Major surface protein 1a effects tick infection and transmission of Anaplasma marginale.

Anaplasma marginale, an ehrlichial pathogen of cattle and wild ruminants, is transmitted biologically by ticks. A developmental cycle of A. marginale occurs in a tick that begins in gut cells followed by infection of salivary glands, which are the site of transmission to cattle. Geographic isolates of A. marginale vary in their ability to be transmitted by ticks. In these experiments we studied transmission of two recent field isolates of A. marginale, an Oklahoma isolate from Wetumka, OK, and a Florida isolate from Okeechobee, FL, by two populations of Dermacentor variabilis males obtained from the same regions. The Florida and Oklahoma tick populations transmitted the Oklahoma isolate, while both tick populations failed to transmit the Florida isolate. Gut and salivary gland infections of A. marginale, as determined by quantitative PCR and microscopy, were detected in ticks exposed to the Oklahoma isolate, while these tissues were not infected in ticks exposed to the Florida isolate. An adhesion-recovery assay was used to study adhesion of the A. marginale major surface protein (MSP) 1a to gut cells from both tick populations and cultured tick cells. We demonstrated that recombinant Escherichia coli expressing Oklahoma MSP1a adhered to cultured and native D. variabilis gut cells, while recombinant E. coli expressing the Florida MSP1a were not adherent to either tick cell population. The MSP1a of the Florida isolate of A. marginale, therefore, was unable to mediate attachment to tick gut cells, thus inhibiting salivary gland infection and transmission to cattle. This is the first report of MSP1a being responsible for effecting infection and transmission of A. marginale by Dermacentor spp. ticks. The mechanism of tick infection and transmission of A. marginale is important in formulating control strategies and development of improved vaccines for anaplasmosis.     

Preliminary studies on the effect of Anaplasma marginale antibodies ingested by Dermacentor andersoni ticks (Acari:Ixodidae) with their blood meal on infections in salivary glands

The effect of Anaplasma marginale antibodies ingested with the tick blood meal was tested on infected male ticks that were allowed to feed on cattle immunized with the erythrocytic stage of A. marginale. The experiments were done in two trials. Trial 1 was done using splenectomized calves (two calves per treated and control groups) while ticks in trial 2 were fed on intact yearling cattle (four cattle per treated and control groups). The cattle were immunized with purified outer membrane proteins of erythrocyte-derived A. marginale using saponin (trial 1) or monophosphoryl lipid-A-trehalose dicorynomycolate adjuvant (trial 2). The corresponding control cattle received adjuvant only. All cattle were challenged using Dermacentor andersoni males infected as adults that were allowed to feed for 7 days. In trial 1, the ticks were allowed to feed a second time on susceptible calves to test whether exposure of ticks to immunized cattle affected their ability to transmit anaplasmosis. Infections in fed ticks were monitored by determining the infection rates in salivary glands with an A. marginale-specific RNA probe and light microscopy. Vaccine-derived antibodies ingested with the tick blood meal did not appear to affect the development of A. marginale in previously infected ticks. The infection rates in the salivary glands were not significantly different among ticks fed on immunized versus adjuvant control cattle. When the vaccine-exposed ticks in trial 1 were allowed to feed a second time on susceptible calves, the resulting clinical symptoms of anaplasmosis were similar to those of the controls. There was no statistically significant effect of tick exposure to the anti-erythrocytic stage antibody on the development of salivary gland infection or transmission of A. marginale by ticks.     

Adaptations of the Tick-Borne Pathogen, Anaplasma marginale, for Survival in Cattle and Ticks

The tick-borne cattle pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) multiplies within membrane-bound inclusions in host cell cytoplasm. Many geographic isolates of A. marginale occur that vary in genotype, antigenic composition, morphology and infectivity for ticks. A tick cell culture system for propagation of A. marginale proved to be a good model for study of tick-pathogen interactions. Six major surface proteins (MSPs) identified on A. marginale from bovine erythrocytes were conserved on A. marginale derived from tick cells. MSP1a and MSP1b were adhesins for bovine erythrocytes, while only MSP1a was found to be an adhesin for tick cells. The tandemly repeated portion of MSP1a was found to be necessary and sufficient for adhesion to both tick cells and bovine erythrocytes. Infectivity of A. marginale isolates for ticks was dependent on the adhesive capacity of the isolate MSP1a, which was found to involve both the adhesive properties and sequence of the repeated peptides. Cattle immunized with A. marginale derived from bovine erythrocytes or tick cells demonstrated a differential antibody response to MSP1a and MSP1b that resulted from the differential expression of these proteins in cattle and ticks cells. MSP2, derived from a multi-gene family, was found to undergo antigenic variation in cattle and ticks and may contribute to establishment of persistent A. marginale infections. MSP1a has been used as a stable genetic marker for geographic isolates because the molecular weight varies due to differing numbers of the tandem repeats. However, phylogenetic studies of A. marginale isolates from North America using MSP1a and MSP4 demonstrated that MSP4 was a good biogeographic marker, while MSP1a varied greatly among and within geographic areas. Infection and development of A. marginale in cattle and tick cells appears to differ and to be mediated by several surface proteins encoded from the small genome. This revised version was published online in July 2006 with corrections to the Cover Date.     

Silencing of genes involved in Anaplasma marginale-tick interactions affects the pathogen developmental cycle in Dermacentor variabilis

Background  

The cattle pathogen, Anaplasma marginale, undergoes a developmental cycle in ticks that begins in gut cells. Transmission to cattle occurs from salivary glands during a second tick feeding. At each site of development two forms of A. marginale (reticulated and dense) occur within a parasitophorous vacuole in the host cell cytoplasm. However, the role of tick genes in pathogen development is unknown. Four genes, found in previous studies to be differentially expressed in Dermacentor variabilis ticks in response to infection with A. marginale, were silenced by RNA interference (RNAi) to determine the effect of silencing on the A. marginale developmental cycle. These four genes encoded for putative glutathione S-transferase (GST), salivary selenoprotein M (SelM), H+ transporting lysosomal vacuolar proton pump (vATPase) and subolesin.     

Preliminary development of a polymerase chain reaction assay forAnaplasma marginale in ticks

Summary Polymerase chain reaction (PCR) was applied for detection ofAnaplasma marginale in tissues of maleDermacentor andersoni. Primer sequences were derived from the gene for the MSP1β surface protein ofA. marginale (Florida isolate). Optimum PCR conditions were used to detectA. marginale in individual bisected ticks and salivary glands; associated control tissues were negative.     

Knockdown of the Rhipicephalus microplus Cytochrome c Oxidase Subunit III Gene Is Associated with a Failure of Anaplasma marginale Transmission

Rhipicephalus microplus is an obligate hematophagous ectoparasite of cattle and an important biological vector of Anaplasma marginale in tropical and subtropical regions. The primary determinants for A. marginale transmission are infection of the tick gut, followed by infection of salivary glands. Transmission of A. marginale to cattle occurs via infected saliva delivered during tick feeding. Interference in colonization of either the tick gut or salivary glands can affect transmission of A. marginale to naïve animals. In this study, we used the tick embryonic cell line BME26 to identify genes that are modulated in response to A. marginale infection. Suppression-subtractive hybridization libraries (SSH) were constructed, and five up-regulated genes {glutathione S-transferase (GST), cytochrome c oxidase sub III (COXIII), dynein (DYN), synaptobrevin (SYN) and phosphatidylinositol-3,4,5-triphosphate 3-phosphatase (PHOS)} were selected as targets for functional in vivo genomic analysis. RNA interference (RNAi) was used to determine the effect of tick gene knockdown on A. marginale acquisition and transmission. Although RNAi consistently knocked down all individually examined tick genes in infected tick guts and salivary glands, only the group of ticks injected with dsCOXIII failed to transmit A. marginale to naïve calves. To our knowledge, this is the first report demonstrating that RNAi of a tick gene is associated with a failure of A. marginale transmission.     

Active surveillance to update county scale distribution of four tick species of medical and veterinary importance in Oklahoma

The incidence of tick‐borne disease continues to increase in humans and companion animals in the United States, yet distribution maps for several tick vectors in Oklahoma, including Dermacentor variabilis, Dermacentor albipictus, Ixodes scapularis, and Amblyomma maculatum, are not available or are outdated. To address this issue, county‐scale tick records from peer‐reviewed literature and passive collections were reviewed for Oklahoma. Additionally, dry ice traps, tick drags, and harvested deer were utilized to actively collect adult ticks throughout the state. Through these methods, D. variabilis, D. albipictus, I. scapularis, and A. maculatum were identified in 88% (68/77), 45.4% (35/77), 66.2% (51/77), and 64.9% (50/77) of the counties in Oklahoma, respectively. Baseline maps were developed for the distribution of D. variabilis and D. albipictus and distribution maps were updated for I. scapularis and A. maculatum. This data confirms that these four species of ticks continue to be widespread within Oklahoma with a western expansion of the range of I. scapularis within the state. These results assist efforts to better understand the epidemiology of the different diseases caused by pathogens transmitted by these tick species within the Great Plains region.     

Tick species establishment in Oklahoma County,Oklahoma, U.S.A., identified by seasonal sampling in residential and non‐residential sites

In recent years, human tick‐borne disease occurrence has risen in Oklahoma, U.S.A., but year‐round data on tick presence in frequently used recreational areas is not widely available. In this study, ticks were collected monthly for one year at residential and non‐residential sites in a suburban area of Oklahoma County, OK, U.S.A. At each trapping site, dry ice traps were used in both woodland and grassland areas and fabric tick drags were used in grassland areas. Four species were collected from each park: Amblyomma americanum, Amblyomma maculatum, Dermacentor variabilis, and Ixodes scapularis. Prior to this study, A. americanum was the only species with an established population in Oklahoma County. Consistent with this, A. americanum was collected in all months of the year and accounted for over 90% of ticks collected at each site. Based on our tick survey, we report that A. maculatum, D. variabilis, and I. scapularis, which were each collected in numbers greater than six within a single sampling occasion, are now each confirmed as established populations in Oklahoma County.     

Differential adhesion of major surface proteins 1a and 1b of the ehrlichial cattle pathogen Anaplasma marginale to bovine erythrocytes and tick cells

Anaplasma marginale is a tick-borne ehrlichial pathogen of cattle for which six major surface proteins (MSPs) have been described. The MSP1 complex, a heterodimer composed of MSP1a and MSP1b, was shown to induce a protective immune response in cattle and both proteins have been identified as putative adhesins for bovine erythrocytes. In this study the role of MSP1a and MSP1b as adhesins for bovine erythrocytes and tick cells was defined. msp1alpha and msp1beta1 genes from the Oklahoma isolate of A. marginale were cloned and expressed in Escherichia coli K-12 under the control of endogenous and tac promoters for both low and high level protein expression. Expression of the recombinant polypeptides was confirmed and localised on the surface of transformed E. coli. The adhesion properties of MSP1a and MSP1b were determined by allowing recombinant E. coli expressing these surface polypetides to react with bovine erythrocytes, Dermacentor variabilis gut cells and cultured tick cells derived from embryonic Ixodes scapularis. Adhesion of the recombinant E. coli to the three cell types was determined using recovery adhesion and microtiter haemagglutination assays, and by light and electron microscopy. MSP1a was shown by all methods tested to be an adhesin for bovine erythrocytes and both native and cultured tick cells. In contrast, recombinant E. coli expressing MSP1b adhered only to bovine erythrocytes and not to tick cells. When low expression vectors were used, single E. coli expressing MSP1a was seen adhered to individual tick cells while reaction of tick cells with the E. coli/MSP1a/high expression vector resulted in adhesion of multiple bacteria per cell. With electron microscopy, fusion of E. coli cell membranes expressing MSP1a or MSP1b with erythrocyte membranes was observed, as well as fusion of tick cell membranes with E. coli membranes expressing MSP1a. These studies demonstrated differential adhesion for MSP1a and MSP1b for which MSP1a is an A. marginale adhesin for both bovine erythrocytes and tick cells while MSP1b is an adhesin only for bovine erythrocytes. The role of the MSP1 complex, therefore, appears to vary among vertebrate and invertebrate hosts.     

Genomic evidence for host-associated differentiation in an animal parasite,Dermacentor variabilis,the American dog tick

The process responsible for the formation of genetically distinct populations associated with different host species is known as host-associated differentiation (HAD). Many insect parasites of plants have been shown to exhibit HAD but there have been fewer studies of HAD in parasites of vertebrate animals. Previous to this study, HAD has been documented in at least three species of ticks. The American dog tick, Dermacentor variabilis (Say) (Acari: Ixodidae) was chosen as the focal species for this study due to its importance as the vector of tularemia and Rocky Mountain spotted fever. Previous population genetic studies of this tick found the existence of various haplotypes but the tick’s host origins were unknown. In this study, ticks were collected from 15 vertebrate host species to test for HAD using single nuclear polymorphisms (SNPs). In total, 136 individual D. variabilis ticks were sequenced using ddRADseq. Genomic evidence was found to point to D. variabilis exhibiting HAD on eight different hosts. A STRUCTURE analysis showed that the highest posterior probability was obtained with a population size of eight and these populations correlated with host species. Pairwise F_ST values were as high as 0.622 and indicated a range of genetic distinction between host groups. In addition, ticks collected from the vegetation appeared as one homogenous distinct genotype suggesting the existence of nidicolous (nest dwelling) and non-nidicolous genotypes. The identification of host race formation occurring in this animal parasite has implications for the understanding of D. variabilis pathogen transmission and targeted control efforts because genetically distinct populations can differ in traits relevant to these applications.     

How Specific are Host-Produced Kairomones to Host-Seeking Ixodid Ticks?

Ixodid ticks respond to host-produced substances (kairomones) that influence the ticks’ host-finding behavior. In the laboratory adult blacklegged ticks, Ixodes scapularis Say, lone star ticks, Amblyomma americanum L., and American dog ticks, Dermacentor variabilis (Say) became akinetic on residues rubbed from their principal hosts (deer for the former two species and dogs for the latter). However, arrestment also occurred when adults of these species were tested using the same method bioassay, but with host substances reversed (i.e., I. scapularis and A. americanum against canine substances, and D. variabilis against deer gland substances). Although adult D. variabilis exhibited arrestant responses to deer substances and are often found along trails used by deer, they apparently make little use of deer as hosts. It is unclear whether responding to deer-produced kairomones may have disadvantages for D. variabilis. Until the active components of host-produced arrestment kairomones are isolated, identified and evaluated in behavioral tests, this host-finding strategy remains only partially understood. This revised version was published online in July 2006 with corrections to the Cover Date.     

Prevalence of tick-borne pathogens in ixodid ticks (Acari: Ixodidae) collected from European wild boar (<Emphasis Type="Italic">Sus scrofa</Emphasis>) and Iberian red deer (<Emphasis Type="Italic">Cervus elaphus hispanicus</Emphasis>) in central Spain

Emerging tick-borne diseases of humans and animals have occurred frequently during the past 30 years. These disease outbreaks appear to result from changes in the distribution of tick and vertebrate hosts, and the introduction of humans and domestic animals into tick–pathogen–wildlife cycles. Use of molecular technologies now available for identification of pathogens in ticks can provide valuable information that allows for risk analysis of emerging tick-borne diseases. In this study, the prevalence of selected pathogens in ticks collected in six locations in central Spain from the major wild ungulate species, European wild boar (Sus scrofa) and Iberian red deer (Cervus elaphus hispanicus), was determined by PCR. Tick species collected included Ixodes ricinus, Dermacentor marginatus, Rhipicephalus bursa and Hyalomma m. marginatum. Pathogens identified in ticks included piroplasmids, Anaplasma spp., Ehrlichia spp. and Rickettsia spp. Piroplasmids were identified in all tick species except I. ricinus. Ehrlichia spp. were detected in all tick species and collection locations, while Rickettsia spp., which proved to be R. slovaca and a recently identified Rickettsia sp. DnS28, were identified only in D. marginatus. A. marginale and A. phagocytophilum were detected in D. marginatus, R. bursa and Hy. m. marginatum. Concurrent infections of these pathogens were frequently observed in ticks. Notably, A. phagocytophilum, which is infective for a broad host range that includes humans and domestic and wild animals, was identified in ticks from all collection locations. The variety of ticks and tick-borne pathogens demonstrated in this study suggests a risk in central Spain for the emergence of tick-borne diseases in humans and domestic animals.     

Functional and Immunological Relevance of Anaplasma marginale Major Surface Protein 1a Sequence and Structural Analysis

Bovine anaplasmosis is caused by cattle infection with the tick-borne bacterium, Anaplasma marginale. The major surface protein 1a (MSP1a) has been used as a genetic marker for identifying A. marginale strains based on N-terminal tandem repeats and a 5′-UTR microsatellite located in the msp1a gene. The MSP1a tandem repeats contain immune relevant elements and functional domains that bind to bovine erythrocytes and tick cells, thus providing information about the evolution of host-pathogen and vector-pathogen interactions. Here we propose one nomenclature for A. marginale strain classification based on MSP1a. All tandem repeats among A. marginale strains were classified and the amino acid variability/frequency in each position was determined. The sequence variation at immunodominant B cell epitopes was determined and the secondary (2D) structure of the tandem repeats was modeled. A total of 224 different strains of A. marginale were classified, showing 11 genotypes based on the 5′-UTR microsatellite and 193 different tandem repeats with high amino acid variability per position. Our results showed phylogenetic correlation between MSP1a sequence, secondary structure, B-cell epitope composition and tick transmissibility of A. marginale strains. The analysis of MSP1a sequences provides relevant information about the biology of A. marginale to design vaccines with a cross-protective capacity based on MSP1a B-cell epitopes.     

Functional genomic studies of tick cells in response to infection with the cattle pathogen, Anaplasma marginale

The coevolution of ticks and the pathogens that they transmit has ensured their mutual survival. In these studies, we used a functional genomics approach to characterize tick genes regulated in response to Anaplasma marginale infection. Differentially regulated genes/proteins were identified by suppression-subtractive hybridization and differential in-gel electrophoresis analyses of cultured IDE8 tick cells infected with A. marginale. Nine of 17 of these genes were confirmed by real-time RT-PCR to be differentially regulated in ticks and/or IDE8 tick cells in response to A. marginale infection. RNA interference was used for functional studies. Six genes, which encode putative selenoprotein W2a, hematopoietic stem/progenitor cells protein-like, proteasome 26S subunit, ferritin, GST, and subolesin control, were found to affect A. marginale infection in IDE8 tick cells. Four genes, which encode putative GST, salivary selenoprotein M, vATPase, and ubiquitin, affected A. marginale infection in different sites of development in ticks. The results of these studies demonstrated that a molecular mechanism occurs by which tick cell gene expression mediates the A. marginale developmental cycle and trafficking through ticks.     

Immunity against Boophilus annulatus induced by the Bm86 (Tick-GARD) vaccine

Friesian cattle were immunized with two inoculations of anti-tick Bm86 (Tick-GARD) vaccine and were challenged 30 or 90 d later with Boophilus annulatus larvae derived from 1.2 g of eggs. No nymphs or adult ticks were found on the immunized cattle during four weeks after challenge. Repeated infestations (2 to 4) with larvae on three other calves during a period of 160 and 390 d after the immunization did not result in development of nymphal and adult stages. In control, non-immunized cattle infested with corresponding batches of larvae 1380 to 4653 replete adult female ticks were collected. Larvae issued from Babesia bovis-infected female ticks transmitted the infection to Bm86-immunized cattle, but the progeny of B. bigemina-infected females did not. Since B. bigemina is transmitted exclusively by nymphal stages of Bo. annulatus these results support the observation that immunity induced by Bm86 affects the larval stage of this tick.     

Genetic variation in the mitochondrial 16S rRNA gene of the American dog tick,Dermacentor variabilis (Acari: Ixodidae)

Genetic variation in the mitochondrial (mt) 16S ribosomal RNA (rRNA) gene was examined for the American dog tick, Dermacentor variabilis (Say, 1821). Nine different haplotypes were detected among 369 adult D. variabilis collected from four localities in Canada. There were eight variable nucleotide positions in the 404 bp sequence alignment. Individuals of haplotype 1 occurred at frequency of >75% at all localities. Five haplotypes were detected at only one of the four localities. High haplotype diversity and low nucleotide diversity, combined with significantly negative F_s values for ticks at three localities, suggest a recent population expansion. Genetic differences were found between populations at different localities, but a Mantel regression analysis revealed no association between genetic differences and geographical distances. There was also no association between tick haplotype and the prevalence of the bacterium, Rickettsia montanensis Weiss and Moulder, 1984, in D. variabilis among localities or on opposite sides of Blackstrap Lake (Saskatchewan). The 16S rDNA haplotypes from Canadian populations of D. variabilis formed a clade with those from the eastern and central U.S.A., to the exclusion of D. variabilis from geographically isolated populations in the western U.S.A. Although sample sizes for D. variabilis in the eastern U.S.A. are small, there may be genetic divergence between populations in Canada and those in the eastern U.S.A., which may have implications for studies on the pathogenic agents transmitted by D. variabilis to its hosts.     

Interstadial and Infestation Level-dependent Variation in the Transmission Efficiency of Borrelia Burgdorferi from Mice to Ixodes Ricinus Ticks

The efficiency with which the spirochaete Borrelia burgdorferi sensu stricto was transmitted from laboratory mice to larval and nymphal Ixodes ricinus ticks was assessed, using the polymerase chain reaction. The transmission efficiency to nymphs was significantly greater than to larvae when both fed together on the same host. Increased tick infestation levels of mice were correlated with significantly greater engorgement weights and higher B. burgdorferi transmission coefficients from mice to nymphs. These observations indicate that both the feeding success of ticks and the transmission coefficients from host to tick may be influenced by the tick infestation level of an infected host. The infestation level and the relative numbers of each life stage of the tick are factors which should be considered in the design of transmission experiments.     

Stability and tick transmission phenotype of gfp-transformed Anaplasma marginale through a complete in vivo infection cycle

We tested the stability and tick transmission phenotype of transformed Anaplasma marginale through a complete in vivo infection cycle. Similar to the wild type, the gfp-transformed A. marginale strain established infection in cattle, a natural reservoir host, and persisted in immune competent animals. The tick infection rates for the transformed A. marginale and the wild type were the same. However, there were significantly lower levels of the transformed A. marginale than of the wild type in the tick. Despite the lower levels of replication, ticks transmitted the transformant. Transformants can serve as valuable tools to dissect the molecular requirements of tick colonization and pathogen transmission.