Isolate of Anaplasma marginale not transmitted by ticks

The tick-borne transmissibility of 2 isolates of Anaplasma marginale was compared. Dermacentor variabilis were exposed to A marginale as nymphs by feeding on 1 of 4 splenectomized calves during periods of ascending parasitemia (maximum 49% to 81% parasitized erythrocytes) induced by injection of a stabilate. Tick-borne transmission was attempted, using 26 to 224 adult ticks within 30 to 220 days after molting. Adult D variabilis did not transmit an Illinois isolate of A marginale in 7 tick-borne transmission experiments (P = 0.0047), including 2 experiments in which calves were inoculated IV with homogenates of adult ticks. In contrast, a Virginia isolate of A marginale was readily transmitted by the same tick colony. Thus, previously reported morphologic and immunologic differences among A marginale isolates may extend to tick-borne transmissibility. The Virginia and Illinois A marginale isolates had an inclusion appendage that was not a marker for tick transmissibility.     

Demonstration of colonies of Cowdria ruminantium in midgut epithelial cells of Amblyomma variegatum

The development of colonies of Cowdria ruminantium was studied in midgut epithelial cells of adult Amblyomma variegatum that had become infected by feeding as nymphs on cattle with experimentally induced heart-water disease. Colonies were not observed in gut tissues obtained from nymphs during the feeding period, but were present in midgut epithelial cells of ticks obtained at 15 days after they were replete through molting to the adult stage. Colonies were small (1 to 10 micron) initially, but as tick development progressed, their diameter increased to as much as 60 micron. With electron microscopy, colonies were observed to be membrane bound and contained pleomorphic organisms that were reticulated. The organisms seemed to be dividing by binary fission. Many colonies contained a large, electron-dense inclusion that was morphologically similar to hemoglobin deposits found in the cytoplasm of midgut epithelial cells of recently fed ticks. Cowdria ruminantium was often observed adhered to these inclusions.     

Detection of colonies of Anaplasma marginale in salivary glands of three Dermacentor spp infected as nymphs or adults

Salivary glands from males of 3 Dermacentor species (D andersoni, D variabilis and D occidentalis) that were infected with either the Virginia or Idaho isolate of Anaplasma marginale as nymphs or adults were examined for colonies of A marginale by use of light and electron microscopy. Prior to dissection of salivary glands, exposed ticks were held at 25 C for 15 to 18 days, followed by a 3-day incubation at 37 C. Ticks of 2 species transmitted A marginale to calves; the third tick species was confirmed infected by demonstration of typical colonies in tick gut cells, but transmission was not attempted; Colonies of A marginale were seen with light microscopy in salivary glands of all 3 species of ticks; they were located in acinar cells that contained simple granules. Colonies varied morphologically from small, compact ones to larger structures that contained distinct organisms and often were adjacent to the host cell nucleus. Electron microscopy confirmed that the colonies were rickettsial organisms. Morphologic features of A marginale varied and included reticulated forms, forms with electron-dense centers, and small particles; these various forms were similar to those described previously in midgut epithelial cells of ticks. We believe that the organism seen within tick salivary glands may replicate in the glands before its transmission to the vertebrate host.     

Anaplasma infection in free-ranging Iberian red deer in the region of Castilla-La Mancha, Spain

Organisms in the genus Anaplasma are obligate intracellular pathogens that multiply in both vertebrate and invertebrate hosts. The type species, Anaplasma marginale, causes bovine anaplasmosis and infects erythrocytes of the vertebrate host and undergoes a complex developmental cycle in ticks which serve as biological vectors. Infected cattle, wild ruminants and ticks can all serve as reservoirs of A. marginale. In this study, hunter killed Iberian red deer (Cervus elaphus hispanicus) from the region of Castilla-La Mancha in southwestern Spain were tested for Anaplasma infection. We found that 10% of the deer examined were seropositive for Anaplasma. Three A. marginale strains were subsequently obtained from salivary glands of Hyalomma marginatum that were removed from these deer, and the sequence of the major surface protein (msp)4 gene was determined for each strain and used for phylogenetic studies. Maximum parsimony analyses of msp4 sequences from H. marginatum ticks in comparison with New World cattle and bison isolates reported previously, suggested different origins for these Spanish A. marginale strains. The results of this study demonstrated that Iberian red deer are naturally infected with Anaplasma, and may therefore serve as a wildlife reservoir of the pathogen. Although the link between deer infection and the strains of A. marginale identified in ticks was not established, H. marginatum and Rhipicephalus bursa were identified as potential biological vectors for A. marginale in this region and may effect transmission of A. marginale between deer and cattle populations.     

Mapping of B-cell epitopes in the N-terminal repeated peptides of Anaplasma marginale major surface protein 1a and characterization of the humoral immune response of cattle immunized with recombinant and whole organism antigens

Major surface protein (MSP) 1a of the genus type species Anaplasma marginale (Rickettsiales: Anaplasmataceae) together with MSP1b forms the MSP1 complex. MSP1a has been shown to be involved in adhesion, infection and tick transmission of A. marginale, as well as to contribute to protective immunity in cattle. A differential antibody response to MSP1a and MSP1b was observed in cattle immunized with A. marginale derived from bovine erythrocytes (anti-MSP1a response) or cultured tick cells (anti-MSP1b response). In this study, we further characterized the MSP1a antibody response of cattle using several immunogens, including recombinant MSP1a (rMSP1a) protein, erythrocyte- or tick cell culture-derived A. marginale, or a combination of tick cell culture-derived A. marginale and rMSP1a. The MSP1a antibody response to all these immunogens was directed primarily against the N-terminal region of MSP1a that contains tandemly repeated peptides, whereas low antibody levels were detected against the C-terminal portion. Linear B-cell epitopes of MSP1a were mapped using synthetic peptides representing the entire sequence of the protein that were prepared by SPOT synthesis technology. Only two peptides in the N-terminal repeats were recognized by sera from immunized cattle. These peptides shared the sequence SSAGGQQQESS, which is likely to contain the linear B-cell epitope that was recognized by the pools of bovine sera. The average differential of antibody titers against MSP1a minus those against MSP1b correlated with lower percent reductions in PCV. A preferential antibody response to MSP1a was observed in cattle immunized with erythrocyte-derived, cell culture-derived plus rMSP1a or rMSP1a alone, and the percent reduction PCV was significantly lower in these cattle as compared with the other immunization groups. These results provide insight into the bovine antibody response against A. marginale and the role of MSP1a in protection of cattle against A. marginale infection.     

Characterization of the functional domain of major surface protein 1a involved in adhesion of the rickettsia Anaplasma marginale to host cells

The major surface protein (MSP) 1a of the genus type species Anaplasma marginale (Rickettsiales: Anaplasmataceae) has been shown to mediate adhesion, infection and transmission of the organism, as well as to contribute to protective immunity in cattle. MSP1a contains a variable number of tandemly repeated peptides in the amino-terminal region, while the remainder of the protein is highly conserved among isolates. The number of repeats varies among geographic isolates of A. marginale but is constant within an isolate and has been used as a stable genetic marker of isolate identity. Because the sequence of the tandem repeats is the most variable part of the protein among isolates, this region of the protein is most likely to be involved in adhesion to host cells, a prerequisite to infection. The purpose of this study was to characterize the organization and function of the MSP1a tandem repeats of A. marginale in adhesion to host cells. We demonstrated by use of recombinant mutant proteins that the tandemly repeated region of MSP1a was necessary and sufficient to mediate adhesion of MSP1a to tick cells and bovine erythrocytes. Synthetic peptides representing the predominant sequences of individual repeats were tested for their adhesive capacity for tick cell extract (TCE). Peptides containing acidic amino acids D or E at position 20 bound to TCE, while peptides with a G as the 20th amino acid were not adhesive to TCE. Antibodies produced in rabbits against a synthetic repeat peptide neutralized A. marginale infection of cultured tick cells, and the neutralization observed was similar to that effected by antibodies produced against the whole MSP1a recombinant protein. Analysis of tandemly repeated MSP1a peptides of several geographic isolates of A. marginale revealed a complex relationship between the msp1alpha genotype and the tick-transmissible phenotype of the isolate and suggested that both the sequence and conformation of the repeated peptides influenced the adhesive properties of MSP1a. These studies demonstrated that the tandemly repeated region of the protein mediates the adhesive function of MSP1a.     

Sequence analysis of the msp4 gene of Anaplasma ovis strains

Anaplasma ovis (Rickettsiales: Anaplasmataceae) is a tick-borne pathogen of sheep, goats and wild ruminants. The genetic diversity of A. ovis strains has not been well characterized due to the lack of sequence information. In this study, we evaluated bighorn sheep (Ovis canadensis) and mule deer (Odocoileus hemionus) from Montana for infection with A. ovis by serology and sequence analysis of the msp4 gene. Antibodies to Anaplasma spp. were detected in 37% and 39% of bighorn sheep and mule deer analyzed, respectively. Four new msp4 genotypes were identified. The A. ovismsp4 sequences identified herein were analyzed together with sequences reported previously for the characterization of the genetic diversity of A. ovis strains in comparison with other Anaplasma spp. The results of these studies demonstrated that although A. ovismsp4 genotypes may vary among geographic regions and between sheep and deer hosts, the variation observed was less than the variation observed between A. marginale and A. phagocytophilum strains. The results reported herein further confirm that A. ovis infection occurs in natural wild ruminant populations in Western United States and that bighorn sheep and mule deer may serve as wildlife reservoirs of A. ovis.     

Analysis of world strains of Anaplasma marginale using major surface protein 1a repeat sequences

Anaplasma marginale is a tick-borne pathogen of cattle that causes the disease bovine anaplasmosis worldwide. Major surface proteins (MSPs) are involved in host-pathogen and tick-pathogen interactions and have been used as markers for the genetic characterization of A. marginale strains and phylogenetic studies. MSP1a is involved in the adhesion and transmission of A. marginale by ticks and varies among geographic strains in the number and sequence of amino-terminal tandem repeats. The aim of this study was to characterize the genetic diversity of A. marginale strains collected from countries in North and South America, Europe, Asia, Africa and Australia, inclusive of all continents. In this study, we characterized 131 strains of A. marginale using 79 MSP1a repeat sequences. These results corroborated the genetic heterogeneity of A. marginale strains in endemic regions worldwide. The phylogenetic analyses of MSP1a repeat sequences did not result in clusters according to the geographic origin of A. marginale strains but provided phylogeographic information. Seventy-eight percent of the MSP1a repeat sequences were present in strains from a single geographic region. Strong (> or =80%) support was found for clusters containing sequences from Italian, Spanish, Chinese, Argentinean and South American strains. The phylogenetic analyses of MSP1a repeat sequences suggested tick-pathogen co-evolution and provided evidence of multiple introductions of A. marginale strains from various geographic locations worldwide. These results contribute to the understanding of the genetic diversity and evolution of A. marginale and tick-pathogen interactions.     

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.     

Prevalence and genetic diversity of Anaplasma marginale strains in cattle in South Africa

Bovine anaplasmosis, caused by the tick-borne rickettsia Anaplasma marginale, is endemic in South Africa and results in considerable economic loss to the cattle industry. This study was designed to characterize strains of A. marginale at the molecular level from cattle raised in communal and commercial farms in the north-eastern and south-western regions of the Free State Province, South Africa, that varied in rainfall and vegetation. Seroprevalence to A. marginale was determined in 755 cattle by an Anaplasma spp. competitive enzyme-linked immunosorbent assay and ranged from 44% to 98% and was similar in both regions. While Anaplasma centrale was not targeted in this study, A. marginale infections were identified by species-specific msp1alpha polymerase chain reaction in 129 of 215 of the samples studied. Similar genetic diversity of A. marginale strains was found in both the north-eastern and south-western regions. The sequences of 29 A. marginalemsp1alpha amplicons from South African strains revealed considerable genetic diversity providing 14 new repeat sequences. However, 42% of MSP1a repeat sequences were not unique to this region. These results indicated the presence of common genotypes between South African, American and European strains of A. marginale. Cattle movement between different parts of South Africa was suggested by the presence of identical A. marginale MSP1a genotypes in north-eastern and south-western regions of the Free State Province. Control strategies for anaplasmosis in South Africa should therefore be designed to be protective against genetically heterogeneous strains of A. marginale.