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Strain composition of the ehrlichia Anaplasma marginale within persistently infected cattle, a mammalian reservoir for tick transmission.

Tick-borne ehrlichial pathogens of animals and humans require a mammalian reservoir of infection from which ticks acquire the organism for subsequent transmission. In the present study, we examined the strain structure of Anaplasma marginale, a genogroup II ehrlichial pathogen, in both an acute outbreak and in persistently infected cattle that serve as a reservoir for tick transmission. Using the msp1alpha genotype as a stable strain marker, only a single genotype was detected in a disease outbreak in a previously uninfected herd. In contrast, a diverse set of genotypes was detected in a persistently infected reservoir herd within a region where A. marginale is endemic. Genotypic diversity did not appear to be rapidly generated within an individual animal, because only a single genotype, identical to that of the inoculating strain, was detected at time points up to 2 years after experimental infection, and only a single identical genotype was found in repeat sampling of individual naturally infected cattle. Similarly, only a single genotype, identical to that of the experimentally inoculated St. Maries or South Idaho strain, was identified in the bloodmeal taken by Dermacentor andersoni ticks, in the midgut and salivary glands of the infected ticks, and in the blood of acutely infected cattle following tick transmission. The results show that mammalian reservoirs harbor genetically heterogeneous A. marginale and suggest that different genotypes are maintained by transmission within the reservoir population.

Amino Acid Sequence↗

Seroprevalence of antibodies that react with Anaplasma phagocytophila, the agent of human granulocytic ehrlichiosis, in different populations in Westchester County, New York.

We determined the frequencies of antibodies to Anaplasma phagocytophila, the agent of human granulocytic ehrlichiosis (HGE), in different groups of adults and children from Westchester County, New York. The groups included 159 adult blood donors and 215 children who were seronegative for Borrelia burgdorferi antibodies, 118 adult patients and 57 children who were seropositive for B. burgdorferi antibodies, and 42 adult patients with culture-confirmed erythema migrans. Eighteen (11.3%) of the blood donors and 11 (5.1%) of the B. burgdorferi-seronegative children were found to have A. phagocytophila antibodies by indirect immunofluorescent-antibody assay (IFA). Nine of 42 (21.4%) patients with culture-confirmed erythema migrans tested at the baseline visit, 42 of 118 (35.6%) adults, and 3 of 57 (5.3%) children whose sera were reactive for B. burgdorferi antibodies also tested positive for A. phagocytophila antibodies. The geometric mean titer ranged from 219 to 315 for all groups, and the differences in titers among the groups were not statistically significant. Only one-third of the healthy blood donors reactive by IFA were confirmed to be positive by immunoblotting. We conclude that a significant proportion of adults and children without clinical evidence of HGE will test positive for A. phagocytophila antibodies when the conventional cutoff titer of 80 is used in the IFA. This information must be considered in interpretation of test results.

Adolescent↗

Analysis of sequences and loci of p44 homologs expressed by Anaplasma phagocytophila in acutely infected patients.

Anaplasma phagocytophila is an obligatory intragranulocytic bacterium that causes human granulocytic ehrlichiosis. Immunodominant 44-kDa outer membrane proteins of A. phagocytophila are encoded by a p44 multigene family. In the present study, expression profiles of p44 genes in the blood of acutely infected patients in the year 2000 were characterized. A single p44 gene was predominantly expressed in peripheral blood leukocytes from one patient, while up to 17 different p44 genes were transcribed without a single majority in the other two patients. The cDNA sequences of the central hypervariable region of several p44 genes were identical among the isolates from the three patients and a 1995 A. phagocytophila isolate. A. phagocytophila was isolated by cell culture from all of the three 2000 patients. Genomic Southern blot analysis of the three 2000 and two 1995 A. phagocytophila isolates with probes specific to the most dominant p44 transcript in each patient showed that the p44 loci in the A. phagocytophila genome were conserved. Analysis of the predicted amino acid sequences of 43 different p44 genes including 19 new sequences found in the present study, revealed that five amino acids were absolutely conserved. The hypervariable region was subdivided into five domains, including three extremely hypervariable central domains. These results suggest that variations in the sequences of p44 are not random but are restricted. Furthermore, several p44 genes are not hypermutatable in nature, based on the conservation of gene sequences and loci among isolates obtained 5 years apart.

Acute Disease↗

Serologic and molecular detection of Ehrlichia chaffeensis and Anaplasma phagocytophila (human granulocytic ehrlichiosis agent) in Korean patients.

Sera from 491 Korean patients with acute febrile diseases were tested for Ehrlichia chaffeensis and Anaplasma phagocytophila antibodies by indirect immunofluorescence assay (IFA), Western blotting, and TaqMan real-time PCR. Overall, 0.4% of sera reacted with E. chaffeensis, and 1.8% reacted with A. phagocytophila in IFAs. This is the first report of detection of antibodies to A. phagocytophila and E. chaffeensis in Korea and suggests the presence of A. phagocytophila and E. chaffeensis or antigenically similar species.

Adult↗

Identification of Anaplasma phagocytophila (formerly Ehrlichia phagocytophila) variants in blood from sheep in Norway.

A total of 41 blood samples were collected from 40 Anaplasma phagocytophila-infected sheep in 11 sheep flocks from four different counties of southern Norway. The presence and nature of the Anaplasma species were identified by microscopic detection of morulae, PCR, reverse line blot hybridization, and 16S rRNA gene sequencing. A. phagocytophila was identified in all of the samples, and sequencing of the 16S rRNA gene revealed the presence of four variants of A. phagocytophila. Two of these variants have been described before, but two were newly identified 16S rRNA variants of this species. A. phagocytophila variant 1 was found in nine flocks, A. phagocytophila variant 2 was found in four flocks, the A. phagocytophila prototype was found in two flocks, and A. phagocytophila variant 5 was found in one flock. In two flocks, some sheep were infected with A. phagocytophila variant 1, whereas others were infected with A. phagocytophila variant 2, and in three animals a double infection with two variants was registered. Analyses of the blood samples revealed that blood from sheep infected with A. phagocytophila variant 2 contained nearly twice as many neutrophils and eight times as many Anaplasma-infected neutrophils as blood from sheep infected with the A. phagocytophila variant 1. Furthermore, only 43% of the A. phagocytophila variant 2-infected sheep displayed antibody responses in an immune fluorescence assay, whereas 93% of the sheep with the A. phagocytophila variant 1-infected sheep were seropositive.

Anaplasma↗

Anaplasma marginale msp1alpha genotypes evolved under positive selection pressure but are not markers for geographic isolates.

Anaplasma marginale (order Rickettsiales, family Anaplasmataceae), a tick-borne pathogen of cattle, is endemic in tropical and subtropical regions of the world. Many geographic isolates of A. marginale occur in the United States and have been identified by major surface protein 1a (MSP1a), which varies in sequence and molecular weight due to different numbers of tandem 28- to 29-amino-acid repeats. The present study was undertaken to examine the genetic variations among isolates of A. marginale obtained during 2001 from infected cattle from east-central Oklahoma, where A. marginale is endemic. The gene and protein sequences of MSP1a and msp4 nucleotide sequences were used to infer the phylogenetic relationships among Oklahoma and New World isolates from Argentina, Brazil, Mexico, and the United States. All 11 A. marginale isolates collected from Oklahoma had different MSP1a sequences but identical MSP4 sequences. The phylogenies of the msp4 sequences of 13 isolates from Oklahoma in comparison with those of 7 Latin American isolates and 12 U.S. isolates by maximum-parsimony (MP) and maximum-likelihood (ML) analyses, with A. centrale and A. ovis sequences used as outgroups, provided strong bootstrap analysis support for a Latin American clade. Isolates of A. marginale from the southern United States (Florida, Mississippi, and Virginia) and the west-central United States (California, Idaho, Illinois, Oregon, Missouri, and Texas) also grouped into two clades. Both clades contained isolates from Oklahoma, suggesting extensive cattle movement. ML analysis of the msp4 sequences of isolates from Oklahoma provided bootstrap analysis support for east-central and north-central clades in Oklahoma, and both clades included isolates from Stillwater, Okla. Analysis of the codon and amino acid changes among the msp4 sequences of isolates with different phylogenies provided evidence that msp4 is not under positive selection pressure. In contrast, the phylogenies of the MSP1a DNA and protein sequences of 13 isolates from Oklahoma in comparison with those of 7 Latin American and 13 isolates from the United States by MP and ML analyses demonstrated no geographic clustering and provided evidence that this gene is under positive selection pressure. The results indicate that msp1alpha is not a marker for the characterization of A. marginale geographic isolates and suggest that the genetic heterogeneity observed among isolates of A. marginale within Oklahoma could be explained by cattle movement and the maintenance of different genotypes by independent transmission events.

Amino Acid Sequence↗

Enzootic transmission of Anaplasma bovis in Nantucket cottontail rabbits.

Serological studies of cottontail rabbits sampled from Nantucket Island, Mass., have suggested exposure to at least two ehrlichiae. The agent of human granulocytic ehrlichiosis (Anaplasma phagocytophilum) is intensely enzootic in rabbits there, but the identity of the other ehrlichial infection remains undescribed. We sampled rabbits over five transmission seasons and tested their blood and tissues for evidence of infection using PCR targeting an Ehrlichia genus-wide 16S rDNA target. Sequence analysis of positive amplicons revealed the presence of Anaplasma bovis, an agent not known to be present in North America. The average annual prevalence of A. bovis within rabbits, as determined by PCR of blood samples, was 18%. Haemaphysalis leporispalustris appears to serve as vector. The public health (human or veterinary) significance of this finding remains speculative.

Anaplasma↗

Transmission of Anaplasma marginale by Boophilus microplus: retention of vector competence in the absence of vector-pathogen interaction.

Whether arthropod vectors retain competence for transmission of infectious agents in the long-term absence of vector-pathogen interaction is unknown. We addressed this question by quantifying the vector competence of two tick vectors, with mutually exclusive tropical- versus temperate-region distributions, for genetically distinct tropical- and temperate-region strains of the cattle pathogen Anaplasma marginale. The tropical cattle tick Boophilus microplus, which has been eradicated from the continental United States for over 60 years, was able to acquire and transmit the temperate St. Maries (Idaho) strain of A. marginale. Similarly, the temperate-region tick Dermacentor andersoni efficiently acquired and transmitted the Puerto Rico strain of A. marginale. There were no significant quantitative differences in infection rate or number of organisms per tick following feeding on cattle with persistent infections of either A. marginale strain. In contrast, the significantly enhanced replication of the Puerto Rico strain in the salivary gland of B. microplus at the time of transmission feeding is consistent with adaptation of a pathogen strain to its available vector. However, the transmission of both strains by B. microplus demonstrates that adaptation or continual interaction between the pathogen and vector is not required for retention of vector competence. Importantly, the results clearly show that reestablishment of acaricide-resistant B. microplus in the United States would be associated with A. marginale transmission.

Anaplasma↗

Stochastic transmission of multiple genotypically distinct Anaplasma marginale strains in a herd with high prevalence of Anaplasma infection.

Multiple genotypically unique strains of the tick-borne pathogen Anaplasma marginale occur and are transmitted within regions where the organism is endemic. In this study, we tested the hypothesis that specific A. marginale strains are preferentially transmitted. The study herd of cattle (n = 261) had an infection prevalence of 29% as determined by competitive inhibition enzyme-linked immunosorbent assay and PCR, with complete concordance between results of the two assays. Genotyping revealed the presence of 11 unique strains within the herd. Although the majority of the individuals (70 of 75) were infected with only a single A. marginale strain, five animals each carried two strains with markedly distinct genotypes, indicating that superinfection does occur with distinct A. marginale strains, as has been reported with A. marginale and A. marginale subsp. centrale strains. Identification of strains in animals born into and infected within the herd during the period from 1998 to 2003 revealed no significant difference from the overall strain prevalence in the herd, results that do not support the occurrence of preferential strain transmission within a population of persistently infected animals and are most consistent with pathogen strain transmission being stochastic.

Anaplasma marginale↗

The effects of war on the control of diseases of livestock in Rhodesia (Zimbabwe).

The disruption of veterinary services in the tribal areas of Rhodesia (now Zimbabwe) during seven years of conflict resulted in serious epidemics of disease. The cessation of dipping was followed by the death of an estimated one million cattle from tick-borne disease. Heavy mortality followed the disruption of control measures for trypanosomiasis. Foot-and-mouth disease and anthrax spread widely in the tribal areas. Rabies, normally restricted to areas bordering Botswana and Mocambique, became widespread. A marked increase in human deaths from anthrax and rabies occurred.

Anaplasmosis↗

A clinical trial of buparvaquone in the treatment of East Coast fever.

A clinical trial was conducted to test buparvaquone (Butalex; Coopers Pitman-Moore) in the treatment of East Coast fever under field conditions in Kenya. Data from 229 cases were analysed following treatment with one (69), two (142) or three (18) doses at 2.5 mg/kg. The majority of cattle (95.2 per cent) were exotic (Bos taurus) or improved (Bos taurus cross Bos indicus) and 39.3 per cent were infected with Anaplasma marginale. There was an overall recovery rate of 85.6 per cent, with 90.1 per cent recovering following one treatment and 75.4 per cent recovering following two treatments. At a follow-up visit three to six months after completion of the trial data was obtained on 224 cases. Thirty had died, 13 of which were reported to have been from East Coast fever, nine had been sold and six slaughtered. Of the remaining 146, 86.3 per cent were in good condition, 13.7 per cent fair and 2.0 per cent in poor condition. A two dose regimen was most effective and should be recommended except in very early cases or those under direct veterinary supervision.

Anaplasmosis↗

Lyme disease: the quest for magic bullets.

Lyme disease represents a growing public health threat. Recent molecular and genetic studies have confirmed that Borrelia burgdorferi, the spirochetal agent of Lyme disease, is one of the most complex bacteria known to man. Affinity for multiple cell types and the presence of non-replicating forms of B. burgdorferi have contributed to persistent infection and failure of simple antibiotic regimens. The controversial clinical science of Lyme disease has impeded reliable diagnosis and effective treatment of this protean illness. Two major clinical hurdles are the absence of a therapeutic endpoint in treating Lyme disease and the presence of tick-borne coinfections that may complicate the course of the illness. New strategies for the diagnosis, treatment and prevention of Lyme disease are urgently needed.

Anaplasmosis↗