RPA-CRISPR/Cas12a assay for the diagnosis of bovine Anaplasma marginale infection.

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.

Unraveling the genetic mechanisms governing the host response to bovine anaplasmosis.

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.

RPA/CRISPR-cas12a as a specific, sensitive and rapid method for diagnosing Ehrlichia canis and Anaplasma platys in dogs in Thailand.

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.

Nucleotide sequence types (ntSTs) of Anaplasma marginale in cattle in Nigeria based on the major surface protein 5 (msp5) gene.

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.

Targeted mutagenesis in Anaplasma marginale to define virulence and vaccine development against bovine anaplasmosis.

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.

Molecular detection and genetic characterization of Anaplasma marginale and Anaplasma platys in cattle in Nigeria.

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.

Recombinase polymerase amplification (RPA) with lateral flow detection for three Anaplasma species of importance to livestock health.

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 × 104 copies/µl = A. marginale, 5.04 × 106 copies/µl = A. ovis, and 4.58 × 103 copies/µl = A. phagocytophilum, and for each multiplex lateral flow or RPA-nfo assays is 8.99 × 103 copies/µl of A. marginale, 5.04 × 103 copies/µl of A. ovis, 4.58 × 103 copies/µl of A. phagocytophilum, and 5.51 × 103 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.

Functional inhibition or genetic deletion of acid sphingomyelinase bacteriostatically inhibits Anaplasma phagocytophilum infection in vivo.

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.

Recombinant expression and characterization of major surface protein 4 from Anaplasma marginale.

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.

[Cytogenetic, kariopathological and morphological abnormalities of spermatozoa and urothelial cells in human granulocytic anaplasmosis depending on polymorphism of the gene of DNA ligase IV].

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.

Development of TEM-1 ß-lactamase based protein translocation assay for identification of Anaplasma phagocytophilum type IV secretion system effector proteins.

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 ß-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.

Molecular detection, characterization of Anaplasma spp. in domestic cats from Rio de Janeiro state.

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.

Molecular epidemiology, associated risk factors, and phylogenetic analysis of anaplasmosis in camel.

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.

Molecular detection of Anaplasma phagocytophilum-like Anaplasma spp. and pathogenic A. Phagocytophilum in cattle from South Korea.

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.

First molecular evidence of equine granulocytic anaplasmosis in Pakistan.

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_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.

Influence of Genetic Background on Hematologic and Histopathologic Alterations during Acute Granulocytic Anaplasmosis in 129/SvEv and C57BL/6J Mice Lacking Type I and Type II Interferon Signaling.

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.

Molecular survey of Anaplasma platys and Ehrlichia canis in dogs from Campo Grande, Mato Grosso do Sul, Brazil.

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.

Serological detection of antibodies to Anaplasma spp., Borrelia burgdorferi sensu lato and Ehrlichia canis and of Dirofilaria immitis antigen in dogs from Costa Rica.

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.

Molecular survey of Anaplasma platys and Ehrlichia canis in dogs from Campo Grande, Mato Grosso do Sul, Brazil

ABSTRACT 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.

Anaplasma marginale: Diversity, Virulence, and Vaccine Landscape through a Genomics Approach.

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.