A hybrid protein containing MSP1a repeats and Omp7, Omp8 and Omp9 epitopes protect immunized BALB/c mice against anaplasmosis.

Anaplasma marginale (A. marginale) has a remarkable impact on livestock production, and an effective vaccine is not currently available due to the inexistence of a small animal model. Recently, BALB/c mice were successfully infected with A. marginale, resulting in an acute and persistent anaplasmosis infection. Here, we designed a hybrid protein containing repeats of polypeptide 1a from major surface protein-1 complex (MSP1a) repeats and common epitopes of outer membrane proteins (OMPs) OMP7, OMP8 and OMP9 expressed in Escherichia coli. Our proof-of-concept assessed vaccinal effectiveness against a challenge with live bacteria. The MSP1a/OMP7/8/9 immunized BALB/C mice exhibited a strong reduction in rickettsemia and had no signs of anaplasmosis or hepatic lesions. In contrast, the non-immunized mice exhibited signs of anaplasmosis and a body weight loss associated with increases in monocyte and neutrophil counts. Furthermore, the non-immunized mice displayed atrophies with chronic inflammatory infiltrates in the spleen and increased binucleation and hydropic degeneration in the hepatocytes. Our findings demonstrated that immunization with our hybrid protein induced a strong reduction in rickettsemia and conferred protection against anaplasmosis. Therefore, given the strong evidence of the protective effect against anaplasmosis, hybrid protein designs are potential candidates for the rational design of vaccinal subunits.

Differential expression analysis for subolesin in Rhipicephalus microplus infected with Anaplasma marginale.

Rhipicephalus microplus (formerly Boophilus microplus) ticks are potential vectors of several pathogens of livestock especially in tropical and subtropical regions where may have substantial effects on economic development. Among tick-borne pathogens, Anaplasma marginale is considered one of the most important in domestic and wild ruminants worldwide. Different molecular mechanisms have been employed by both ticks and these intracellular pathogens, in order to be able to adapt and survive. Subolesin, originally called 4D8, is an evolutionarily well-preserved protein among ixodid tick species. This new antigen was found to be protective against tick infestations when used as a vaccine, as it has an essential role in tick blood digestion, development and infection of host cells by A. marginale. Recent studies have demonstrated that infection of both tick and vertebrate host cells with this microorganism changed gene expression. Therefore, the main objective of this study was to investigate subolesin expression in uninfected and A. marginale-infected R. microplus salivary glands by real-time reverse transcriptase (RT)-PCR. To analyze the differential expression of the recombinant protein subolesin, the gene was previously expressed from ticks infected with A. marginale. Results from this study revealed that, the expression of subolesin was significantly higher in salivary glands of infected R. microplus in comparison to uninfected ones.

Anaplasma marginale Outer Membrane Protein A Is an Adhesin That Recognizes Sialylated and Fucosylated Glycans and Functionally Depends on an Essential Binding Domain.

Anaplasma marginale causes bovine anaplasmosis, a debilitating and potentially fatal tick-borne infection of cattle. Because A. marginale is an obligate intracellular organism, its adhesins that mediate entry into host cells are essential for survival. Here, we demonstrate that A. marginale outer membrane protein A (AmOmpA; AM854) contributes to the invasion of mammalian and tick host cells. AmOmpA exhibits predicted structural homology to OmpA of A. phagocytophilum (ApOmpA), an adhesin that uses key lysine and glycine residues to interact with α2,3-sialylated and α1,3-fucosylated glycan receptors, including 6-sulfo-sialyl Lewis x (6-sulfo-sLex). Antisera against AmOmpA or its predicted binding domain inhibits A. marginale infection of host cells. Residues G55 and K58 are contributory, and K59 is essential for recombinant AmOmpA to bind to host cells. Enzymatic removal of α2,3-sialic acid and α1,3-fucose residues from host cell surfaces makes them less supportive of AmOmpA binding. AmOmpA is both an adhesin and an invasin, as coating inert beads with it confers adhesiveness and invasiveness. Recombinant forms of AmOmpA and ApOmpA competitively antagonize A. marginale infection of host cells, but a monoclonal antibody against 6-sulfo-sLex fails to inhibit AmOmpA adhesion and A. marginale infection. Thus, the two OmpA proteins bind related but structurally distinct receptors. This study provides a detailed understanding of AmOmpA function, identifies its essential residues that can be targeted by blocking antibody to reduce infection, and determines that it binds to one or more α2,3-sialylated and α1,3-fucosylated glycan receptors that are unique from those targeted by ApOmpA.

Anaplasma marginale Actively Modulates Vacuolar Maturation during Intracellular Infection of Its Tick Vector, Dermacentor andersoni.

UNLABELLED: Tick-borne transmission of bacterial pathogens in the order Rickettsiales is responsible for diverse infectious diseases, many of them severe, in humans and animals. Transmission dynamics differ among these pathogens and are reflected in the pathogen-vector interaction. Anaplasma marginale has been shown to establish and maintain infectivity within Dermacentor spp. for weeks to months while escaping the complex network of vacuolar peptidases that are responsible for digestion of the tick blood meal. How this prolonged maintenance of infectivity in a potentially hostile environment is achieved has been unknown. Using the natural vector Dermacentor andersoni, we demonstrated that A. marginale-infected tick vacuoles (AmVs) concurrently recruit markers of the early endosome (Rab5), recycling endosome (Rab4 and Rab11), and late endosome (Rab7), are maintained near neutral pH, do not fuse with lysosomes, exclude the protease cathepsin L, and engage the endoplasmic reticulum and Golgi apparatus for up to 21 days postinfection. Maintenance of this safe vacuolar niche requires active A. marginale protein synthesis; in its absence, the AmVs mature into acidic, protease-active phagolysosomes. Identification of this bacterially directed modeling of the tick midgut endosome provides a mechanistic basis for examination of the differences in transmission efficiency observed among A. marginale strains and among vector populations. IMPORTANCE: Ticks transmit a variety of intracellular bacterial pathogens that cause significant diseases in humans and animals. For successful transmission, these bacterial pathogens must first gain entry into the tick midgut digestive cells, avoid digestion, and establish a replicative niche without harming the tick vector. Little is known about how this replicative niche is established and maintained. Using the ruminant pathogen A. marginale and its natural tick vector, D. andersoni, this study characterized the features of the A. marginale niche in the tick midgut and demonstrates that A. marginale protein synthesis is required for the maintenance of this niche. This work opens a new line of inquiry about the pathogen effectors and their targets within the tick that mediate tick-pathogen interactions and ultimately serve as the determinants of pathogen success.

Molecular diagnosis of the tick-borne pathogen Anaplasma marginale in cattle blood samples from Nigeria using qPCR.

Tick-borne diseases (TBDs) are some of the most important animal health and management problems in Africa, including Nigeria. This study aims to determine the prevalence of an important TBD, anaplasmosis, in a North-central region of Nigeria. Blood samples were collected from cattle and stored on Whatman FTA® cards. Information on village, age and sex associated with each cattle was also recorded. The packed red blood cell volume (PCV) for each blood sample was determined. After DNA extraction, pathogen presence was evaluated by TaqMan® based qPCR of which 75.9 % of the cattle tested positive for Anaplasma marginale. Statistical analysis revealed that the presence of A. marginale infection differed significantly between cattle age groups. However, there was no significant difference in the prevalence of this pathogen between the sexes or among cattle grouped by PCV level. Finally, using a highly sensitive molecular method our pioneer study contributes to the improvement of the current knowledge regarding tick-borne pathogens that seriously affect animal health in specific areas of Nigeria.

Correlations among Anaplasma marginale parasitemia and markers of oxidative stress in crossbred calves.

The present study was designed to determine the correlations among Anaplasma marginale parasitemia and markers of oxidative stress in crossbred calves. Blood was collected from 11 crossbred calves infected with A. marginale along with 11 healthy crossbred calves as controls for determination of hematology and oxidative stress indicators. Percentage of parasitemia in infected calves varied from 0.8% to 6.0%. The values of hematological indicators and antioxidant enzymes were decreased, whereas erythrocytic lipid peroxidation (LPO) and plasma nitrate (NO) level were significantly (p < 0.05) augmented in A. marginale-infected animals over healthy group. Parasitemia was positively correlated (p < 0.01) with erythrocytic LPO and plasma NO and negatively correlated (p < 0.01) with hematological indicators and antioxidant enzymes. In addition, erythrocytic LPO was negatively correlated (p < 0.01) with the hemoglobin, erythrocyte count, and packed cell volume. From the present study, it can be concluded that anaplasmosis in crossbred calves is associated with a parasitic load-dependent oxidative damage as indicated by poor antioxidant status and enhanced oxidative stress, which are contributed to severe anemia.

Identification of Anaplasma marginale proteins specifically upregulated during colonization of the tick vector.

The transition between infection of the mammalian host and colonization of an arthropod vector is required for the ongoing transmission of a broad array of pathogens, from viruses to protozoa. Understanding how this transition is mediated provides opportunities to disrupt transmission through either chemotherapy or immunization. We used an unbiased proteomic screen to identify Anaplasma marginale proteins specifically upregulated in the tick compared to the mammalian host. Comparative mass spectrometric analysis of proteins separated by two-dimensional gel electrophoresis of uninfected and infected ISE6 cells and infected mammalian cells identified 15 proteins exclusively expressed or upregulated in tick cells. All 15 had originally been annotated as hypothetical proteins. We confirmed quantitative upregulation and expression in situ within the midgut epithelial and salivary gland acinar cells of vector ticks during successful transmission. The results support the hypothesis that A. marginale gene expression is regulated by the specific host environment and, in a broader context, that the core genome evolved in the arthropod vector with differential regulation, allowing adaptation to mammalian hosts. Furthermore, the confirmation of the in situ expression of candidates identified in ISE6 cell lines indicates that this approach may be widely applicable to bacteria in the genera Anaplasma and Ehrlichia, removing a major technical impediment to the identification of new targets for vaccine and chemotherapeutic blocking of transmission.

Differential expression of genes in salivary glands of male Rhipicephalus (Boophilus)microplus in response to infection with Anaplasma marginale.

BACKGROUND: Bovine anaplasmosis, caused by the rickettsial tick-borne pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae), is vectored by Rhipicephalus (Boophilus)microplus in many tropical and subtropical regions of the world. A. marginale undergoes a complex developmental cycle in ticks which results in infection of salivary glands from where the pathogen is transmitted to cattle. In previous studies, we reported modification of gene expression in Dermacentor variabilis and cultured Ixodes scapularis tick cells in response to infection with A. marginale. In these studies, we extended these findings by use of a functional genomics approach to identify genes differentially expressed in R. microplus male salivary glands in response to A. marginale infection. Additionally, a R. microplus-derived cell line, BME26, was used for the first time to also study tick cell gene expression in response to A. marginale infection. RESULTS: Suppression subtractive hybridization libraries were constructed from infected and uninfected ticks and used to identify genes differentially expressed in male R. microplus salivary glands infected with A. marginale. A total of 279 ESTs were identified as candidate differentially expressed genes. Of these, five genes encoding for putative histamine-binding protein (22Hbp), von Willebrand factor (94Will), flagelliform silk protein (100Silk), Kunitz-like protease inhibitor precursor (108Kunz) and proline-rich protein BstNI subfamily 3 precursor (7BstNI3) were confirmed by real-time RT-PCR to be down-regulated in tick salivary glands infected with A. marginale. The impact of selected tick genes on A. marginale infections in tick salivary glands and BME26 cells was characterized by RNA interference. Silencing of the gene encoding for putative flagelliform silk protein (100Silk) resulted in reduced A. marginale infection in both tick salivary glands and cultured BME26 cells, while silencing of the gene encoding for subolesin (4D8) significantly reduced infection only in cultured BME26 cells. The knockdown of the gene encoding for putative metallothionein (93 Meth), significantly up-regulated in infected cultured BME26 cells, resulted in higher A. marginale infection levels in tick cells. CONCLUSIONS: Characterization of differential gene expression in salivary glands of R. microplus in response to A. marginale infection expands our understanding of the molecular mechanisms at the tick-pathogen interface. Functional studies suggested that differentially expressed genes encoding for subolesin, putative von Willebrand factor and flagelliform silk protein could play a role in A. marginale infection and multiplication in ticks. These tick genes found to be functionally relevant for tick-pathogen interactions will likely be candidates for development of vaccines designed for control of both ticks and tick-borne pathogens.

Epidemiology and genetic diversity of Anaplasma marginale in Zamora-Chinchipe, Ecuador.

Bovine anaplasmosis, caused by the tick-borne pathogen Anaplasma marginale, is a hemolytic disease that constitutes a major constraint to cattle production in tropical and subtropical regions including Ecuador. However, the epidemiological situation of this hemoparasitosis in Ecuador is poorly characterized. The present study was aimed to determine the prevalence and genetic diversity of A. marginale in cattle of Ecuador. A cross-sectional study was carried out covering several farms from six out nine cantons of the Zamora-Chinchipe province. A total of 185 cattle were randomly selected and blood samples were collected from the animals. The studied group of animals included six breeds, three age groups, and both sexes. The molecular diagnostic was performed based on a nPCR assay targeting the A. marginale msp5 gene. Anaplasma marginale prevalence was 63.8 % and the bacteria were detected in all the cantons studied. Thirteen representative strains were selected and genetically characterized based on the msp1α gene. Genetic diversity analysis revealed that different strains circulate in the bovine herds studied. The results suggest that cattle movement may contribute to the circulation of common strains in the area. The results demonstrate a high prevalence of A. marginale in the region which should be considered by the sanitary authorities. The epidemiological surveillance for this disease should increase to anticipate acute disease outbreaks with high mortality. Bovine anaplasmosis outbreaks can cause economic losses and the death of several animals; therefore, measures for the prevention and control of this disease are required.

Quantitative analysis of Anaplasma marginale acquisition and transmission by Dermacentor andersoni fed in vitro.

In this study, we describe a new in vitro tick feeding system that facilitates the study of ticks and tick-borne pathogens. To optimize the system, we used Dermacentor andersoni and Anaplasma marginale as a tick-pathogen interaction model. Ticks were fed on bovine blood containing 10-fold dilutions of the pathogen to determine the effect of dose on tick infection rate. After feeding on infected blood, ticks were transferred to uninfected blood to stimulate bacterial replication within the tick vector. During stimulation feeding, blood samples were collected daily to determine if infected ticks secreted viable A. marginale. The results demonstrated similar attachment rates between the first and second tick feeding. Tick midgut and salivary glands were infected with A. marginale. However, salivary gland infection rates decreased as the percentage of parasitized erythrocytes decreased during tick acquisition feeding. Bacteria recovered from the in vitro system were able to infect a naïve bovine host. Using the highly transmissible A. marginale St. Maries strain, we demonstrated that the artificial tick feeding system is a suitable tool to study tick-pathogen interactions and that A. marginale tick salivary gland infection is dose dependent. This work demonstrates the utility of an artificial tick feeding system to directly study the association between the number of acquired pathogens and transmissibility by ticks.

Epidemiology and genotyping of Anaplasma marginale and co-infection with piroplasms and other Anaplasmataceae in cattle and buffaloes from Egypt.

BACKGROUND: Anaplasma marginale is an obligate intracellular bacterium and the main cause of bovine anaplasmosis in tropical and subtropical regions. In Egypt, data regarding the prevalence of A. marginale in ruminant hosts and of the circulating genotypes is lacking. This study therefore aimed to (i) investigate the presence, epidemiology and genotypes of A. marginale in cattle and buffaloes in Egypt, (ii) to evaluate suitable diagnostic tools and (iii) to identify co-infections of A. marginale with other selected tick-borne pathogens. METHODS: Blood samples were collected from 394 animals (309 cattle and 85 buffaloes) from three different areas in Egypt. For the detection of A. marginale infection, several tests were compared for their sensitivity and specificity: blood smear analysis, enzyme-linked immunosorbent assay (ELISA), PCR, real-time PCR and reverse line blot (RLB) assay. Co-infections with A. marginale, piroplasms and other Anaplasmataceae were surveyed by RLB while A. marginale genotypes were identified by amplifying and sequencing the partial msp1α gene. RESULTS: Anaplasma marginale DNA was amplified by qPCR in 68.3% of cattle and 29.4% of buffaloes. RLB showed infection with A. marginale in 50.2% of cattle and 42.5% of buffaloes. Blood smear analysis detected this agent in 16.2% of cattle and 2.4% of buffaloes. ELISA showed specific antibodies against A. marginale in 54.9% of cattle. Anaplasma marginale was associated, in cattle and buffaloes, with several tick-borne pathogens (Theileria annulata, Babesia bovis, Babesia bigemina, Babesia occultans and Anaplasma platys). A significant difference of A. marginale infection level was noticed in cattle, where animals between 3-5-years-old had a higher prevalence (79.2%) compared to those older than 5 years (36.4%) and younger than 3 years (59.7%) and one year (64.5%), respectively (P = 0.002281). Microsatellite analysis identified 15 different genotypes. CONCLUSIONS: The epidemiological findings revealed high prevalence of A. marginale in cattle and buffaloes in all the investigated areas. The circulation of diverse genotypes was observed, most of these A. marginale genotypes being specific for Egypt. The qPCR assay was confirmed to be the most sensitive tool for detection of A. marginale in cattle and buffaloes even in the carrier state, highlighting the importance of using suitable diagnostic tests.

The co-infection with Ehrlichia minasensis, Anaplasma marginale and Anaplasma platys is not associated with anemia in beef cattle in the Brazilian Pantanal.

The Anaplasmataceae family is composed of obligatory intracellular Gram-negative bacteria transmitted by arthropod vectors. In Brazil, with the exception of Anaplasma marginale, little is known about the occurrence of other Anaplasma and Ehrlichia species infecting cattle. The present study aimed at investigating the occurrence of Anaplasma spp. and Ehrlichia spp. in beef cattle (Bos indicus) sampled in the Brazilian Pantanal, an area prone to periodic flooding and endemic for bovine trypanosomiasis. Blood samples from 400 cattle were collected and screened by PCR assays based on rrs and dsb genes from Anaplasma spp. and Ehrlichia spp., respectively. Positive samples for Anaplasma spp. were subjected to qPCR assays based on the msp-2 gene and nPCR based on the groEL gene. As a result, 4.75% (19/400) and 48.12% (167/347) were positive for Anaplasma platys and Ehrlichia minasensis, respectively. Besides, positivity of 56.75% (227/400) for A. marginale and seropositivity of 90.75% (363/400) for Trypanosoma vivax were found. A high rate of co-infection was observed (67.25%), from which the co-infection by A. marginale and E. minasensis was more frequently found in calves than cows. Interestingly, none of the animals presenting co-infection showed anemia or other clinical signs. The present study showed, for the first time, the occurrence of A. platys and E. minasensis in beef cattle in the southern Pantanal, as well as a high rate of co-infection by A. marginale, E. minasensis and T. vivax in the sampled animals.

Independence of Anaplasma marginale strains with high and low transmission efficiencies in the tick vector following simultaneous acquisition by feeding on a superinfected mammalian reservoir host.

Strain superinfection occurs when a second pathogen strain infects a host already carrying a primary strain. Anaplasma marginale superinfection occurs when the second strain carries a variant repertoire different from that of the primary strain, and the epidemiologic consequences depend on the relative efficiencies of tick-borne transmission of the two strains. Following strain superinfection in the reservoir host, we tested whether the presence of two A. marginale (sensu lato) strains that differed in transmission efficiency altered the transmission phenotypes in comparison to those for single-strain infections. Dermacentor andersoni ticks were fed on animals superinfected with the Anaplasma marginale subsp. centrale vaccine strain (low transmission efficiency) and the A. marginale St. Maries strain (high transmission efficiency). Within ticks that acquired both strains, the St. Maries strain had a competitive advantage and replicated to significantly higher levels than the vaccine strain. The St. Maries strain was subsequently transmitted to naïve hosts by ticks previously fed either on superinfected animals or on animals singly infected with the St. Maries strain, consistent with the predicted transmission phenotype of this strain and the lack of interference due to the presence of a competing low-efficiency strain. The vaccine strain was not transmitted by either singly infected or coinfected ticks, consistent with the predicted transmission phenotype and the lack of enhancement due to the presence of a high-efficiency strain. These results support the idea that the strain predominance in regions of endemicity is mediated by the intrinsic transmission efficiency of specific strains regardless of occurrence of superinfection.

Propagation of a Brazilian isolate of Anaplasma marginale with appendage in a tick cell line (BME26) derived from Rhipicephalus (Boophilus) microplus.

Anaplasma marginale is a tick-borne pathogen of cattle responsible for the disease anaplasmosis. Data suggest that Rhipicephalus (Boophilus) microplus and R. annulatus may be the major tick vectors of A. marginale in tropical and subtropical regions of the world. In this work we demonstrated the first infection and propagation of a Brazilian isolate of A. marginale (UFMG1) in the BME26 cell line derived originally from embryos of R. (Boophilus) microplus. The establishment of A. marginale infection in a cell line derived from R. (Boophilus) microplus is relevant for studying the A. marginale/tick interface.

Comparison of the efficiency of biological transmission of Anaplasma marginale (Rickettsiales: Anaplasmataceae) by Dermacentor andersoni Stiles (Acari: Ixodidae) with mechanical transmission by the horse fly, Tabanus fuscicostatus Hine (Diptera: Muscidae).

Mechanical transmission ofAnaplasma marginale by horse flies (Tabanidae) is thought to be epidemiologically significant in some areas of the United States. We compared the relative efficiencies of mechanical transmission of Anaplasma marginale by the horse fly, Tabanus fuscicostatus Hine, during acute infection (approximately 10(7) to approximately 10(9) infected erythrocytes [IE]/ml blood) with biological transmission by Dermacentor andersoni Stiles in the persistent phase of infection (approximately 10(2.5) to approximately 10(6) IE/ml). Transmission of A. marginale was not observed when horse flies were partially fed on an acutely infected donor calf and immediately transferred to susceptible calves to complete their blood meal. Ticks that were acquisition fed on the same donor host after it reached the persistent phase of infection successfully transmitted A. marginale when transferred to the same recipient calves that failed to acquire infection after fly feeding. Failure of fly-borne mechanical transmission at a rickettsemia >240-fold higher than that from which ticks transmitted with 100% efficiency shows that tick-borne biological transmission is at least two orders of magnitude more efficient than mechanical transmission by horse flies.

Evaluation of sequential coinfection with Anaplasma phagocytophilum and Anaplasma marginale in cattle.

OBJECTIVE: To determine whether sequelae of infection differed among single versus double infection with Anaplasma phagocytophilum or Anaplasma marginale, with and without tick salivary extract, in cattle. ANIMALS: Eighteen 13-month old steers. PROCEDURES: Treatment groups of 3 cattle each included A marginale inoculated ID followed on day 35 by A phagocytophilum without tick saliva, A phagocytophilum followed on day 10 by A marginale without tick saliva, A marginale followed on day 35 by A phagocytophilum with tick saliva, A phagocytophilum followed on day 10 by A marginale with tick saliva, tissue culture control injection, and tick saliva control injection. Infection was monitored via clinical observations, CBC, serologic testing, and PCR analysis of blood and tissues. RESULTS: Infected cattle had significantly reduced weight gain. Anemia occurred 25 to 32 days after A marginale infection, which was attenuated by tick saliva. Parasitism was greater if cattle had not previously been inoculated with A phagocytophilum. Nine of the 12 treated cattle had positive results of PCR analysis for A phagocytophilum from at least 1 blood sample. Five tissue samples had positive results of PCR analysis for A phagocytophilum; PCR results for A marginale were positive in spleen, lung, lymph node, heart, and ear skin of infected cattle. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated an important biological interaction between A marginale and A phagocytophilum infection as well as with tick saliva in disease kinetics and severity in cattle, which may be important for interpretation of diagnostic tests and management of disease in areas where both pathogens occur.

Silencing expression of the defensin, varisin, in male Dermacentor variabilis by RNA interference results in reduced Anaplasma marginale infections.

Antimicrobial peptides, including defensins, are components of the innate immune system in ticks that have been shown to provide protection against both gram-negative and gram-positive bacteria. Varisin, one of the defensins identified in Dermacentor variabilis, was shown to be produced primarily in hemocytes but transcript levels were also expressed in midguts and other tick cells. In this research, we studied the role of varisin in the immunity of ticks to the gram-negative cattle pathogen, Anaplasma marginale. Expression of the varisin gene was silenced by RNA interference (RNAi) in which male ticks were injected with varisin dsRNA and then allowed to feed and acquire A. marginale infection on an experimentally-infected calf. Silencing expression of varisin in hemocytes, midguts and salivary glands was confirmed by real time RT-PCR. We expected that silencing of varisin would increase A. marginale infections in ticks, but the results demonstrated that bacterial numbers, as determined by an A. marginale msp4 quantitative PCR, were significantly reduced in the varisin-silenced ticks. Furthermore, colonies of A. marginale in ticks used for RNAi were morphologically abnormal from those seen in elution buffer injected control ticks. The colony shape was irregular and in some cases the A. marginale appeared to be free in the cytoplasm of midgut cells. Some ticks were found to be systemically infected with a microbe that may have been related to the silencing of varisin. This appears to be the first report of the silencing of expression of a defensin in ticks by RNAi that resulted in reduced A. marginale infections.

Epidemiology and risk factors associated with Anaplasma marginale infection of cattle in Peninsular Malaysia.

Bovine anaplasmosis is a major concern to cattle farming in most parts of the world. Anaplasmosis negatively impacts the profitability of cattle farming by reducing the production, reproduction, and draft ability of cattle. Here, we report results from a one-year cross sectional study to determine the epidemiology and the risk factors for Anaplasma marginale infection of cattle in Peninsular Malaysia. Examination of one thousand and forty five blood samples of apparently healthy cattle from forty-three farms in all the states of Peninsular Malaysia by polymerase chain reaction (PCR) assay revealed an overall prevalence of A. marginale infection of cattle of 72.6%, showing high endemicity of this heamoprotozoan among cattle in the country. Cattle breeds, production type, herd owner, herd size, management system, farm size, farm age, prophylactic treatment against blood parasites, presence of ticks, frequency of deticking, zones, closeness to forest, closeness to waste area, closeness to human settlement and closeness to body of water were the risk factors significantly associated (P < 0.05) with the detection of A. marginale in cattle. Results of this first molecular study on the epidemiology and risk factors for A. marginale infection of cattle from all the states of Peninsular Malaysia suggest policies and strategies for the prevention and control of the parasite to improve profitability of cattle farming in the country.

Molecular epidemiology of Babesia species, Theileria parva, and Anaplasma marginale infecting cattle and the tick control malpractices in Central and Eastern Uganda.

East Coast fever, babesiosis, and anaplasmosis are the major tick-borne diseases affecting cattle productivity in Uganda. The emergence of acaricide-resistant ticks is suspected to have caused a rise in hemoparasites. This study sought to detect and characterize hemoparasites among farms in acaricide-failure hotspots of central as compared to the acaricide-failure naïve areas in Eastern Uganda. Nested PCR assays were performed to determine the prevalences of Babesia bovis, Babesia bigemina, Theileria parva, and Anaplasma marginale in cattle blood samples sourced from randomly selected farms. Randomly selected isolates were sequenced to determine the genetic diversity of the parasites using the following marker genes: B. bovis spherical body protein 4, B. bigemina rhoptry-associated protein 1a, T. parva 104 kDa microneme-rhoptry antigen, and A. marginale major surface protein 5. Furthermore, partially and fully engorged adult ticks were collected for taxonomy, and tick-control practices were assessed using a semi-structured questionnaire. The prevalences of B. bigemina, T. parva, and A. marginale in cattle were 17.2, 65.1, and 22.0%, and 10.0, 26.5, and 3% in the central and eastern region, respectively. Whilst, B. bovis was not detected in the farms involved. The sequences for B. bigemina, T. parva, and A. marginale from the central region showed 99% identity with those from the eastern region. Of the 548 ticks collected, 319, 147, 76, and 6 were Rhipicephalus (Boophilus) decoloratus, Rhipicephalus appendiculatus, Amblyomma variegatum, and Rhipicephalus evertsi evertsi, respectively. The Rhipicephalus ticks were more abundant in the central region, whereas A. variegatum ticks were more abundant in the eastern region. Tick control malpractices were found in both Central and Eastern Uganda, and 42 of the 56 surveyed farms lacked appropriate restraining facilities and so they utilized either ropes or a 'boma' (enclosure). In summary, B. bigemina, T. parva, A. marginale and their co-infections were more prevalent in the central than eastern region; even though, tick control malpractices were observed in both regions. Therefore, an urgent tick and TBD control strategy is needed.

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.