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Anaplasma marginale infection is one of the most common tick-borne diseases, causing a substantial loss in the beef and dairy production industries. Once infected, the pathogen remains in the cattle for life, allowing the parasites to spread to healthy animals. Since clinical manifestations of anaplasmosis occur late in the disease, a sensitive, accurate, and affordable pathogen identification is crucial in preventing and controlling the infection. To this end, we developed an RPA-CRISPR/Cas12a assay specific to A. marginale infection in bovines targeting the msp4 gene. Our assay is performed at one moderately high temperature, producing fluorescent signals or positive readout of a lateral flow dipstick, which is as sensitive as conventional PCR-based DNA amplification. This RPA-CRISPR/Cas12a assay can detect as few as 4 copies/μl of Anaplasma using msp4 marker without cross-reactivity to other common bovine pathogens. Lyophilized components of the assay can be stored at room temperature for an extended period, indicating its potential for field diagnosis and low-resource settings of anaplasmosis in bovines., (© 2024. The Author(s).)

Rickettsial pathogens including Ehrlichia canis and Anaplasma platys are bacteria that cause parasitic infections in dogs such as canine monocytic ehrlichiosis (CME) and canine cyclic thrombocytopenia (CCT), respectively affecting mortality and morbidity worldwide. An accurate, sensitive, and rapid method to diagnose these agents is essential for effective treatment. In this study, a recombinase polymerase amplification (RPA) coupled with CRISPR-Cas12a methods was established to detect E. canis and A. platys infection in dogs based on the 16S rRNA. The optimal condition for DNA amplification by RPA was 37 °C for 20 min, followed by CRISPR-Cas12a digestion at 37 °C for one hour. A combination of RPA and the cas12a detection method did not react with other pathogens and demonstrated strong sensitivity, detecting as low as 100 copies of both E. canis and A. platys. This simultaneous detection method was significantly more sensitive than conventional PCR. The RPA-assisted cas12a assay provides specific, sensitive, rapid, simple and appropriate detection of rickettsial agents in canine blood at the point-of-care for diagnostics, disease prevention and surveillance., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Bovine anaplasmosis caused by Anaplasma marginale is a tick-borne disease of livestock with widespread prevalence and huge economic implications. In order to get new insights into modulation of host gene expression in response to natural infections of anaplasmosis, this study is the first attempt that compared the transcriptome profiles of peripheral blood mononuclear cells (PBMCs) of A. marginale infected and healthy crossbred cattle. Transcriptome analysis identified shared as well as unique functional pathways in the two groups. Translation and structural constituent of ribosome were the important terms for the genes abundantly expressed in the infected as well as healthy animals. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the differentially expressed genes revealed that immunity and signal transduction related terms were enriched for the up-regulated genes in the infected animals. The over-represented pathways were cytokine-cytokine receptor interaction and signaling pathways involving chemokines, Interleukin 17 (IL17), Tumour Necrosis Factor (TNF), Nuclear Factor Kappa B (NFKB) etc. Interestingly, many genes previously associated with parasite-borne diseases such as amoebiasis, trypanosomiasis, toxoplasmosis, and leishmaniasis were profusely expressed in the dataset of the diseased animals. High expression was also evident for the genes for acute phase response proteins, anti-microbial peptides and many inflammatory cytokines. Role of cytokines in mediating communication between immune cells was the most conspicuous gene network identified through the Ingenuity Pathway Analysis. This study provides comprehensive information about the crosstalk of genes involved in host defense as well as parasite persistence in the host upon infection with A. marginale., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Bovine anaplasmosis caused by Anaplasma marginale is an important endemic disease that exerts negative impact on livestock production with huge socioeconomic consequences in most developing countries. Genetic studies have reported the existence of diverse ntSTs of A. marginale with varying pathogenicity in different countries. Continuous studies to obtain accurate information on disease etiologies is desirable for the formulation of cost-effective control measures. To this end, 582 blood samples from cattle were collected from 10 out of the 36 States of Nigeria from April 2021 to March 2022 and analyzed based on the major surface protein 5 (msp5) gene to determine the ntSTs of A. marginale in Nigeria. In all, 38 out of the 582 samples (6.5%) from cattle in the different Agro-ecological Zones (AEZs) of Nigeria were positive. The Nigerian A. marginale nucleotide sequences were 96.7 to 100% identical to sequences from other countries and were placed in distinct clusters with other A. marginale sequences deposited in GenBank. Network analysis revealed three ntSTs (#2, #4 & #8) of A. marginale from Nigeria with a nucleotide sequence type diversity (Hd) of 0.8, nucleotide diversity (Pi) of 0.015 and average number of nucleotide differences (k) of 7.09. Two different amino acid substitution sites were found in Nigerian and worldwide sequences at positions 148 and 160. This is the first nationwide report on the ntST diversity and genetic characterization of A. marginale in cattle in Nigeria based on the msp5 gene. Bovine anaplasmosis is widespread in Nigeria and deserves further attention., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Bovine anaplasmosis poses serious challenge to profitable livestock production in the tropics. Accurate information on the prevalence, distribution and genetic characteristics of Anaplasma spp. infections of cattle is invaluable for the design of cost-effective control measures. Blood samples from 275 cattle in Nigeria were screened for the DNA of Anaplasma spp. using species-specific primers and nucleotide sequence analysis. The DNA of Anaplasmataceae was detected based on 16S rRNA gene in 135 out of the 275 (49.1%) individuals examined, with 31 (23.0%) and 21(15.6%) being positive for Anaplasma marginale based on msp4 and msp2 genes, respectively. DNA of Anaplasma platys was detected in 62 (45.9%) based on groEL gene and in 27 (20.0%) using the A. platys species-specific primers. Presence of Anaplasma spp. DNA was significantly associated (p = 0.011) with the breed of the animals. Anaplasma nucleotide sequences of one group of the infected samples showed high identities of 99.0 to 100% (16S rRNA gene) and 99.6% (groEL gene) with reference sequences of A. platys, while those of another group matched to A. marginale references (msp2 with 98.9% and msp4 with 99.1%). Furthermore, phylogenetic analysis clustered the nucleotide sequences in this study with A. platys and A. marginale sequences in GenBank, confirming these relationships. For the first time, this study revealed the presence of mixed haplotypes in both A. platys and A. marginale in cattle in Nigeria. More studies are needed to elucidate the epidemiology and veterinary and public health significance of Anaplasma spp. infections in cattle in Nigeria., (Copyright © 2022. Published by Elsevier GmbH.)

Tick-borne Anaplasma species are obligate, intracellular, bacterial pathogens that cause important diseases globally in people, agricultural animals, and dogs. Targeted mutagenesis methods are yet to be developed to define genes essential for these pathogens. In addition, vaccines conferring protection against diseases caused by Anaplasma species are not available. Here, we describe a targeted mutagenesis method for deletion of the phage head-to-tail connector protein (phtcp) gene in Anaplasma marginale. The mutant did not cause disease and exhibited attenuated growth in its natural host (cattle). We then assessed its ability to confer protection against wild-type A. marginale infection challenge. Additionally, we compared vaccine protection with the mutant to that of whole cell A. marginale inactivated antigens as a vaccine (WCAV) candidate. Upon infection challenge, non-vaccinated control cattle developed severe disease, with an average 57% drop in packed cell volume (PCV) between days 26-31 post infection, an 11% peak in erythrocytic infection, and apparent anisocytosis. Conversely, following challenge, all animals receiving the live mutant did not develop clinical signs or anemia, or erythrocyte infection. In contrast, the WCAV vaccinees developed similar disease as the non-vaccinees following A. marginale infection, though the peak erythrocyte infection reduced to 6% and the PCV dropped 43%. This is the first study describing targeted mutagenesis and its application in determining in vivo virulence and vaccine development for an Anaplasma species pathogen. This study will pave the way for similar research in related Anaplasma pathogens impacting multiple hosts., Competing Interests: The authors have declared that no competing interests exist.

Anaplasma marginale, A. ovis, and A. phagocytophilum are the causative agents of bovine anaplasmosis, ovine anaplasmosis, and granulocytic anaplasmosis, respectively. The gold standard for diagnosis of post-acute and long-term persistent infections is the serological cELISA, which does not discriminate between Anaplasma species and requires highly equipped laboratories and trained personnel. This study addresses the development of a rapid, isothermal, sensitive, species-specific RPA assays to detect three Anaplasma species in blood and cELISA A. marginale-positive serum samples. Three RPA primer and probe sets were designed targeting msp4 genes of each Anaplasma species and the internal control (GAPDH gene) for each assay. The limit of detection of gel-based or RPA-basic assays is 8.99 × 10 4 copies/µl = A. marginale, 5.04 × 10 6 copies/µl = A. ovis, and 4.58 × 10 3 copies/µl = A. phagocytophilum, and for each multiplex lateral flow or RPA-nfo assays is 8.99 × 10 3 copies/µl of A. marginale, 5.04 × 10 3 copies/µl of A. ovis, 4.58 × 10 3 copies/µl of A. phagocytophilum, and 5.51 × 10 3 copies/µl of internal control (GAPDH). Although none of the 80 blood samples collected from Oklahoma cattle were positive, the RPA-nfo assays detected all A. marginale cattle blood samples with varying prevalence rates of infection, 83% of the 24 cELISA A. marginale-positive serum samples, and all A. phagocytophilum cell culture samples. Overall, although early detection of three Anaplasma species was not specifically addressed, the described RPA technique represents an improvement for detection of three Anaplasma in regions where access to laboratory equipment is limited., (© 2021. The Author(s).)

Anaplasma phagocytophilum infects neutrophils to cause granulocytic anaplasmosis. It poorly infects mice deficient in acid sphingomyelinase (ASM), a lysosomal enzyme critical for cholesterol efflux, and wild-type mice treated with desipramine that functionally inhibits ASM. Whether inhibition or genetic deletion of ASM is bacteriostatic or bactericidal for A. phagocytophilum and desipramine's ability to lower pathogen burden requires a competent immune system were unknown. Anaplasma phagocytophilum-infected severe combined immunodeficiency disorder (SCID) mice were administered desipramine or PBS, followed by the transfer of blood to naïve wild-type mice. Next, infected wild-type mice were given desipramine or PBS followed by transfer of blood to naïve SCID mice. Finally, wild-type or ASM-deficient mice were infected and blood transferred to naïve SCID mice. The percentage of infected neutrophils was significantly reduced in all desipramine-treated or ASM-deficient mice and in all recipients of blood from these mice. Infection was markedly lower in ASM-deficient and desipramine-treated wild-type mice versus desipramine-treated SCID mice. Yet, infection was never ablated. Thus, ASM activity contributes to optimal A. phagocytophilum infection in vivo, pharmacologic inhibition or genetic deletion of ASM impairs infection in a bacteriostatic and reversible manner and A. phagocytophilum is capable of co-opting ASM-independent lipid sources., (© The Author(s) 2020. Published by Oxford University Press on behalf of the American Society for Nutrition.)

Anaplasma marginale is the rickettsia which causes the bovine anaplasmosis. The distribution of A. marginale is both tropical and subtropical regions of the world. The major surface protein 4 (MSP4) of this parasite was identified as an immunodominant protein. In this study, the full length of DNA encoding A. marginale MSP4 (AmMSP4) was cloned from the parasites. The open reading frame of msp4 coding sequence of Thailand strain is 849 bp. Phylogenetic analysis revealed that the msp4 coding sequence of A. marginale was highly conserved when compared with Anaplasma phagocytophilum. The recombinant plasmid was further transformed into the BL21-CodonPlus (DE3)-RIPL competent cells for over-expression of the recombinant major surface protein 4 of A. marginale (rAmMSP4). Sera from rabbit immunized with rAmMSP4 and from cattle infected with A. marginale were used to study the antigenicity of rAmMSP4 (35 kDa) and AmMSP4 (31 kDa). Both rAmMSP4 and AmMSP4 were recognized by these sera showing that recombinant and native AmMSP4 have conserved epitopes. Localization of Anaplasma parasites by immunofluorescence showed these parasites are distributed on both the membrane and the outside of infected erythrocytes. Regarding antigenicity, recombinant MSP4 could be used for immunodiagnostic purposes and as a possible vaccine candidate against anaplasmosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Aim: to assess the role of human granulocytic anaplasmosis (HGA) caused by Anaplasma phagocytophilum in the induction of cytogenetic damage of spermatozoa and karyopathological abnormalities of urothelial cells depending on the polymorphism of the gene of enzyme, DNA ligase IV., Material and Methods: A total of 129 male patients with HGA and 84 otherwise healthy donors were examined. The samples of both semen and urothelial cells were obtained from each individuals for microscopic analysis. The diagnosis was confirmed by cytological (microscopic) method, enzyme immunoassay and polymerase chain reaction (PCR). An analysis of the frequencies of damaged spermatozoa and urothelial cells in all participants was carried out. In addition, a molecular cytogenetic study of spermatozoa by fluorescent in situ hybridization (FISH) was carried out using an AneuVysion multicolour for chromosomes 18 and 21 ("Abbott", USA) to determine the frequency of aneuploidy in spermatozoa. The level of DNA fragmentation was studied by SCD (Sperm Chromatin Dispertion Test) using a commercial Halosperm kit ("Halotech DNA", Spain)., Results: The cytological analysis revealed the significant increase in the proportion of spermatozoa with cytogenetic abnormalities and urothelial cells with karyopathological damage in the HGA patients. The most significant damage to nuclear structures of cells was determined in the patients with Ile/Ile genotype. The significant effects of HGA in DNA damage and cytogenetic abnormalities in patients were verified by the increased frequency of spermatozoa with DNA fragmentation, monosomy and disomy in 21 and 18 chromosomes, as well as the appearance of urothelial cells with karyopathological abnormalities. In addition, the increased frequencies of pathospermia with pathological abnormalities of head, neck and tail of spermatozoa in HGA patients were found., Conclusion: According to our results, the cytological analysis in the patients with HGA demonstrated the significant increase in the frequencies of spermatozoa with head, neck and tail defects and DNA fragmentation, monosomy and trisomy of the 18th and 21st chromosomes, as well as the increase in the frequencies of urothelial cells with karyopathological abnormalities. The genetic polymorphism of the effects of HGA was revealed, and the most significant cytogenetical damage was found in the patients carrying the Ile/Ile genotype of the LIG4 Thr9Ile gene.

Anaplasma phagocytophilum, the aetiologic agent of human granulocytic anaplasmosis (HGA) is an obligate intracellular Gram-negative bacterium with the genome size of 1.47 megabases. The intracellular life style and small size of genome suggest that A. phagocytophilum has to modulate a multitude of host cell physiological processes to facilitate its replication. One strategy employed by A. phagocytophilum is through its type IV secretion system (T4SS), which translocates bacterial effectors into target cells to disrupt normal cellular activities. In this study we developed a TEM-1 β-lactamase based protein translocation assay and applied this assay for identification of A. phagocytophilum T4SS effectors. An A. phagocytophilum hypothetical protein, APH0215 is identified as a T4SS effector protein and found interacting with trans-Golgi network in transfected cells. Hereby, this protein translocation assay developed in this study will facilitate the identification of A. phagocytophilum T4SS effectors and elucidation of HGA pathogenesis.

Species of the genus Anaplasma, in the family Anaplasmatacae, are responsible to vector-borne diseases that affecting animals and humans. Feline anaplasmosis is poorly reported in Brazil. This study aimed at investigating the occurrence of Anaplasma spp. in domestic cats from Greater Rio de Janeiro, and evaluating hematological changes associated with this rickettsial infection. Were sampled 216 cats, we performed nested PCR (nPCR) and real-time PCR (qPCR) assays targeting A. platys-16S-rDNA, A. platys-gltA and A. phagocytophilum-msp2 sequences. As evaluated with gltA-qPCR the frequency of cats positive for A. platys was 3.7% (n = 8/216) and by 16S-rDNA nested-PCR it was 0.9% (n = 2/216). No cats were positive to msp2-qPCR to A. phagocytophilum. The sequences of A. platys presented 100% similarity with previously described isolates around the world and Brazil. Two cats that were positive in the gltA-qPCR reactions have platelet inclusions in the microscopic examination. However, no significant (p > 0.05) hematological changes were observed, probably due to low parasite load. This study showed that A. platys occur in domestic cats from Greater Rio de Janeiro. Further studies are needed to more precisely characterize these organisms., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Camel Anaplasmosis is caused by members of family Anaplasmatacae, a tick transmitted, obligate intracellular bacteria. The etiological bacteria are transmitted by ixodid tick species. The species have multi host range distribution that is why it is crucial to diagnose it timely. The aim of present study was to investigate the molecular epidemiology i.e. prevalence and risk factors analysis of camel anaplasmosis. Furthermore, variations in hematological standards were also evaluated. The study found an overall 13.33% prevalence in camels. The confirmation of PCR positive samples for Anaplasma spp. was made through sequencing, the study isolatesshowed high homology with Iranian, Chinese, Philippines and South African isolates of Anaplasmatacae (Accession numbers'; KX765882, KP062964, KY242456, LC007100 and U54806) on BLAST queries. The phylogenetic analysis revealedthree study isolates of present study clustered with each other and the cluster was found closer to Chinese isolate of A. phagocytophilum (KY242456), A. marginale (KU586048), and Mongolian isolates of A. ovis (LC194134). Two of the isolates resembled Iranian isolate of Candidatus Anaplasmacamelii (KX765882), while one isolate resembled with Chinese isolates of A. Platys (KX987336) and Croatian isolates of A. Platys (KY114935). The key risk factors odds ratio (OR>1) identified for occurrence of camel anaplasmosis using regression model found sex and age of animal, previous tick history, tick infestation and tick control status, housing type, cracks in walls, rearing system and other species in surrounding as the key risk factors. The hematological parameters like lymphocytes, monocytes, granulocytes and platelets count were significantly decreased (p < 0.05) in diseased camels than healthy. This is the first ever molecular data on camel anaplasmosis in Pakistan. The disease should be monitored unceasingly as the etiologies have multi host distribution. Prompt attention should be offered to animals because neutropenia, lymphopenia and thrombocytopenia can exacerbate the disease by making the animal predisposed to otherdiseases., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Anaplasma phagocytophilum is the causative agent of human granulocytic anaplasmosis and tick-borne fever in domestic ruminants. Differential diagnosis of zoonotic and pathogenic tick-borne diseases like granulocytic anaplasmosis is important for the efficient implementation of control programs. Thus, the differentiation of pathogenic A. phagocytophilum from non-pathogenic A. phagocytophilum-like (APL) Anaplasma spp. is essential. Recent molecular analyses of APL revealed its distinct phylogenetic position from A. phagocytophilum. This study was conducted to detect A. phagocytophilum and genetically related strains in 764 cattle in South Korea using PCR and restriction fragment length polymorphism assays. APL clade A and A. phagocytophilum were identified in 20 (2.6%) and 16 (2.1%) cattle, respectively, with 16 cattle (2.1%) displaying co-infection. The 16S rRNA sequences of APL clade A were similar (98.3-99.9%) to those clustered in the APL clade A from eastern Asia. The A. phagocytophilum 16S rRNA sequence shared 98.6-100% identity to those of the A. phagocytophilum group. We used PCR to amplify the groEL and msp2 genes from the 20 samples positive for the 16S rRNA gene and found that 16 were positive for the groEL sequences in the APL clade A, which showed identity (82.8-84.4%) to those clustered in the APL clade A from Japan. Amplification of msp2 was unsuccessful. The co-infection results suggested sequence diversity in Anaplasma spp. Till date, both A. phagocytophilum and APL have been reported to be distributed separately in several animals throughout South Korea. This report is the first co-detection of A. phagocytophilum and APL in Korean cattle using molecular methods. Further studies are needed to provide additional molecular background and trace the evolutionary tree of Anaplasma species in animals and ticks., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Anaplasma phagocytophilum (A. phagocytophilum) is an obligate intracellular bacterium that causes equine granulocytic anaplasmosis (EGA) disease in equines. This pathogen has zoonotic potential, which makes it very important to be detected and controlled as early as possible. This study was aimed to assess the molecular prevalence, associated risk factors of EGA along with its effects on various hematological parameters. This study revealed an overall 10.67% prevalence in equine. Horses showed highest prevalence followed by mules and donkeys presenting 11.86, 10.53 and 9.43% prevalence, respectively. The samples were confirmed for anaplasmosis through sequencing. The BLAST queries confirmed very high homology of our isolates with Chinese and Japanese isolates of A. phagocytophilum (Accession no's; KX505303, KY242456 and LC002836). The phylogenetic analysis found the study isolates clustered with each other and this cluster closely resembled Chinese isolate of A. bovis (FJ169957), A. phagocytophilum (HQ872464) and A. phagocytophilum (NR&#95;044762) human isolate from northern Minnesota and Wisconsin. The key risk factors identified for occurrence of EGA in equine species on the basis of univariable analysis were sex of animal, housing type, tick infestation, previous tick history and tick control status, type of acaricides used, rearing system and farm hygiene, respectively. The hematological parameters like Hemoglobin (Hb), Total Leukocyte Count (TLC), Total Erythrocytes Count (TEC), and granulocytes were decreased in diseased animals. The mules showed no typical hematological variations which make sense for its nature as carrier of infection to the susceptible species. This is the first molecular evidence of EGA in Pakistan. The disease needs to be handled seriously as it has zoonotic potential. The animals should be properly attended in disease conditions as leukopenia, neutropenia and lymphopenia can aggravate the condition by making the animal prone to secondary infections., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In a study in Costa Rica 314 serum samples from dogs throughout all seven provinces were tested using a commercial kit for the detection of circulating antibodies against Anaplasma spp., Borrelia burgdorferi sensu lato and Ehrlichia canis, and of circulating antigen of Dirofilaria immitis. A total of 6.4% (20/314) and 38.2% (120/314) were positive for Anaplasma spp. (An) and E. canis (Ec) antibodies. Overall, 8.0% (25/314) were positive for D. immitis (Di) antigen. One single dog reacted positive with B. burgdorferi s.l. (Bb) antigen (0.3%, 1/314). E. canis positive dogs were detected in all provinces (highest percentages in Guanacaste, Puntarenas [both significantly different compared to the overall] and Limón). Guanacaste and Puntarenas also showed the highest prevalences of Anaplasma spp. (both significantly different compared to the overall). The highest prevalence of D. immitis was detected in Puntarenas (significantly different compared to the overall). Double pathogen exposure (Ec plus An; Ec plus Di; Ec plus Bb) were recorded in 8.9% (28/314). Two dogs showed a triple pathogen exposure (0.6%, 2/314; An, Ec and Di). There was a significant difference between male (11.5%, 18/156) and female (4.4%, 7/158) animals for D. immitis positive results. There was also a significant difference between breed and no breed dogs regarding the characteristics of a general positive test, as well as seropositivity to the single pathogens of Anaplasma spp., E. canis and D. immitis. Finally there was a significant difference in the presence of clinical signs again regarding the characteristics of a general positive test, as well as seropositivity to Anaplasma spp., E. canis and D. immitis. Practitioners in Costa Rica should be aware of the canine vector-borne diseases mentioned as dogs are at risk of becoming infected. Concerning the positive B. burgdorferi s.l. dog, an autochthonous occurrence cannot be confirmed due to a history of adoption and an unusual tattoo number. Veterinary advice to protect dogs and limit transmission of vector-borne pathogens, also to humans, by using prophylactic measures is strongly recommended., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The role of host type I IFN signaling and its interaction with other immune pathways during bacterial infections is incompletely understood. Type II IFN signaling plays a key role during numerous bacterial infections including granulocytic anaplasmosis (GA) caused by Anaplasma phagocytophilum infection. The function of combined type I and type II IFN signaling and their potential synergism during GA and similar tick-borne diseases is a topic of current research investigation. The goal of this study was to evaluate 2 mouse models of absent type I/type II IFN signaling in experimental A. phagocytophilum infection to determine the effects of background strain. Mice lacking both type I and type II IFN receptor signaling (IFNAR-/-/IFNGR-/-) on either the 129/SvEv or C57BL/6J genetic background were evaluated at days 0, 6, 8, and 12 of infection. Pathogen burden in multiple organs was largely similar between strains of infected mice, with few significant differences. Background strain influenced the immune response to infection. Mice of the 129/SvEv strain developed more severe hematologic abnormalities, particularly more severe leukocytosis with marked neutrophilia and lymphocytosis, throughout acute infection. Histopathologic changes occurred in infected mice of both strains and varied in severity by organ. 129/SvEv mice developed more severe pathologic changes in spleen and bone marrow, whereas C57BL/6J mice developed more severe renal pathology. This work highlights the importance of mouse background strain in dictating pathophysiologic response to infection and informs future work regarding the loss of type I and type II IFN signaling on the immune response during GA.

This study investigated the frequency of infection by Anaplasma platys and Ehrlichia canis in dogs submitted to animal health centers in Campo Grande, state of Mato Grosso do Sul, Brazil. E. canis and A. platys showed infection frequencies of 55.75% and 16.96%, respectively. The identity of the two species was confirmed by DNA sequencing.

In order to understand the genetic diversity of A. marginale, several efforts have been made around the world. This rickettsia affects a significant number of ruminants, causing bovine anaplasmosis, so the interest in its virulence and how it is transmitted have drawn interest not only from a molecular point of view but also, recently, some genomics research have been performed to elucidate genes and proteins with potential as antigens. Unfortunately, so far, we still do not have a recombinant anaplasmosis vaccine. In this review, we present a landscape of the multiple approaches carried out from the genomic perspective to generate valuable information that could be used in a holistic way to finally develop an anaplasmosis vaccine. These approaches include the analysis of the genetic diversity of A. marginale and how this affects control measures for the disease. Anaplasmosis vaccine development is also reviewed from the conventional vaccinomics to genome-base vaccinology approach based on proteomics, metabolomics, and transcriptomics analyses reported. The use of these new omics approaches will undoubtedly reveal new targets of interest in the near future, comprising information of potential antigens and the immunogenic effect of A. marginale proteins.

Anaplasma phagocytophilum is an emerging pathogen that causes human granulocytic anaplasmosis. Infection with this zoonotic pathogen affects cell function in both vertebrate host and the tick vector, Ixodes scapularis. Global tissue-specific response and apoptosis signaling pathways were characterized in I. scapularis nymphs and adult female midguts and salivary glands infected with A. phagocytophilum using a systems biology approach combining transcriptomics and proteomics. Apoptosis was selected for pathway-focused analysis due to its role in bacterial infection of tick cells. The results showed tissue-specific differences in tick response to infection and revealed differentiated regulation of apoptosis pathways. The impact of bacterial infection was more pronounced in tick nymphs and midguts than in salivary glands, probably reflecting bacterial developmental cycle. All apoptosis pathways described in other organisms were identified in I. scapularis, except for the absence of the Perforin ortholog. Functional characterization using RNA interference showed that Porin knockdown significantly increases tick colonization by A. phagocytophilum. Infection with A. phagocytophilum produced complex tissue-specific alterations in transcript and protein levels. In tick nymphs, the results suggested a possible effect of bacterial infection on the inhibition of tick immune response. In tick midguts, the results suggested that A. phagocytophilum infection inhibited cell apoptosis to facilitate and establish infection through up-regulation of the JAK/STAT pathway. Bacterial infection inhibited the intrinsic apoptosis pathway in tick salivary glands by down-regulating Porin expression that resulted in the inhibition of Cytochrome c release as the anti-apoptotic mechanism to facilitate bacterial infection. However, tick salivary glands may promote apoptosis to limit bacterial infection through induction of the extrinsic apoptosis pathway. These dynamic changes in response to A. phagocytophilum in I. scapularis tissue-specific transcriptome and proteome demonstrated the complexity of the tick response to infection and will contribute to characterize gene regulation in ticks.

Human granulocytic anaplasmosis (HGA) is caused by the obligate intracellular bacterium Anaplasma phagocytophilum. Our data previously demonstrated that A. phagocytophilum induces an immunopathologic response by activating IFN-γ production through the Stat1 signaling pathway. In this study, we investigated the broader role of Stat1 signaling in the host response to infection with A. phagocytophilum. In Stat1 knockout (KO) compared with wild-type mice, A. phagocytophilum infection was more highly pathogenic as characterized by the unanticipated development of clinical signs in mice including markedly increased splenomegaly, more severe inflammatory splenic and hepatic histopathology, >100-fold higher blood and splenic bacterial loads, and more elevated proinflammatory cytokine/chemokine responses in serum. CD4(+) and CD8(+) T lymphocyte populations were significantly expanded in spleens of A. phagocytophilum-infected Stat1 KO mice compared with wild-type mice. The leukocyte infiltrates in the livers and spleens of A. phagocytophilum-infected Stat1 KO mice also contained expansions in neutrophil and monocyte/macrophage populations. Importantly, A. phagocytophilum-infected Stat1 KO mice did not demonstrate induction of inducible NO synthase in splenocytes. These results show that Stat1 plays an important role in controlling bacterial loads but also by unexpectedly providing an undefined mechanism for dampening of the immunopathologic response observed with A. phagocytophilum infection., (Copyright © 2014 by The American Association of Immunologists, Inc.)

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.

Arthropod vectors transmit a diversity of animal and human pathogens, ranging from RNA viruses to protozoal parasites. Chemotherapeutic control of pathogens has classically focused either on insecticides that kill the vector itself or antimicrobials for infected patients. The limitation of the former is that it targets both infected and uninfected vectors and selects for resistant populations while the latter requires prompt and accurate diagnosis. An alternative strategy is to target vector molecules that permit the pathogen to establish itself, replicate, and/or develop within the vector. Using the rickettsial pathogen Anaplasma marginale and its tropical tick vector, Rhipicephalus microplus, as a model, we tested whether silencing specific gene targets would affect tick infection rates (the % of fed ticks that are infected with the pathogen) and pathogen levels within infected ticks. Silencing of three R. microplus genes, CK187220, CV437619 and TC18492, significantly decreased the A. marginale infection rate in salivary glands, whereas gene silencing of TC22382, TC17129 and TC16059 significantly increased the infection rate in salivary glands. However in all cases of significant difference in the infection rate, the pathogen levels in the ticks that did become infected, were not significantly different. These results are consistent with the targeted genes affecting the pathogen at early steps in infection of the vector rather than in replication efficiency. Identifying vector genes and subsequent determination of the encoded functions are initial steps in discovery of new targets for inhibiting pathogen development and subsequent transmission.

Live vaccination with Anaplasma marginale subsp. centrale (synonym for Anaplasma centrale) induces protection against severe disease upon challenge with A. marginale sensu stricto strains. Despite over a century of field use, the targets of protective immunity remained unknown. Using a broad proteomic approach, we identified the proteins in a challenge sensu stricto strain that were bound by the relevant antibody isotype induced by live vaccination with Anaplasma marginale subsp. centrale. A core of 15 proteins was identified in vaccinated animals across multiple major histocompatibility complex (MHC) haplotypes. This core separated into two structural/functional classes: "housekeeping" proteins involved in replication and metabolism and outer membrane proteins (OMPs). Orthologous proteins of both classes were identified within the vaccine strain and among sensu stricto strains. In contrast to the broad conservation among strains in the sequences of the housekeeping proteins, there was significantly greater divergence in the OMPs and greater divergence in both OMP sequences and the encoding locus structure between the vaccine strain and the sensu stricto strains than among the sensu stricto strains. The OMPs bound by live vaccine-induced antibody overlapped with OMPs that were immunogenic in animals vaccinated with inactivated vaccines and subsequently protected against bacteremia and disease. The identification of this core set of OMPs is consistent with the hypothesis that "subdominant" immunogens are required for vaccine-induced protection against A. marginale and provides clear direction for development of a safer, more effective vaccine.

Anaplasma and Mycobacterium species are among the most prevalent bacterial pathogens in European red deer (Cervus elaphus) in south-central Spain and are known to modify gene expression in ruminants. In this study, we used microarray hybridization and real-time RT-PCR analyses to characterize global gene expression profiles in red deer in response to Anaplasma ovis and A. ovis/Mycobacterium bovis/Mycobacterium avium sub. paratuberculosis (MAP) infections, compare the expression of immune response genes between red deer infected with A. ovis, M. bovis and A. ovis/M. bovis/MAP, and characterize the differential expression of immune response genes identified in red deer in cattle infected with M. bovis and Anaplasma marginale. Global gene differential expression in A. ovis- and A. ovis/M. bovis/MAP-infected deer resulted in the modification of common and pathogen-specific cellular biological processes. The differential expression of host immune response genes showed pathogen and host-specific signatures and the effect of infection with multiple pathogens on deer immune response. These results suggested that intracellular bacteria from Anaplasma and Mycobacterium genera produce similar genes expression patterns in infected ruminants. However, pathogen and host-specific differences could contribute to disease diagnosis and treatment in ruminants., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

In the United States, Ixodes scapularis ticks overwinter in the Northeast and Upper Midwest and transmit the agent of human granulocytic anaplasmosis, Anaplasma phagocytophilum, among other pathogens. We now show that the presence of A. phagocytophilum in I. scapularis ticks increases their ability to survive in the cold. We identified an I. scapularis antifreeze glycoprotein, designated IAFGP, and demonstrated via RNAi knockdown studies the importance of IAFGP for the survival of I. scapularis ticks in a cold environment. Transfection studies also show that IAFGP increased the viability of yeast cells subjected to cold temperature. Remarkably, A. phagocytophilum induced the expression of iafgp, thereby increasing the cold tolerance and survival of I. scapularis. These data define a molecular basis for symbiosis between a human pathogenic bacterium and its arthropod vector and delineate what we believe to be a new pathway that may be targeted to alter the life cycle of this microbe and its invertebrate host.

Background: The tick-borne intracellular pathogen, Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae) causes human granulocytic anaplasmosis after infection of polymorphonuclear leucocytes. The human Sp110 gene is a member of the nuclear body (NB) components that functions as a nuclear hormone receptor transcriptional coactivator and plays an important role in immunoprotective mechanisms against pathogens in humans. In this research, we hypothesized that Sp110 may be involved in the infection of human promyelocytic HL-60 cells with A. phagocytophilum., Methods: The human Sp110 and A. phagocytophilum msp4 mRNA levels were evaluated by real-time RT-PCR in infected human HL-60 cells sampled at 0, 12, 24, 48, 72 and 96 hours post-infection. The effect of Sp110 expression on A. phagocytophilum infection was determined by RNA interference (RNAi). The expression of Sp110 was silenced in HL-60 cells by RNAi using pre-designed siRNAs using the Nucleofector 96-well shuttle system (Amaxa Biosystems, Gaithersburg, MD, USA). The A. phagocytophilum infection levels were evaluated in HL-60 cells after RNAi by real-time PCR of msp4 and normalizing against human Alu sequences., Results: While Sp110 mRNA levels increased concurrently with A. phagocytophilum infections in HL-60 cells, the silencing of Sp110 expression by RNA interference resulted in decreased infection levels., Conclusion: These results demonstrated that Sp110 expression is required for A. phagocytophilum infection and multiplication in HL-60 cells, and suggest a previously undescribed mechanism by which A. phagocytophilum modulates Sp110 mRNA levels to facilitate establishment of infection of human HL-60 cells.

Human anaplasmosis is an emerging infectious disease transmitted by ticks that can be potentially fatal in the immunocompromised and the elderly. The mechanisms of defense against the causative agent, Anaplasma phagocytophilum, are not completely understood; however, interferon (IFN)-gamma plays an important role in pathogen clearance. Here, we show that IFN-gamma is regulated through an early IL-12/23p40-dependent mechanism. Interleukin (IL)-12/23p40 is regulated in macrophages and dendritic cells after activation by microbial agonists and cytokines and constitutes a subunit of IL-12 and IL-23. IL-12/23p40-deficient mice displayed an increased A. phagocytophilum burden, accelerated thrombocytopenia and increased neutrophil numbers in the spleen at day 6 postinfection. Infection of MyD88- and mitogen-activated kinase kinase 3 (MKK3)-deficient mice suggested that the early susceptibility due to IL-12/23p40 deficiency was not dependent on signaling through MyD88 or MKK3. The lack of IL-12/23p40 reduced IFN-gamma production in both CD4(+) and CD8(+) T cells although the effect was more pronounced in CD4(+) T cells. Our data suggest that the immune response against A. phagocytophilum is a multifactorial and cooperative process. The IL-12/23p40 subunit drives the CD4(+) Th1 immune response in the early phase of infection and IL-12/23p40-independent mechanisms ultimately contribute to pathogen elimination from the host.

Persistence of Anaplasma spp. in the animal reservoir host is required for efficient tick-borne transmission of these pathogens to animals and humans. Using A. marginale infection of its natural reservoir host as a model, persistent infection has been shown to reflect sequential cycles in which antigenic variants emerge, replicate, and are controlled by the immune system. Variation in the immunodominant outer-membrane protein MSP2 is generated by a process of gene conversion, in which unique hypervariable region sequences (HVRs) located in pseudogenes are recombined into a single operon-linked msp2 expression site. Although organisms expressing whole HVRs derived from pseudogenes emerge early in infection, long-term persistent infection is dependent on the generation of complex mosaics in which segments from different HVRs recombine into the expression site. The resulting combinatorial diversity generates the number of variants both predicted and shown to emerge during persistence.

Outer membrane protein preparations have been demonstrated to elicit protective immunity for Anaplasma marginale. Attempts to recreate this protective immunity using known surface proteins have been unsuccessful; therefore, novel outer membrane proteins have been searched for using a proteomic/genomic approach. Annotation of the whole genome sequence identified 62 outer membrane protein candidates based on sequence similarity to known surface proteins. In a proteomics approach for the identification of immunostimulatory outer membrane proteins, outer membrane preparations that were separated on 2D gels were used to immunize calves . Antisera from the calves were used to detect immunoreactive proteins, which were then selected and subjected to mass spectrometric analyses. These data were mapped back to the annotated A. marginale genome and have identified several new outer membrane proteins that are vaccine candidates.

Anaplasma marginale (Rickettsiales: Anaplasmataceae), a tick-borne pathogen of cattle, is endemic in tropical and subtropical regions of the world. Many geographic isolates of A. marginale occur worldwide that have been identified by major surface protein (MSP) 1a, which varies in sequence and molecular weight owing to different numbers of tandem 28-29 amino acid repeats. Although serologic tests have identified American bison, Bison bison, as being infected with A. marginale, the present studies were undertaken to confirm A. marginale infection in bison, to characterize bison isolates, and to compare the phylogenetic relationship of the bison isolates with other A. marginale isolates from North America. Nine A. marginale isolates derived from Canadian bison possessed identical msp4 sequences with one characteristic silent nucleotide change. The sequence of MSP1a was determined for one Canadian and two U.S. bison isolates of A. marginale, and these isolates contained 4 and 5 tandem repeats, respectively. One U.S. bison isolate tested for infectivity proved to be infective for cattle and transmitted by Dermacentor variabilis ticks. the results of this study demonstrated that these A. marginale isolates obtained from bison were similar to ones derived from naturally infected cattle.

Recent advances in genomic technology have focused many veterinary researchers on the possibility of producing one multivalent recombinant vaccine against all the haemoparasites that infect cattle in the tropics. Before such a vaccine is developed it is essential to define target cattle populations as well as the range of anti-pathogen vaccines required in order to control disease. To further this objective, we have evaluated a reverse line blot (RLB) assay, which simultaneously detects the principal tick transmitted protozoan and rickettsial cattle pathogens, in different epidemiological scenarios in Uganda. A critical question is the sensitivity, particularly in relation to detecting carrier animals. As Theileria parva is considered to be the most important pathogen in the region, we assessed the sensitivity of the RLB assay for T. parva and showed that 1-2 x 10(3) parasites per ml of blood could be detected-a level comparable with previously developed PCR methods and well below conventional microscopic detection. We applied the RLB assay to evaluate the differences in pathogen profiles between crossbred and indigenous cattle and show that there were different profiles, with a low prevalence of T. parva and Theileria taurotragi in the indigenous cattle compared to a high prevalence in the crossbred cattle. In contrast, we show higher prevalences of Theileria mutans and Theileria velifera in the indigenous compared to the crossbred cattle. Interestingly Anaplasma marginale, Babesia bovis and Babesia bigemina were of low prevalence but a high prevalence of Ehrlichia bovis was seen, raising the question of whether this rickettsial species could be pathogenic in cattle. Analysis of animals with clinical symptoms of East Coast Fever showed that, while T. parva is a major cause of these symptoms, T. mutans and possibly T. taurotragi and T. velifera, may also cause clinical disease. Overall, the results presented here highlight the complexity of tick-borne pathogen infections in cattle in Uganda.

Bovine anaplasmosis presents a worldwide distribution. However, specific models for studying the epidemiology of the disease are not available. Epidemiological modeling encounters some difficulties due to a lack of culturing techniques for Anaplasma marginale, the causative agent, as well as for the lack of typing techniques to characterize strains. The chronic carrier state and the population dynamics of mechanical and biological vectors also create difficulties. In addition, conventional serology and blood smear diagnostic techniques fail to detect all chronic carriers. Fortunately the needs for the accurate typing of isolates and for detecting chronic carriers made it possible to encourage the development of new tools based on molecular epidemiology principles. A. marginale isolates can now be typed by using panels of monoclonal antibodies, and the genes coding for some major surface proteins can be expressed or analyzed by looking at the nucleotide arrangement level. In the same manner, the latest techniques for detecting A. marginale chronic infections use DNA and RNA probes, and PCR-based methods to detect A. marginale DNA from bovine blood samples with extremely low rickettsaemias. Currently all these new epidemiological tools are being incorporated to experimental models to analyze their applicability for epidemiological studies in the near future.

Anaplasma marginale is a rickettsial parasite of bovine erythrocytes causing world-wide economic losses in livestock production. Despite its importance, little is known about this rickettsia at a molecular level because it has not been cultured in vitro, and there is no small-animal model. Although several genes have been cloned and sequenced, the gross genome structure of the organism has not yet been well characterized. We separated intact bovine erythrocytes from leucocytes, and determined the genome size of A. marginale by use of restriction endonuclease cleavage and pulsed-field gel electrophoresis (PFGE). A value of 56 mol% G+C was obtained for this genome by spectral analysis. Undigested A. marginale DNA failed to migrate under several different electrophoretic conditions, indicating a circular genome. Digestions of intact A. marginale DNA were performed using restriction endonucleases NotI, SfiI and PacI. Complete digestion with SfiI resulted in 12 distinct bands ranging in size from 14 to 170 kbp. Total size determined by addition of SfiI-digested fragments was approximately 1200 kbp. PacI cleaved the A. marginale genome from three different isolates into just three fragments, of 598, 557 and 97 kbp. Incomplete digestion produced a band measuring 1250 kbp. These results indicate that A. marginale has a circular genome between 1200 and 1260 kbp, with a G+C content of 56 mol%.