First detection of Ehrlichia chaffeensis, Ehrlichia canis, and Anaplasma ovis in Rhipicephalus bursa ticks collected from sheep, Turkey.

Anaplasmosis and ehrlichiosis are important tick-borne rickettsial diseases of medical and veterinary importance that cause economic losses in livestock. In this study, the prevalence of Anaplasma ovis, Ehrlichia canis and Ehrlichia chaffeensis was investigated in ticks collected from sheep in various farms in Van province, which is located in the Eastern Anatolian Region of Turkey. The ticks used in this study were collected by random sampling in 26 family farm business in 13 districts of Van province. A total of 688 ticks were collected from 88 sheep and 88 tick pools were created. All ticks identified morphologically as Rhipicephalus bursa. Phylogenetic analysis of Chaperonin and 16S rRNA gene sequences confirmed A. ovis, E. canis and E. chaffeensis in this study. Of the 88 tick pools tested, 28.41% (25/88) were positive for at least one pathogen. Anaplasma DNA was detected in five of the 88 pools (5.68%), E. canis DNA was detected in 19 of the 88 pools (21.59%), and E. chaffeensis DNA was detected in one of the 88 pools (1.14%) of R. bursa ticks. To our knowledge, this is the first report describing the presence of A. ovis, E. canis, and E. chaffeensis in R. bursa ticks collected from sheep in Turkey. Further studies are needed to investigate other co-infections in sheep in Turkey.

Molecular and haematological investigations on Anaplasma ovis infection in goats in Ahvaz, Iran: Insights into infection rate, haemolytic effect and breed influences.

BACKGROUND: Anaplasma ovis (A. ovis) is the predominant causative agent of anaplasmosis in goats and sheep in most tropical and subtropical regions of the world. However, there is considerable variation in reported infection rates, breed susceptibility, and controversial findings regarding the haemolytic effects of A. ovis infection in goats. OBJECTIVES: Thus, we investigated the molecular and haematological aspects of A. ovis infection in goats from Ahvaz city. METHODS: One hundred and fifty apparently healthy goats (74 blacks and 76 Najdi goats) were randomly sampled from six flocks in the Ahvaz suburb during ticks' activity season. Haematological evaluation, smear microscopic (SM) examination and PCR assay were performed to assess A. ovis infection. Additionally, the percentage of parasitemia was determined from blood smears. RESULTS: SM examination revealed that 25.7% of the goats displayed erythrocyte Anaplasma-like inclusion bodies. PCR analysis indicated that 54% of the goats were positive for A. ovis infection (44.6% of blacks and 63.2% of Najdi goats). No significant difference in haematological values was observed between healthy and infected goats based on PCR testing. However, a significant difference in haematological indices was observed between the group with parasitemia level of 0.01-0.02% (SM and PCR positive) compared to the healthy goats (SM and PCR negative), particularly concerning Hb, PCV and RBC count (p < 0.01). CONCLUSIONS: When the parasitemia exceeds 0.01%, A. ovis infection may disrupt haematological parameters in infected goats. The high prevalence of A. ovis infection (54%) among the studied goats underscores the importance of giving special attention to implementing necessary measures for disease control in the Ahvaz suburb.

Molecular characterization of Anaplasma ovis Msp4 protein in strains isolated from ticks in Turkey: A multi-epitope synthetic vaccine antigen design against Anaplasma ovis using immunoinformatic tools.

Tick-borne pathogens increasingly threaten animal and human health as well as cause great economic loss in the livestock industry. Among these pathogens, Anaplasma ovis causing a decrease in meat and milk yield is frequently detected in sheep in many countries including Turkey. This study aimed to reveal potential vaccine candidate epitopes in Msp4 protein using sequence data from Anaplasma ovis isolates and then to design a multi-epitope protein to be used in vaccine formulations against Anaplasma ovis. For this purpose, Msp4 gene was sequenced from Anaplasma ovis isolates (n:6) detected in ticks collected from sheep in Turkey and the sequence data was compared with previous sequences from different countries in order to detect the variations of Msp4 gene/protein. Potential vaccine candidate and diagnostic epitopes were predicted using various immunoinformatics tools. Among the discovered vaccine candidate epitopes, antigenic and conserved were selected, and then a multi-epitope protein was designed. The designed vaccine protein was tested for the assessment of TLR-2, IgG, and IFN-g responses by molecular docking and immune simulation analyses. Among the discovered epitopes, EVASEGSGVM and YQFTPEISLV epitopes with properties of high antigenicity, non-allergenicity, and non-toxicity were proposed to be used for Anaplasma ovis in further serodiagnostic and vaccine studies.

Long-lasting infection with Anaplasma ovis in sheep.

Ovine anaplasmosis is an emerging vector-borne disease in Europe caused by Anaplasma ovis. The infection has spread quickly in recent years, causing moderate to severe outbreaks in sheep flocks, leading to relevant economic losses in sheep farming. This wider spread has been associated with global warming and climate change, favouring the maintenance and life cycle of their main vector, the ticks. However, another epidemiological aspect could favour this quick spread. Long persistence infection of Anaplasma ovis has been proposed as a hypothesis in several articles but never scientifically proven. The results of the present study demonstrate that eight adult sheep, both naturally or experimentally infected, maintain Anaplasma ovis load in blood during their whole productive life (4 to 6 years), being permanently infected. In addition, the results suggest that A. ovis bacterial load can be constant or suffer fluctuations, as has been demonstrated in other Anaplasma species. Both aspects can be determinants in the epidemiology and the transmission of the infection.

Molecular prevalence and phylogeny of Anaplasma marginale, Anaplasma ovis and Theileria ovis in goats and sheep enrolled from a hill station in Punjab, Pakistan.

Anaplasma marginale (A. marginale), Anaplasma ovis (A. ovis) and Theileria ovis (T. ovis) are among the most commonly reported intracellular tick borne pathogens that infect ruminants across the globe causing huge economic losses. This study aims to report the prevalence and phylogenetic evaluation of these three pathogens infecting sheep and goats (n = 333) that were enrolled from Fort Munro region in Pakistan by using msp1b, msp4 and 18S rRNA genes for A. marginale, A. ovis and T. ovis respectively. Results revealed almost similar infection rates in sheep and goats with an overall prevalence of 11% for A. marginale, 28% for A. ovis and 3% for T. ovis. Concurrent infection was also recorded, however, the number of animals infected with two pathogens (n = 24; 7.2%) was higher than infection with three pathogens (n = 2; 0.6%). Risk factor analysis revealed that sheep reared in small herds had higher A. marginale (P = 0.03) and A. ovis (P = 0.04) infection rates compared to those from large herds. In addition, it was observed that bucks (P ≤ 0.05) and tick-free goats (P ≤ 0.05) exhibited higher A. ovis infection rates than nannies. Phylogenetic analysis of all three pathogens showed that Pakistani isolates were clustered together and were closely related to previously deposited Pakistani isolates as well as with those that were reported from worldwide countries. In conclusion, we are reporting that Pakistani sheep and goats have A. marginale, A. ovis and T. ovis mediated infections and control measures should be taken against them to improve the productivity of the livestock sector.

Epidemiology and Phylogeny of Anaplasma ovis with a Note on Hematological and Biochemical Changes in Asymptomatic Goats Enrolled from Four Districts in Punjab, Pakistan.

Background: Anaplasma ovis is an intra-erythrocytic gram negative rickettsial bacterium that infects small ruminants, resulting in huge economic losses worldwide. Materials and Methods: The present investigation aims at reporting the molecular prevalence of A. ovis in 1200 asymptomatic goats that were enrolled from 4 districts (Layyah, Lohdran, Dera Ghazi Khan, and Rajanpur) in Punjab, Pakistan by targeting the msp4 gene of bacterium. Risk factors associated with the prevalence of A. ovis and phylogeny of bacterium were also documented. Results: 184 out of 1200 (15%) goat blood samples were infected with A. ovis. The prevalence of the pathogen varied with the sampling sites (p = 0.005), and the highest prevalence was detected in goats from Layyah (19%) followed by Rajanpur (17%), Dera Ghazi Khan (15%), and Lohdran district (9%). The represented partial msp4 gene amplicon was confirmed by Sanger sequencing and deposited to GenBank (OP225957-59). Phylogenetic analysis revealed that the amplified isolates resembled the msp4 sequences reported from Iran, Mangolia, Sudan, and the United States. Sex and age of goats, herd composition and size, and the presence of ticks on goats and dogs associated with herds were the rick factors associated with the prevalence of A. ovis. Red blood cells, lymphocytes (%), neutrophils (%), hemoglobin, and hematocrit levels in blood and Aspartate amino transferase, urea, and creatinine levels in serum were disturbed in A. ovis infected goats when compared with uninfected animals. Conclusion: We are reporting the prevalence of A. ovis in Pakistani goats from four districts of Punjab and these data will help in developing the integrated control policies against this tick-borne pathogen that is infecting our goat breeds.

Detection of Rickettsia spp. and Anaplasma ovis in Melophagus ovinus from southern Xinjiang, China.

Melophagus ovinus is a hematophagous insect that is distributed worldwide and plays a crucial role in transmitting disease-causing pathogens. From June 2021 to March 2022, a total of 370 M. ovinus were collected from 11 sampling points in southern Xinjiang, China. The specimens were identified using morphological and molecular analyses. Rickettsia spp. and Anaplasma ovis were detected from all the samples using seven Rickettsia-specific genetic markers and the msp-4 gene of A. ovis. Approximately 11% of the M. ovinus specimens were positive for Rickettsia spp., and Candidatus Rickettsia barbariae was the most predominant species (35/41; 85.4%), while R. massiliae was least prevalent (6/41; 14.6%). Approximately 10.5% (39/370) of the M. ovinus specimens were positive for A. ovis of genotype III, which was co-detected with Candidatus R. barbariae in M. ovinus (3/370; 0.8%). To the best of our knowledge, this is the first report of the detection of R. massiliae and Candidatus R. barbariae in M. ovinus globally. The detection and control of insect-borne diseases originating from M. ovinus should be strengthened in southern Xinjiang, an area important to animal husbandry and production.

Distribution and Molecular Epidemiology of Anaplasma ovis in Melophagus ovinus and Small Ruminants in Border Regions of South Xinjiang, China.

Background: Anaplasma ovis are obligate intracellular bacteria that can endanger human and animal health, and they can be transmitted by arthropod vectors, such as Melophagus ovinus and ticks. Materials and Methods: In this study, 433 specimens, including 370 M. ovinus and 63 sheep blood samples, were collected from nine districts of South Xinjiang to investigate the distribution and molecular epidemiology of A. ovis in M. ovinus and small ruminant. Results: DNA of A. ovis was detected in 109 (25.2%, 109/433) of the 433 samples using PCR and sequencing. The analysis of A. ovis msp4 sequences revealed four different genotypes, including genotype III (47.7%; 52/109), GB3 (34.0%; 37/109), AoGOv3 (15.6%; 17/109), and XJ9 (2.8%; 3/109). Conclusions: To the best of our knowledge, A. ovis genotypes GB3, AoGOv3, and XJ9 detected in this study are the first to be reported in M. ovinus, and our data indicate that XJ9 is a novel A. ovis genotype presented herein for the first time. These findings provide important references for the new understanding and prevention of A. ovis in border counties in China.

Molecular epidemiological survey, genetic characterization and phylogenetic analysis of Anaplasma ovis infecting sheep in Northern Egypt.

Anaplasma ovis is the most common etiologic agent of ovine anaplasmosis, mainly transmitted by ticks. The present study aimed to determine the molecular prevalence of A. ovis in sheep from Egypt and assessed the associated risk factors. The study was conducted, between January and December 2020, in four governorates situated in Northern Egypt. Blood samples from 355 asymptomatic sheep were collected and examined by the use of PCR specific to A. ovis. Diversity analysis and phylogenetic study based on partial msp4 gene sequence were performed on revealed A. ovis DNA. Overall, the molecular prevalence rate of A. ovis was 15.5% and the highest rate was observed in Kafr ElSheikh governorate (16.8%). Statistical analysis revealed that A. ovis infection was significantly related to sheep gender and to tick infestation. The risk factors that were found to be associated with A. ovis infection in exposed sheep were: female sex (OR=2.6, 95%CI: 1.13-6.12), and infestation with ticks (OR=2.1, 95%CI: 1.11-3.79). The analysis of A. ovis msp4 sequences revealed two different genotypes classified in the Old World sub-cluster with other Egyptian isolates. Investigation on prevalence, risk factors and genetic variability of A. ovis in sheep reported in this study is important for the implementation of control programs. Further studies are needed to determine the vectors and reservoirs of A. ovis in Egyptian small ruminants and to identify the real economic impact of A. ovis infection on the country.

Anaplasma ovis infection in sheep from Iran: molecular prevalence, associated risk factors, and spatial clustering.

The aim of this cross-sectional study was to determine the molecular prevalence and associated risk factors in sheep populations of Iran. To this end, between March 2017 and February 2018 jugular vein blood samples were collected from 1842 apparently healthy sheep from 327 herds in nine provinces in four ecological zones of Iran. A specific nested-PCR targeting the msp4 gene of A. ovis was employed. Fourteen variables were subjected to logistic regression analyses (univariate and multivariate) to specify the potential risk factors for infection. Statistically significant variables in univariate analyses (P ≤ 0.20) were assessed by multivariable logistic regression to control the confounding factors. Anaplasma ovis DNA was detected in 51.1% of herds (167/327) and 28.3% of animals (521/1842). Among geographical zones, herd and animal prevalence was highest in the Persian-Gulf zone (P < 0.001), and among provinces, Lorestan (in west) and Khuzestan (in south-west) had the highest prevalence (P < 0.001). Analysis of factors associated with A. ovis infection revealed that distance from other farms (OR = 2.52, P < 0.001), presence of other animal species in the farm (OR = 2.03, P = 0.046), season (OR = 1.40, P = 0.005), breed (OR = 3.762, P < 0.001), and age of sheep (OR = 1.20, P = 0.049) are potential risks in Iran. The spatial scan statistic in SaTScan recognized two high risks clusters for A. ovis infection in central (Semnan province) and the Persian-Gulf (Khuzestan province) zones amongst the study areas (P < 0.001). Sequence and phylogenetic analysis of the msp4 gene confirmed the detection of A. ovis. This research is the largest study focusing on ovine anaplasmosis in Iran and shows that infected sheep are present in all geographic zones, bioclimatic areas, and provinces.

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.

Identification and evaluation of midgut protein RL12 of Dermacentor silvarum interacting with Anaplasma ovis VirD4.

Anaplasma ovis, a tick-borne intra-erythrocytic Gram-negative bacterium, is a causative agent of ovine anaplasmosis. It is known that Dermacentor ticks act as biological vectors for A. ovis. VirD4 is the machine component of Type IV Secretion System of A. ovis. To better understand the pathogen-vector interaction, VirD4 was used as a bait protein for screening midgut proteins of Dermacentor silvarum via yeast two-hybrid mating assay. As a result, a ribosomal protein RL12 was identified from the midgut cDNA library of D. silvarum. For further validation, using in vitro Glutathione S-transferase (GST) pull-down assay, interaction between the proteins, GST-RL12 and HIS-VirD4, was observed in Western blot analysis. The study is first of its kind reporting a D. silvarum midgut protein interaction with VirD4 from A. ovis. Functional annotations showed some important cellular processes are attributed to the protein, particularly in the stringent response and biogenesis. The results of the study suggest the involvement of the VirD4-RL12 interaction in the regulation of signaling pathways, which is a tool for understanding the pathogen-vector interaction.

A report on tick burden and molecular detection of tick-borne pathogens in cattle blood samples collected from four regions in Saudi Arabia.

Babesiosis, theileriosis and anaplasmosis are among the most commonly reported tick-borne diseases in cattle and are associated with significant economic losses. Through the present study the researchers aimed to report the presence of various pathogens that cause babesiosis, theileriosis and anaplasmosis in cattle collected from different provinces in Saudi Arabia and to report their phylogenetic relationship. A total of 362 blood samples of cattle along with ticks that were present on the cattle were collected from four regions (Riyadh, Al-Kharj, Al-Hasa and Al-Qassim) of Saudi Arabia. Blood samples were screened by polymerase chain reaction (PCR) for the presence of various Babesia, Theileria and Anaplasma species by amplification of their 18S rRNA and/or 23S rRNA genes. A total of 541 ticks were collected and identified from the cattle. These included Hyalomma anatolicum, Hyalomma dromedarii, Hyalomma impeltatum, Hyalomma excavatum, Rhipicephalus annulatus and Rhipicephalus turanicus. Regarding tick-borne pathogens, the overall prevalence was 1.9 % (7/362) for Theileria annulata, (2/362) 0.6 % for Theileria and (21/362) 5.8 % for Anaplasma ovis. Four of the cattle were found to be co-infected with more than one pathogen (1.1 %). We did not detect any Babesia species in the blood of the studied cattle. Prevalence of the Theileria and Anaplasma species was highest in cattle that resided in Riyadh, followed by cattle from Al-Hasa and Al-Qassim. Representative amplified partial-gene sequences of T. annulata (GenBank accession numbers MK826137-39) and A. ovis (GenBank acc. no. MK 880224) were submitted to GenBank. The presence of ticks on cattle was found to be associated with a high prevalence of Theileria spp. (P = 0.02) and Anaplasma ovis (P < 0.001). We report novel genotypes of T. annulata and A. ovis from cattle in Saudi Arabia and we recommend that molecular surveys are undertaken throughout the country to address the prevalence and geographical distribution of tick-borne infections for their effective diagnosis and treatment.

Molecular detection of tick-borne pathogens harbored by ticks collected from livestock in the Xinjiang Uygur Autonomous Region, China.

Ticks carry and transmit a wide range of pathogens (bacteria, viruses, and protozoa) that are of importance to humans and animals globally. However, information about the tick-borne pathogens harbored by ticks in the Xinjiang Uygur Autonomous Region (XUAR), northwestern China, is scarce. This study investigated the occurrence of tick species of domestic animals and tick-borne pathogens by using morphological molecular identification and sequence analysis in Turpan, Qitai, Altay, Hejing, Nileke, and Zhaosu counties (XUAR). A total of 5822 adult ticks (females and males) from 12 tick species were identified from 5 animal species (cattle, goats, sheep, camels, and horses) in 6 counties in the XUAR. Collected tick species included Dermacentor marginatus (24.7 %), Dermacentor nuttalli (20.8 %), Hyalomma anatolicum (13.7 %), Dermacentor niveus (13.1 %), Haemaphysalis punctata (10.7 %), Dermacentor silvarum (7.1 %), Dermacentor pavlovskyi (3.9 %), Hyalomma asiaticum (2.2 %), Rhipicephalus pumilio (1.9 %), Rhipicephalus sanguineus sensu lato (0.7 %), Rhipicephalus turanicus (0.6 %), and Hyalomma asiaticum kozlovi (0.6 %). Furthermore, 750 partially engorged adult ticks (females and males), including H. anatolicum (250), D. nuttalli (250), and D. marginatus (250), were individually separated according to species and sampling site, used for DNA extraction, and then screened for tick-borne pathogens. The most common pathogen was Rickettsia raoultii (36.80 %), followed by Brucella sp. (26.2 %), Anaplasma ovis (22.4 %), Babesia caballi (14.8 %), Theileria equi (8.7 %), and Theileria ovis (8.5 %). The sequencing of 6 genes showed a 96-100 % nucleotide identity between the sequences in this study and those deposited in GenBank. This study provides a scientific reference for the prevention and control of tick-borne diseases in the XUAR.

First detection of Anaplasma ovis in sheep and Anaplasma platys-like variants from cattle in Menoufia governorate, Egypt.

Tick-borne diseases are of global economic importance, especially due to the costs associated with disease treatment and productivity losses in livestock. In this study, 244 livestock animals (cattle N = 92, buffaloes N = 86 and sheep N = 66) from Menoufia, Egypt were tested for Anaplasma, Ehrlichia and Babesia species using PCR. Results revealed detection of A. ovis (9.1%) in sheep while Anaplasma spp. (14.1%), A. marginale (15.2%), B. bigemina (6.5%) and B. bovis (5.4%) in cattle. On the other hand, Anaplasma spp. (1.2%), A. marginale (1.2%) and B. bovis (1.2%), were detected in buffaloes. Significantly higher detection rates were observed in cattle for Anaplasma spp. (P = .020), A. marginale (P = .001) and B. bigemina (P = .022) than in buffaloes. Sequence analysis of Anaplasma spp. isolates from cattle, revealed A. platys-like strains. Phylogenetic analyses of the A. platys-like isolates revealed variation among the strains infecting cattle. The A. marginale buffalo isolate, on the other hand, showed some level of divergence from the cattle isolates. This study reports the first detection of A. ovis in sheep and A. platys-like strains in cattle in Menoufia and Egypt at large. The results of the current study provide valuable information on the epidemiology and genetic characteristics of tick-borne pathogens infecting livestock in Egypt.

Molecular phylogeny and genetic diversity based on msp1a, groEL and gltA genes of Anaplasma ovis Tunisian isolates compared to available worldwide isolates and strains.

Anaplasma ovis, the causative agent of ovine anaplasmosis in tropical and subtropical countries, is a tick-borne obligatory intraerythrocytic bacterium of sheep, goats and wild ruminants. In Tunisia, data about the molecular phylogeny and the genetic diversity of A. ovis isolates are limited to the analysis of msp4 and groEL genes. The aim of this study was to genetic characterize 40 A. ovis isolates infecting 28 goats, 10 sheep, one camel and one Rhipicephalus turanicus tick located in different geographic regions of Tunisia on the basis of 3 partial genes (gltA, groEL and msp1a). Sequence analysis revealed 6 and 17 different genotypes in the partial gltA and groEL genes, respectively. Phylogenetic analysis revealed, as expected for the groEL gene, that sequences from small ruminants and their infesting ticks clustered separately from those isolated from camels. The analysis of amino-acid Msp1a sequences identified 18 novel genotypes of Msp1a repeats from 20 A. ovis isolates. These Msp1a repeats were highly variable with 33-47 amino-acids, and the number of repeats is one for 19 isolates infecting 18 goats and one R. turanicus tick, and 4 for a single isolate found in one sheep. Phylogenetic trees based on Msp1a partial sequences revealed that the N-terminal region of Msp1a protein appear to be relatively more informative phylogeographically compared to other markers especially according to countries. The presented data give a more detailed knowledge regarding the molecular phylogeny and the genetic diversity of A. ovis isolates occurring in different animal species and their associated ticks in Tunisia.

Molecular Detection of Anaplasma marginale and Anaplasma ovis (Rickettsiales: Anaplasmataceae) in Ixodid Tick Species in Iran.

The present study was conducted as the first molecular detection of Anaplasma species in tick samples based on the sequencing of major surface proteins 4 (msp4) gene fragments in different parts of Iran. A total of 130 tick specimens were collected from Hormozgan, Lorestan, and Guilan, Iran, within 2015 to 2017. Hyalomma asiaticum, Hyalomma dromedarii, Rhipicephalus sanguineus, and Rhipicephalus (Boophilus) species were identified in different geographical regions. An amplicon of 464-bp msp4 of Anaplasma was amplified using polymerase chain reaction in various tick species. Three sequences, including one Anaplasma marginale from R. (Boophilus) species and two Anaplasma ovis from Rhipicephalus sanguineus, were obtained after sequencing. It is concluded that bovine and ovine anaplasmosis agents are present in tick samples in Iran. The use of the gene families of six major surface proteins for the detection of various Anaplasma species is recommended.

Identification and evaluation of UL36 protein from Dermacentor silvarum salivary gland and its interaction with Anaplasma ovis VirB10.

BACKGROUND: Anaplasma ovis is a gram-negative, tick-borne obligate intraerythrocytic pathogen, which causes ovine anaplasmosis in small ruminants worldwide. VirB10 of A. ovis is an integral component of the Type IV Secretion System (T4SS). The T4SS is used by bacteria to transfer DNA and/or proteins undeviatingly into the host cell to increase their virulence. To more thoroughly understand the interaction between A. ovis and Dermacentor silvarum, a vector containing the virb10 gene of A. ovis was used as a bait plasmid to screen interacting proteins from the cDNA library of the D. silvarum salivary gland using the yeast two-hybrid system. METHODS: The cDNA of the D. silvarum salivary gland was cloned into the pGADT7-SmaI vector (prey plasmid) to construct the yeast two-hybrid cDNA library. The virb10 gene was cloned into the pGBKT7 vector to generate a bait plasmid. Any gene auto-activation or toxicity effects in the yeast strain Y2HGold were excluded. The screening was performed by combining the bait and prey plasmids in yeast strains to identify positive preys. The positive preys were then sequenced, and the obtained sequences were subjected to further analyses using Gene Ontology, UniProt, SMART, and STRING. Additionally, the interaction between the bait and the prey was evaluated using the glutathione S-transferase (GST) pull-down assay. RESULTS: A total of two clones were obtained from the cDNA library using the yeast two-hybrid system, and the sequence analysis showed that both clones encoded the same large tegument protein, UL36. Furthermore, the proteins GST-UL36 and His-VirB10 were successfully expressed in vitro and the interaction between the two proteins was successfully demonstrated by the GST pull-down assay. CONCLUSIONS: To our knowledge, this study is the first to screen for D. silvarum salivary gland proteins that interact with A. ovis VirB10. The resulting candidate, UL36, is a multi-functional protein. Further investigations into the functionality of UL36 should be carried out, which might help in identifying novel prevention and treatment strategies for A. ovis infection. The present study provides a base for exploring and further understanding the interactions between A. ovis and D. silvarum.

Molecular investigation of Anaplasma species in sheep from Heilongjiang Province, northeast China identified four Anaplasma species and a novel genotype of Anaplasma capra.

Anaplasmosis poses a great threat to the livestock industry and human health in most tropical and subtropical regions of the world. This study investigated the presence of Anaplasma in sheep from Heilongjiang Province, northeastern China. A total of 341 blood samples were detected by PCR with species-specific primers based on the msp4 gene of Anaplasma ovis, 16S rRNA gene of Anaplasma phagocytophilum and Anaplasma bovis and gltA gene of Anaplasma capra. The results showed that Anaplasma infection was found in 103 (30.2%) of 341 sheep. The infection rates were 2.6%, 8.8%, 15.8% and 10.0% for A. ovis, A. phagocytophilum, A. bovis and A. capra in sheep, respectively. Co-infection involving two Anaplasma species was found in 25 sheep (8.0%), which were usually A. phagocytophilum and A. bovis (72.0%). Co-infection involving A. phagocytophilum, A. capra, A. ovis with zoonotic potential, was found in one sheep. Sequence analysis revealed that the isolates of A. ovis, A. bovis and A. phagocytophilum identified in sheep were closely related to those previously reported in ticks and other animal hosts. Phylogenetic analysis showed that A. capra could be classified into two distinct clusters based on the gltA gene and the isolates identified in sheep from this study were clustered in the A. capra genotype II, which was clearly distinct with the human isolates. The findings in this study report four Anaplasma species and a novel A. capra genotype in sheep from northeastern China, and improve our knowledge of Anaplasma, contributing to the control of ovine anaplasmosis.

Anaplasma ovis as the suspected cause of mortality in a neonatal elk calf.

Anaplasma ovis infection is known to occur in elk experimentally, but without clinical signs or significant clinicopathologic changes. An elk farm in southern Indiana experienced the death of 3 neonates. Gross findings suggested hemolytic anemia as the cause of death. Splenic impression smears revealed numerous intra-erythrocytic parasites compatible with Anaplasma spp. Products of a semi-nested PCR targeting the msp4 gene of A. ovis were sequenced and had 100% identity with published A. ovis sequences. Given the clinical presentation, vertical transmission of A. ovis was suspected. Pathologic and molecular findings confirmed that natural A. ovis infection occurred in an elk calf.