High prevalence of Anaplasma marginale in the Crioula Lageana cattle.

INTRODUCTION: Bovine anaplasmosis is caused by the bacterium Anaplasma marginale; its transmission occurs through vectors such as ticks. Crioula Lageana is a native cattle breed from the South of Brazil used for beef production, with excellent meat quality. There are no studies of the epidemiology of this disease in Crioula Lageana even though tick damage is known to be frequent. METHODOLOGY: Blood samples were collected from 311 Crioula Lageana cattle and subjected to DNA extraction and polymerase chain reaction (PCR) using specific primers for the Major Surface Protein 5 (msp5) gene for the detection of the bovine anaplasmosis agent. The animals were classified according to the gender, the category and the presence or absence of ticks at the time of collection. The animal owners completed an epidemiological questionnaire to determine factors that might be associated with anaplasma infection. RESULTS: The prevalence of A. marginale was 79.9%. The following factors were found to be protective against infection: I) the breeding objectives (whether animals were destined for beef production and trade or solely for beef production), II) tick control rate; and III) pregnant and lactating cows and calves as the categories least affected by the hemoparasite. The main risk factor for hemoparasite acquisition was the use of organophosphates and avermectins as acaricides. CONCLUSIONS: Crioula Lageana cattle are in a situation of enzootic stability, with a high prevalence of A. marginale infection. The factors associated with the infection were: I) breeding objectives, II) tick control rate, III) the acaricides used, and IV) the most tick-parasitized categories of cattle.

A vaccine using Anaplasma marginale subdominant type IV secretion system recombinant proteins was not protective against a virulent challenge.

Anaplasma marginale is the most prevalent tick-borne livestock pathogen with worldwide distribution. Bovine anaplasmosis is a significant threat to cattle industry. Anaplasmosis outbreaks in endemic areas are prevented via vaccination with live A. centrale produced in splenectomized calves. Since A. centrale live vaccine can carry other pathogens and cause disease in adult cattle, research efforts are directed to develop safe recombinant subunit vaccines. Previous work found that the subdominant proteins of A. marginale type IV secretion system (T4SS) and the subdominant elongation factor-Tu (Ef-Tu) were involved in the protective immunity against the experimental challenge in cattle immunized with the A. marginale outer membrane (OM). This study evaluated the immunogenicity and protection conferred by recombinant VirB9.1, VirB9.2, VirB10, VirB11, and Ef-Tu proteins cloned and expressed in E. coli. Twenty steers were randomly clustered into four groups (G) of five animals each. Cattle from G1 and G2 were immunized with a mixture of 50 µg of each recombinant protein with Quil A® or Montanide™ adjuvants, respectively. Cattle from G3 and G4 (controls) were immunized with Quil A and Montanide adjuvants, respectively. Cattle received four immunizations at three-week intervals and were challenged with 107 A. marginale-parasitized erythrocytes 42 days after the fourth immunization. After challenge, all cattle showed clinical signs, with a significant drop of packed cell volume and a significant increase of parasitized erythrocytes (p<0.05), requiring treatment with oxytetracycline to prevent death. The levels of IgG2 induced in the immunized groups did not correlate with the observed lack of protection. Additional strategies are required to evaluate the role of these proteins and their potential utility in the development of effective vaccines.

Segmental Variation in a Duplicated msp2 Pseudogene Generates Anaplasma marginale Antigenic Variants.

Anaplasma marginale is a prototypical highly antigenically variant bacterial pathogen dependent on the sequential generation of major surface protein 2 (Msp2) outer membrane variants to establish persistent infection. Msp2 is encoded by a single expression site, and diversity is achieved by gene conversion of chromosomally encoded msp2 pseudogenes. Analysis of the full complement of msp2 pseudogenes in the St. Maries strain revealed identical sequences in different loci. The Florida strain shared the same locus structure, but in the loci where the St. Maries strain had two identical pseudogenes, the Florida strain had one whose sequence was identical to the St. Maries sequences, while the sequence of the second pseudogene differed. Consequently, we hypothesized that the msp2 pseudogene repertoire arose via gene duplication, allowing structural variation to occur in one copy but the utility of the other to be retained. Using comparative genomics, we first established that duplication of msp2 pseudogenes is common among A. marginale strains: all seven examined strains had at least one duplicate pair in which either the genes in the pair were maintained as identical copies or the genes contained segmental changes. We then demonstrated that a minimal segmental change in a duplicated pseudogene locus is sufficient for immune escape from the broad antibody response generated in a natural host, as is a completely divergent pseudogene sequence in an otherwise conserved locus. The results support a model in which a locus first duplicates, resulting in a second identical copy, and then progressively incorporates changes to generate an msp2 repertoire capable of generating sufficient antigenic variants to escape immunity and establish persistent infection.

Evaluation of oxidative stress and antioxidant status, serum trace mineral levels and cholinesterases activity in cattle infected with Anaplasma marginale.

This study was undertaken to assess the influence of an Anaplasma marginale infection on oxidative stress and antioxidant status, trace elements and cholinesterase as markers of the inflammatory process and biomarkers of oxidative imbalance. An infected group comprised of 35 crossbred Holstein cattle, about 2-3 years old, naturally infected with Anaplasma marginale, were divided into 4 subgroups according to their parasitemia rates (<1%, 1-10%, 10-20%, >20%) and also 10 healthy cattle as control were selected. Blood samples were taken and hematological parameters, activities of antioxidant enzymes including erythrocyte glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT), glucose-6-phosphate dehydrogenase (G6PD), total antioxidant capacity (TAC), median corpuscularfragility (MCF) as well as acetylcholinesterase (AChE), and serum concentrations of antioxidant trace minerals (copper, iron, zinc, manganese, and selenium) and butyrylcholinesterase (BChE) were determined. In addition, as an index of lipid peroxidation, the level of malondialdehyde (MDA) was measured. The results revealed a significant decrease (P < 0.05) in RBC count, packed cell volume (PCV) and Hb concentration as well as the activities of erythrocyte GSH-Px, SOD, CAT, G6PD, TAC, MCF and AChE and serum concentrations of Cu, Zn, Mn, Se and BchE in the infected cattle. In contrast, significantly increased (P < 0.05) levels of MDA and erythrocyte osmotic fragility as well as serum concentration of iron were recorded in the infected animals. The significant decrease in antioxidant enzyme activities and substantial elevated levels of lipid peroxidation and erythrocyte osmotic fragility associated with the notable increase in parasitemia indicate increased exposure of RBCs to oxidative damage. Furthermore, decrease of cholinesterase in infection by A. marginale can and directly or indirectly lead to increase acetylcholine levels potent anti-inflammatory molecules, thereby inhibiting inflammation.

Prevalence, risk factors, and genetic diversity of veterinary important tick-borne pathogens in cattle from Rhipicephalus microplus-invaded and non-invaded areas of Benin.

Babesiosis, theileriosis, anaplasmosis, and heartwater are tick-borne diseases (TBD) that threaten livestock production in sub-Saharan Africa including Benin. This country has been faced with an invasion of Rhipicephalus microplus, a major vector for babesiosis, theileriosis, and anaplasmosis over the last decade. Yet, data on TBD and the impact of the invasive ticks are lacking, making risk level evaluation and disease control arduous. In this study, epidemiological features of Babesia bovis, B. bigemina, Theileria spp., Anaplasma marginale and Ehrlichia ruminantium infections in Benin cattle were investigated in R. microplus-invaded and non-invaded areas. Detection of pathogens was based on species-specific PCR assays and resulting data were used to identify risk factors. Genetic diversity and phylogenies were then evaluated using several markers. Out of 207 samples examined, 170 (82.1%), 109 (52.7%), 42 (20.3%) 24 (11.6%) and 1 (0.5%) were positive for T. mutans, A. marginale, B. bigemina, B. bovis and E. ruminantium, respectively. Animal gender (for B. bovis), exposure to R. microplus (for B. bigemina and A. marginale), animal age (for B. bigemina and A. marginale) and cattle breed and/or antiprotozoal treatment (for T. mutants) significantly modulated pathogen occurrence. In addition, R. microplus exposure was significantly related to co-infection patterns and cases of clinical theileriosis and/or anaplasmosis were recorded among cattle highly exposed to the tick. In the genetic characterization, Theileria spp. and E. ruminantium sequences were conserved. Babesia spp. and A. marginale, however, showed high sequence polymorphisms that indicate the presence of several strains and may be linked to R. microplus invasion. Taken together, these results ascertain the endemicity of tick-borne infections in Benin and suggest that the characteristics of Babesia spp. and A. marginale infections in R. microplus-invaded and non-invaded areas are different.

Ticks and Tick-Borne Pathogens of the Caribbean: Current Understanding and Future Directions for More Comprehensive Surveillance.

Ticks are obligate hematophagous arthropods of significant importance to human and veterinary medicine. They transmit a vast array of pathogens, including bacteria, viruses, protozoa, and helminths. Most epidemiological data on ticks and tick-borne pathogens (TBPs) in the West Indies are limited to common livestock pathogens such as Ehrlichia ruminantium, Babesia spp. (i.e., B. bovis and B. bigemina), and Anaplasma marginale, and less information is available on companion animal pathogens. Of note, human tick-borne diseases (TBDs) remain almost completely uncharacterized in the West Indies. Information on TBP presence in wildlife is also missing. Herein, we provide a comprehensive review of the ticks and TBPs affecting human and animal health in the Caribbean, and introduce the challenges associated with understanding TBD epidemiology and implementing successful TBD management in this region. In particular, we stress the need for innovative and versatile surveillance tools using high-throughput pathogen detection (e.g., high-throughput real-time microfluidic PCR). The use of such tools in large epidemiological surveys will likely improve TBD prevention and control programs in the Caribbean.

Infection-derived lipids elicit an immune deficiency circuit in arthropods.

The insect immune deficiency (IMD) pathway resembles the tumour necrosis factor receptor network in mammals and senses diaminopimelic-type peptidoglycans present in Gram-negative bacteria. Whether unidentified chemical moieties activate the IMD signalling cascade remains unknown. Here, we show that infection-derived lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and 1-palmitoyl-2-oleoyl diacylglycerol (PODAG) stimulate the IMD pathway of ticks. The tick IMD network protects against colonization by three distinct bacteria, that is the Lyme disease spirochete Borrelia burgdorferi and the rickettsial agents Anaplasma phagocytophilum and A. marginale. Cell signalling ensues in the absence of transmembrane peptidoglycan recognition proteins and the adaptor molecules Fas-associated protein with a death domain (FADD) and IMD. Conversely, biochemical interactions occur between x-linked inhibitor of apoptosis protein (XIAP), an E3 ubiquitin ligase, and the E2 conjugating enzyme Bendless. We propose the existence of two functionally distinct IMD networks, one in insects and another in ticks.

Structural Basis for Recombinatorial Permissiveness in the Generation of Anaplasma marginale Msp2 Antigenic Variants.

Sequential expression of outer membrane protein antigenic variants is an evolutionarily convergent mechanism used by bacterial pathogens to escape host immune clearance and establish persistent infection. Variants must be sufficiently structurally distinct to escape existing immune effectors yet retain the core structural elements required for localization and function within the outer membrane. We examined this balance using Anaplasma marginale, which generates antigenic variants in the outer membrane protein Msp2 using gene conversion. The overwhelming majority of Msp2 variants expressed during long-term persistent infection are mosaics, derived by recombination of oligonucleotide segments from multiple alleles to form unique hypervariable regions (HVR). As a result, the mosaics are not under long-term selective pressure to encode a functional protein; consequently, we hypothesized that the Msp2 HVR is structurally permissive for mosaic expression. Using an integrated approach of predictive modeling with determination of the native Msp2 protein structure and function, we demonstrate that structured elements, most notably, ß-sheets, are significantly concentrated in the highly conserved N- and C-terminal domains. In contrast, the HVR is overwhelmingly a random coil, with the structured α-helices and ß-sheets being confined to the genomically defined structural tethers that separate the antigenically variable microdomains. This structure is supported by the surface exposure of the HVR microdomains and the slow diffusion-type porin function in native Msp2. Importantly, the predominance of the random coil provides plasticity for the formation of functional HVR mosaics and realization of the full potential of segmental gene conversion to dramatically expand the variant repertoire.

The Pathogen-Occupied Vacuoles of Anaplasma phagocytophilum and Anaplasma marginale Interact with the Endoplasmic Reticulum.

The genus Anaplasma consists of tick-transmitted obligate intracellular bacteria that invade white or red blood cells to cause debilitating and potentially fatal infections. A. phagocytophilum, a human and veterinary pathogen, infects neutrophils to cause granulocytic anaplasmosis. A. marginale invades bovine erythrocytes. Evidence suggests that both species may also infect endothelial cells in vivo. In mammalian and arthropod host cells, A. phagocytophilum and A. marginale reside in host cell derived pathogen-occupied vacuoles (POVs). While it was recently demonstrated that the A. phagocytophilum-occupied vacuole (ApV) intercepts membrane traffic from the trans-Golgi network, it is unclear if it or the A. marginale-occupied vacuole (AmV) interacts with other secretory organelles. Here, we demonstrate that the ApV and AmV extensively interact with the host endoplasmic reticulum (ER) in endothelial, myeloid, and/or tick cells. ER lumen markers, calreticulin, and protein disulfide isomerase, and the ER membrane marker, derlin-1, were pronouncedly recruited to the peripheries of both POVs. ApV association with the ER initiated early and continued throughout the infection cycle. Both the ApV and AmV interacted with the rough ER and smooth ER. However, only derlin-1-positive rough ER derived vesicles were delivered into the ApV lumen where they localized with intravacuolar bacteria. Transmission electron microscopy identified multiple ER-POV membrane contact sites on the cytosolic faces of both species' vacuoles that corresponded to areas on the vacuoles' lumenal faces where intravacuolar Anaplasma organisms closely associated. A. phagocytophilum is known to hijack Rab10, a GTPase that regulates ER dynamics and morphology. Yet, ApV-ER interactions were unhindered in cells in which Rab10 had been knocked down, demonstrating that the GTPase is dispensable for the bacterium to parasitize the ER. These data establish the ApV and AmV as pathogen-host interfaces that directly engage the ER in vertebrate and invertebrate host cells and evidence the conservation of ER parasitism between two Anaplasma species.

The bacterial microbiome of Dermacentor andersoni ticks influences pathogen susceptibility.

Ticks are of medical importance owing to their ability to transmit pathogens to humans and animals. The Rocky Mountain wood tick, Dermacentor andersoni, is a vector of a number of pathogens, including Anaplasma marginale, which is the most widespread tick-borne pathogen of livestock. Although ticks host pathogenic bacteria, they also harbor bacterial endosymbionts that have a role in tick physiology, survival, as well as pathogen acquisition and transmission. The goal of this study was to characterize the bacterial microbiome and examine the impact of microbiome disruption on pathogen susceptibility. The bacterial microbiome of two populations of D. andersoni with historically different susceptibilities to A. marginale was characterized. In this study, the microbiome was disrupted and then ticks were exposed to A. marginale or Francisella novicida to determine whether the microbiome correlated with pathogen susceptibility. Our study showed that an increase in proportion and quantity of Rickettsia bellii in the microbiome was negatively correlated to A. marginale levels in ticks. Furthermore, a decrease in Francisella endosymbionts was associated with lower F. novicida infection levels, demonstrating a positive pathogen-endosymbiont relationship. We demonstrate that endosymbionts and pathogens have varying interactions, and suggest that microbiome manipulation may provide a possible method for biocontrol by decreasing pathogen susceptibility of ticks.

Identification of a vertically transmitted strain from Anaplasma marginale (UFMG3): Molecular and phylogenetic characterization, and evaluation of virulence.

Bovine anaplasmosis is a disease caused by the intraerythrocytic rickettsia species Anaplasma marginale and results in great economic losses in tropical and subtropical regions. Vertical transmission is an important phenomenon that contributes to the persistence of different strains of the agent within the same herd. The identification of new strains and genetic characterization studies are essential to understanding their epidemiology and virulence and for vaccine development. The aim of this study was to perform molecular and phylogenetic characterizations of a new vertically transmitted strain from A. marginale and to evaluate its virulence by experimental inoculation of rickettsia-free calves. Thirty newborn Holstein calves were subjected to molecular tests for the detection of A. marginale, Babesia bovis and Babesia bigemina. Calves positive for A. marginale (n=3) were splenectomized and monitored for the clinical manifestations of anaplasmosis. Blood samples from one of the calves that presented rickettsemia of 42.8% and spontaneous recovery of clinical parameters were used for molecular and phylogenetic characterization (msp1a gene), and inoculum production was used for the evaluation of virulence. This strain was identified as UFMG3. Three tandem repeat forms (13 and MGI19) were identified from the analysis of the msp1a gene, in which the form MGI19 appeared twice. Analysis of these repeats revealed the presence of the sequences QASTSS and SSASGQQQESS and of aspartic acid (D) at position 20 of both repeats. Phylogenetic analysis showed a close relationship among the UFMG3, MGI19 and UFMG2 strains. For virulence evaluation, six Holstein calves were inoculated intravenously with 2×10(7)A. marginale UFMG3-infected erythrocytes. The calves showed maximum rickettsemia of 5.1%, a moderate decrease in packed cell volume and spontaneous recovery of clinical parameters without the need for treatment. The results of experimental inoculation suggest that the strain A. marginale UFMG3 has low virulence and potential application for use as a live vaccine against A. marginale.

Reduced Infectivity in cattle for an outer membrane protein mutant of Anaplasma marginale.

Anaplasma marginale is the causative agent of anaplasmosis in cattle. Transposon mutagenesis of this pathogen using the Himar1 system resulted in the isolation of an omp10 operon insertional mutant referred to as the omp10::himar1 mutant. The work presented here evaluated if this mutant had morphological and/or growth rate defects compared to wild-type A. marginale. Results showed that the morphology, developmental cycle, and growth in tick and mammalian cell cultures are similar for the mutant and the wild type. Tick transmission experiments established that tick infection levels with the mutant were similar to those with wild-type A. marginale and that infected ticks successfully infected cattle. However, this mutant exhibited reduced infectivity and growth in cattle. The possibility of transforming A. marginale by transposon mutagenesis coupled with in vitro and in vivo assessment of altered phenotypes can aid in the identification of genes associated with virulence. The isolation of deliberately attenuated organisms that can be evaluated in their natural biological system is an important advance for the rational design of vaccines against this species.

Breadth of the CD4+ T cell response to Anaplasma marginale VirB9-1, VirB9-2 and VirB10 and MHC class II DR and DQ restriction elements.

MHC class II molecules influence antigen-specific CD4+ T lymphocyte responses primed by immunization and infection. CD4+ T cell responses are important for controlling infection by many bacterial pathogens including Anaplasma marginale and are observed in cattle immunized with the protective A. marginale outer membrane (OM) vaccine. Immunogenic proteins that comprise the protective OM vaccine include type IV secretion system (T4SS) proteins VirB9-1, VirB9-2 and VirB10, candidates for inclusion in a multiepitope vaccine. Our goal was to determine the breadth of the VirB9-1, VirB9-2 and VirB10 T cell response and MHC class II restriction elements in six cattle with different MHC class II haplotypes defined by DRB3, DQA and DQB allele combinations for each animal. Overlapping peptides spanning each T4SS protein were tested in T cell proliferation assays with autologous antigen-presenting cells (APC) and artificial APC expressing combinations of bovine DR and DQ molecules. Twenty immunostimulatory peptides were identified; three representing two or more epitopes in VirB9-1, ten representing eight or more epitopes in VirB9-2 and seven representing seven or more epitopes in VirB10. Of the eight DRA/DRB3 molecules, four presented 15 peptides, which was biased as DRA/DRB3*1201 presented ten and DRA/DRB3*1101 presented four peptides. Four DQA/DQB molecules composed of two intrahaplotype and two interhaplotype pairs presented seven peptides, of which five were uniquely presented by DQ molecules. In addition, three functional mixed isotype (DQA/DRB3) restriction elements were identified. The immunogenicity and broad MHC class II presentation of multiple VirB9-1, VirB9-2 and VirB10 peptide epitopes justify their testing as a multiepitope vaccine against A. marginale.

Adaptive immunity to Anaplasma pathogens and immune dysregulation: implications for bacterial persistence.

Anaplasma marginale is an obligate intraerythrocytic bacterium that infects ruminants, and notably causes severe economic losses in cattle worldwide. Anaplasma phagocytophilum infects neutrophils and causes disease in many mammals, including ruminants, dogs, cats, horses, and humans. Both bacteria cause persistent infection - infected cattle never clear A. marginale and A. phagocytophilum can also cause persistent infection in ruminants and other animals for several years. This review describes correlates of the protective immune response to these two pathogens as well as subversion and dysregulation of the immune response following infection that likely contribute to long-term persistence. I also compare the immune dysfunction observed with intraerythrocytic A. marginale to that observed in other models of chronic infection resulting in high antigen loads, including malaria, a disease caused by another intraerythrocytic pathogen.

Temporal characterisation of the organ-specific Rhipicephalus microplus transcriptional response to Anaplasma marginale infection.

Arthropods transmit important infectious diseases of humans and animals. Importantly, replication and the development of pathogen infectivity are tightly linked to vector feeding on the mammalian host; thus analysis of the transcriptomes of both vector and pathogen during feeding is fundamental to understanding transmission. Using Anaplasma marginale infection of Rhipicephalus microplus as the experimental model, we tested three hypotheses exploring the temporal and organ-specific nature of the tick midgut and salivary gland transcriptomes during feeding and in response to infection. Numerous R. microplus genes were regulated in response to feeding and were differentially regulated between the midgut and salivary gland; additionally, there was a progression in regulated gene expression in the salivary gland over time. In contrast, relatively few tick genes were specifically regulated in response to A. marginale infection and these genes were predominantly annotated as hypothetical or were of unknown function. Notable among the genes with informative annotation was that several ribosomal proteins were down-regulated, suggesting that there may be a corresponding decrease in translation. The hypotheses that R. microplus midgut and salivary gland genes are differentially regulated and that the salivary gland transcriptome is dynamic over time were accepted. This is consistent with, and important for understanding the roles of, the two organs, the midgut serving as an initial site of uptake and replication while the salivary gland serves as the final site of replication and secretion. The nominal effect of A. marginale on the tick transcriptome in terms of numbers of regulated genes and fold of regulation supports the view that the vector-pathogen relationship is well established with minimal deleterious effect on the tick. The small set of predominantly hypothetical genes regulated by infection suggests that A. marginale is affecting a novel set of tick genes and may provide new opportunities for blocking transmission from the tick.

Tristeza parasitária bovina no Sertão da Paraíba / Cattle tick fever in the semiarid region of the Brazilian state of Paraiba

Descrevem-se 24 surtos de tristeza parasitária bovina no sertão paraibano, sendo 18 de anaplasmose por Anaplasma margimale, dois de babesiose por Babesia bigemina, dois por Babesia não identificada e dois por infecção mista de A. marginale e Babesia sp. Os surtos ocorreram entre agosto de 2007 a outubro de 2009, porém, com uma concentração dos surtos no final do período chuvoso e início do período seco de cada ano, sendo 22 em animais adultos e dois em bezerros de aproximadamente 11 meses. Dois surtos ocorreram em bovinos da raça Nelore, um em animais da raça Gir e os 21 restantes ocorreram em animais das raças Holandês, Pardo Suiço e mestiços das mesmas com zebuínos. Conclui-se que no sertão da Paraíba há áreas de instabilidade enzoótica, ocorrendo surtos de tristeza no final da época de chuvas, principalmente nas áreas de planaltos e serras da região da Borborema e em áreas úmidas como a Bacia do Rio do Peixe, Rio Piranhas e Rio Espinharas em que há a formação de microclimas favoráveis à sobrevivência do carrapato.

Twenty four outbreaks of cattle tick fever are reported in the semiarid region of Paraiba known as Sertão. Eighteen outbreaks were caused by Anaplasma. marginale, two by Babesia bigemina, and two by mixed infection of A. marginale and Babesia sp. In other two outbreaks of babesiosis the species of Babesia was not identified. Outbreaks occurred from August 2007 to October 2009, however with a concentration of the outbreaks at the end of the rainy period and beginning of the dry period in each year. Twenty two outbreaks affected adult cattle and two affected calves approximately 11 months-old. Three outbreaks affected Bos taurus indicus cattle, of the Nelore and Gir breeds. In 21 outbreaks Holstein, Brown Swiss and crossbred of these breeds with Bos taurus indicus cattle were affected. It is concluded that in the sertão of Paraíba there are areas of enzootic instability for cattle tick fever occurring outbreaks at the end of the rainy season, mainly in hilly areas of the Borborema region, and in wet areas of some river basins, including Rio do Peixe, Rio Piranhas and Rio Espinharas.

A review of bovine anaplasmosis.

Bovine anaplasmosis, caused by Anaplasma marginale, is an infectious but non-contagious disease. It is spread through tick bites or by the mechanical transfer of fresh blood from infected to susceptible cattle from biting flies or by blood-contaminated fomites including needles, ear tagging, dehorning and castration equipment. Transplacental transmission of A. marginale may contribute to the epidemiology of bovine anaplasmosis in some regions. Bovine anaplasmosis occurs in tropical and subtropical regions worldwide. Cattle of all ages are susceptible to infection with A. marginale, but the severity of disease increases with age. Once cattle of any age become infected with A. marginale, they remain persistently infected carriers for life. Diagnosis of bovine anaplasmosis can be made by demonstration of A. marginale on stained blood smears from clinically infected animals during the acute phase of the disease, but it is not reliable for detecting infection in pre-symptomatic or carrier animals. In these instances, the infection is generally diagnosed by serologic demonstration of antibodies with confirmation by molecular detection methods. The susceptibility of wild ruminants to infection by A. marginale and the role of wild ruminants in the epidemiology of bovine anaplasmosis are incompletely known owing to lack of published research, lack of validation of diagnostic tests for these species and cross-reaction of Anaplasma spp. antibodies in serologic tests. Control measures for bovine anaplasmosis vary with geographical location and include maintenance of Anaplasma-free herds, vector control, administration of antibiotics and vaccination.

Anaplasma marginale infection with persistent high-load bacteremia induces a dysfunctional memory CD4+ T lymphocyte response but sustained high IgG titers.

Control of blood-borne infections is dependent on antigen-specific effector and memory T cells and high-affinity IgG responses. In chronic infections characterized by a high antigen load, it has been shown that antigen-specific T and B cells are vulnerable to downregulation and apoptosis. Anaplasma marginale is a persistent infection of cattle characterized by acute and chronic high-load bacteremia. We previously showed that CD4(+) T cells primed by immunization with an A. marginale outer membrane protein were rapidly deleted following infection. Furthermore, peripheral blood T cell responses to bacteria were not observed after acute infection was controlled, suggesting dysfunctional T cell priming to other A. marginale antigens. The current study more closely investigated the kinetics of A. marginale-specific CD4(+) T cell responses primed during infection. Frequent sampling of peripheral blood and spleens revealed that antigen-specific CD4(+) T cell responses were first detected at 5 to 7 weeks, but the responses were sporadic and transient thereafter. A similar pattern was observed in animals sampled weekly for nearly 1 year. Paradoxically, by 2 weeks of infection, cattle had developed high titers of A. marginale-specific IgG, which remained high throughout persistent infection. This dysfunctional CD4(+) T cell response to infection is consistent with continual downregulation or deletion of newly primed effector T cells, similar to what was observed for immunization-induced T cells following A. marginale infection. The failure to establish a strong memory T cell response during A. marginale infection likely contributes to bacterial persistence.

Association of pathogen strain-specific gene transcription and transmission efficiency phenotype of Anaplasma marginale.

Efficient transmission of pathogens by an arthropod vector is influenced by the ability of the pathogen to replicate and develop infectiousness within the arthropod host. While the basic life cycle of development within and transmission from the arthropod vector are known for many bacterial and protozoan pathogens, the determinants of transmission efficiency are largely unknown and represent a significant gap in our knowledge. The St. Maries strain of Anaplasma marginale is a high-transmission-efficiency strain that replicates to a high titer in the tick salivary gland and can be transmitted by <10 ticks. In contrast, A. marginale subsp. centrale (Israel vaccine strain) has an identical life cycle but replicates to a significantly lower level in the salivary gland, with transmission requiring >30-fold more ticks. We hypothesized that strain-specific genes expressed in the tick salivary gland at the time of transmission are linked to the differences in the transmission efficiency phenotype. Using both annotation-dependent and -independent analyses of the complete genome sequences, we identified 58 strain-specific genes. These genes most likely represent divergence from common ancestral genes in one or both strains based on analysis of synteny and lack of statistical support for acquisition as islands by lateral gene transfer. Twenty of the St. Maries strain-specific genes and 16 of the strain-specific genes in the Israel strain were transcribed in the tick salivary gland at the time of transmission. Although associated with the transmission phenotype, the expression levels of strain-specific genes were equal to or less than the expression levels in infected erythrocytes in the mammalian host, suggesting that function is not limited to salivary gland colonization.

Protection in the absence of exclusion between two Brazilian isolates of Anaplasma marginale in experimentally infected calves.

This study investigated whether a low pathogenicity isolate of Anaplasma marginale with an appendage (UFMG1) could protect calves from infection with a pathogenic A. marginale isolate (UFMG2). Two groups of five Friesian calves were each inoculated with UFMG1 by intravenous injections of either A. marginale-infected tick cell cultures (group 1) or blood stabilates (group 2); a third (control) group was injected with saline. All animals were inoculated with a blood stabilate containing a high pathogenicity A. marginale isolate (UFMG2) 75 days after the UFMG1 inoculation. After infection with UFMG2, animals in groups 1 and 2 presented low rickettsaemia, but no clinical signs and no reduction in packed cell volume (PCV). Control animals became sick, with high rickettsaemia (16% infected erythrocytes) and a reduction in PCV (71%), resulting in 60% deaths. Up to 2 weeks after the UFMG2 inoculation, msp1α UFMG1 sequences were detected in groups 1 and 2. Four weeks after UFMG2 inoculation, UFMG2 sequences were detected in these animals, along with a new msp1α genotype sequence, closely related to that of the UFMG2 isolate. Control animals had UFMG2 msp1α sequences up to 4weeks after inoculation with UFMG2 and the new msp1α genotype sequence could be detected on the sixth week. The origin of the new A. marginale genotype was unknown, but may represent the first example of MSP1a antigenic variation in infected cattle. The results confirmed the low pathogenicity of the UFMG1 isolate, which provided clinical protection against the highly pathogenic A. marginale UFMG2. Infection with UFMG1 did not prevent the establishment of a second isolate, suggesting protection without infection-exclusion among A. marginale isolates.