMed2006

A Canadian bison isolate of Anaplasma marginale (Rickettsiales: Anaplasmataceae) is not transmissible by Dermacentor andersoni (Acari: Ixodidae), whereas ticks from two Canadian D. andersoni populations are competent vectors of a U.S. strain.

Anaplasma marginale Theiler is a tick-borne rickettsial pathogen of cattle with a global distribution in both temperate and tropical regions. The pathogen is endemic in regions within the United States, whereas the Canadian cattle population is considered to be free ofA. marginale. Farmed bison, Bison bison L., in central Saskatchewan have been found to be infected with A. marginale; however, there is no evidence of transmission from bison to cattle. We tested a Saskatchewan bison isolate of A. marginale (SB1) to determine whether it is transmissible by the Rocky Mountain wood tick, Dermacentor andersoni Stiles. Colonized D. andersoni from the United States and Canada failed to transmit SB1. A separate transmission trial using D. andersoni adults reared from ticks collected in Alberta and British Columbia showed that ticks from these populations could successfully transmit the St. Maries, Idaho, strain of A. marginale. Although the Saskatchewan bison isolate of A. marginale seems not to be transmissible by D. andersoni, in the event of the introduction of a tick-transmissible strain, Canadian D. andersoni are likely to be competent vectors.

Anaplasma marginale↗
PubMed2003

Detection of Borrelia burgdorferi, Ehrlichia chaffeensis, and Anaplasma phagocytophilum in ticks (Acari: Ixodidae) from a coastal region of California.

A study was conducted in Santa Cruz County to estimate the prevalence and distribution of the agents of Lyme disease, human granulocytic (HGE), and human monocytic (HME) ehrlichiosis in 1,187 adult ixodid ticks collected from eight public-use recreation areas over a 2-yr period. Borrelia burgdorferi, the causative agent of Lyme disease, was detected by a polymerase chain reaction (PCR) in 44 of 776 (5.67%) Ixodes pacificus ticks and in 3 of 58 (5.17%) Dermacentor variabilis ticks. Anaplasma phagocytophilum, the causative agent of HGE, was detected by PCR in 48 (6.19%) I. pacificus ticks and 5 (8.62%) D. variabilis ticks. Ehrlichia chaffeensis, the causative agent of HME, was detected by nested PCR in just five (0.64%) I. pacificus ticks and four (6.9%) D. variabilis ticks. Interestingly, eight (1.03%) I. pacificus ticks were co-infected with B. burgdorferi and A. phagocytophilum, and just one (0.12%) tick was co-infected with B. burgdorferi and E. chaffeensis. Less than 1% of 353 Dermacentor occidentalis ticks showed evidence of infection with any of the agents tested. To our knowledge, this is the first reported identification of A. phagocytophilum and E. chaffeensis in D. occidentalis ticks from California This study represents the first extensive survey of Lyme and the ehrlichial diseases across multiple areas of Santa Cruz County, and suggests that prevalence of B. burgdorferi in Santa Cruz County may be higher than other areas of the state.

Anaplasma phagocytophilum↗
PubMed2004

Effects of Anaplasma phagocytophilum infection on the molting success of Ixodes scapularis (Acari: Ixodidae) larvae.

We assessed the effects of sympatric (occupying the same or overlapping geographic areas) and allopatric (occurring in separate geographic areas) isolates of Anaplasma phagocytophilum on the survival of Ixodes scapularis Say larvae that were derived from ticks collected in Bridgeport, CT. Seven isolates of A. phagocytophilum, originating from different geographic regions of the United States, were tested: four isolates from the northeast (Bridgeport, Dawson, Gaillard, and NY-8), two from the Midwest (Webster and Sp-Is), and one from California (MRK). BALB/c mice were infected with each of the seven isolates via exposure to infected I. scapularis nymphs, whereas uninfected nymphs fed upon control mice. Both infected and control mice were infested with uninfected larvae at 1, 2, 3, 4, 6, and 9 wk after nymphal infestation. The molting success in cohorts of infected and uninfected ticks was calculated as the percentage of larvae successfully molting into nymphal stage, and the prevalence of infection in molted nymphs was determined by polymerase chain reaction. In ticks that became infected with the Bridgeport or Sp-Is isolates, the molting success decreased with an increase in the prevalence of infection. Ticks that fed upon mice infected with six allopatric isolates (Dawson, Gaillard, NY-8, Sp-Is, Webster, and MRK) showed significantly lower levels of survival than those fed upon control mice, regardless of the prevalence of infection, whereas in ticks fed upon mice infected with a sympatric isolate (Bridgeport), the overall molting success was similar to the control. Thus, some but not all of the A. phagocytophilum isolates have adverse effects on ticks. Ticks exposed to harmful isolates may experience higher levels of bacterial metabolism, and/or reduced quality of their blood meal, thereby reducing their survival. Noted differences between isolates may be due to the origin of a particular isolate and/or the degree of coadaptation between the pathogen and its vector on the population level.

Anaplasma↗
PubMedDate not supplied

Acute clinical, hematologic, serologic, and polymerase chain reaction findings in horses experimentally infected with a European strain of Anaplasma phagocytophilum.

Six horses were experimentally infected by administration of horse blood containing a Swedish strain of Anaplasma phagocytophilum. The polymerase chain reaction (PCR) signal was consistently detected 2-3 days before appearance of clinical signs and persisted 4-9 days beyond abatement of clinical signs, whereas diagnostic inclusion bodies were 1st noted on average 2.6 +/- 1.5 (SD) days after onset of fever. Clinical signs and hematologic changes were largely indistinguishable from those previously reported for diseases caused by A phagocytophilum (formerly Ehrlichia equi--"Californian agent") and the human-derived human granulocytic ehrlichiosis agent. Horses 1st demonstrated antibody response 12-16 days after inoculation, 2 cases of which were still febrile, and serotiters rapidly peaked within 3-7 days of clinical illness. One horse died during the acute stage of disease, but initial clinical signs and hematologic changes were similar to those of other infected horses. This report shows that, despite minor genetic differences, a European equine-derived strain of A. phagocytophilum may be similar in pathogenicity to the Californian agent. The PCR used holds promise to widen the diagnostic window and would also be diagnostic during the initial days of clinical disease when inclusions in neutrophils in blood smears are not yet apparent.

Anaplasma phagocytophilum↗
PubMed1990

Immunodiagnosis of and immunoprophylaxis against the haemoparasites Babesia sp. and Anaplasma sp. in domestic animals.

The haemoparasites Babesia bovis and Anaplasma marginale are extremely important pathogens of cattle, affecting more than 300 million animals worldwide. Species of less importance affect cattle, dogs, horses and sheep. No vaccine is available for widespread usage. This review summarises existing immunodiagnostic and immunoprophylactic methods currently available for these diseases and reports on future immunoprophylactic methods.

Anaplasma↗
PubMed2005

Seroprevalence of antibodies against Borrelia burgdorferi and Anaplasma phagocytophilum in cats.

OBJECTIVE: To determine whether cats in the northeastern United States develop serum antibodies against antigens of Borrelia burgdorferi and Anaplasma phagocytophilum and whether coinfection with the 2 organisms occurs. SAMPLE POPULATION: Serum samples from 84 healthy cats and 9 cats with lameness, fever, anorexia, or fatigue. PROCEDURE: Serum antibodies against B. burgdorferi and A. phagocytophilum were measured with an ELISA incorporating a whole-cell preparation or purified recombinant antigens, by means of Western blot analysis, or indirect fluorescent antibody (IFA) staining. RESULTS: ELISA results indicated that 44 of 93 (47%) sera contained antibodies against > or = 3 B. burgdorferi antigens, whereas 43 (46%) were reactive to whole-cell B. burgdorferi. Serum reactivity to protein 35, VlsE, and outer surface proteins A and F was most common. Seropositivity to > or = 3 antigens occurred at the same rate (5/9) in the 9 ill cats as in the 84 healthy cats (46% [39/84]). Of 13 sera reactive to recombinant antigens, 9 were seropositive as measured by Western blot testing with whole-cell antigen. Seropositivity rates of 30% and 38% were detected for antibodies against A phagocytophilum via IFA and ELISA testing, respectively. Fifteen (16%) sera had antibodies against both pathogens. CONCLUSIONS AND CLINICAL RELEVANCE: Cats living in areas infested by Ixodes scapularis ticks are exposed to B. burgdorferi and A. phagocytophilum and, in some instances, may be coinfected. Most cats appeared healthy. An ELISA incorporating specific recombinant antigens may be used adjunctively with Western blot and other assays to confirm B. burgdorferi and A. phagocytophilum infection in cats.

Anaplasma phagocytophilum↗