Anaplasma phagocytophilum effector EgeA facilitates infection by hijacking TANGO1 and SCFD1 from ER-Golgi exit sites to pathogen-occupied inclusions.

The obligatory intracellular bacterium Anaplasma phagocytophilum causes human granulocytic anaplasmosis, an emerging zoonosis. Anaplasma has limited biosynthetic and metabolic capacities, yet it effectively replicates inside of inclusions/vacuoles of eukaryotic host cells. Here, we describe a unique Type IV secretion system (T4SS) effector, ER-Golgi exit site protein of Anaplasma (EgeA). In cells infected by Anaplasma, secreted native EgeA, EgeA-GFP, and the C-terminal half of EgeA (EgeA-C)-GFP localized to Anaplasma-containing inclusions. In uninfected cells, EgeA-C-GFP localized to cis-Golgi, whereas the N-terminal half of EgeA-GFP localized to the ER. Pull-down assays identified EgeA-GFP binding to a transmembrane protein in the ER, Transport and Golgi organization protein 1 (TANGO1). By yeast two-hybrid analysis, EgeA-C directly bound Sec1 family domain-containing protein 1 (SCFD1), a host protein of the cis-Golgi network that binds TANGO1 at ER-Golgi exit sites (ERES). Both TANGO1 and SCFD1 localized to the Anaplasma inclusion surface. Furthermore, knockdown of Anaplasma EgeA or either host TANGO1 or SCFD1 significantly reduced Anaplasma infection. TANGO1 and SCFD1 prevent ER congestion and stress by facilitating transport of bulky or unfolded proteins at ERES. A bulky cargo collagen and the ER-resident chaperon BiP were transported into Anaplasma inclusions, and several ER stress marker genes were not up-regulated in Anaplasma-infected cells. Furthermore, EgeA transfection reduced collagen overexpression-induced BiP upregulation. These results suggest that by binding to the two ERES proteins, EgeA redirects the cargo-adapted ERES to pathogen-occupied inclusions and reduces ERES congestion, which facilitates Anaplasma nutrient acquisition and reduces ER stress for Anaplasma survival and proliferation.

Identification of Anaplasma marginale adhesins for entry into Dermacentor andersoni tick cells using phage display.

Anaplasma marginale is an obligate, intracellular, tick-borne bacterial pathogen that causes bovine anaplasmosis, an often severe, production-limiting disease of cattle found worldwide. Methods to control this disease are lacking, in large part due to major knowledge gaps in our understanding of the molecular underpinnings of basic host-pathogen interactions. For example, the surface proteins that serve as adhesins and, thus, likely play a role in pathogen entry into tick cells are largely unknown. To address this knowledge gap, we developed a phage display library and screened 66 A. marginale proteins for their ability to adhere to Dermacentor andersoni tick cells. From this screen, 17 candidate adhesins were identified, including OmpA and multiple members of the Msp1 family, including Msp1b, Mlp3, and Mlp4. We then measured the transcript of ompA and all members of the msp1 gene family through time, and determined that msp1b, mlp2, and mlp4 have increased transcript during tick cell infection, suggesting a possible role in host cell binding or entry. Finally, Msp1a, Msp1b, Mlp3, and OmpA were expressed as recombinant protein. When added to cultured tick cells prior to A. marginale infection, all proteins except the C-terminus of Msp1a reduced A. marginale entry by 2.2- to 4.7-fold. Except OmpA, these adhesins lack orthologs in related pathogens of humans and animals, including Anaplasma phagocytophilum and the Ehrlichia spp., thus limiting their utility in a universal tick transmission-blocking vaccine. However, this work greatly advances efforts toward developing methods to control bovine anaplasmosis and, thus, may help improve global food security.

Epidemiological, clinical, and laboratory characteristics of human granulocytic anaplasmosis in North India.

Human granulocytic anaplasmosis (HGA) is an emerging, rickettsial tick-borne disease caused by Anaplasma phagocytophilum. Sero-epidemiological data demonstrate that this pathogen has a worldwide distribution. The diagnosis of HGA requires a high index of clinical suspicion, even in endemic areas. In recent years, HGA has increasingly been reported from Asia and described in China, Japan, and Korea. We serologically and molecularly screened 467 patients with clinical suspicion of Anaplasmosis. The present study describes the epidemiology, clinical, and laboratory details of 6 confirmed and 43 probable cases of human granulocytic anaplasmosis. One of the HGA patients developed secondary invasive opportunistic Aspergillus fumigatus and Acinetobacter baumanii infection during the illness, which resulted in a fatal infection. The HGA patients without severe complications had excellent treatment responses to doxycycline. The emergence of this newly recognized tick-borne zoonotic HGA in North India is a significant concern for public health and is likely underdiagnosed, underreported, and untreated. Hence, it is also essential to establish a well-coordinated system for actively conducting tick surveillance, especially in the forested areas of the country.IMPORTANCEThe results of the present study show the clinical and laboratory evidence of autochthonous cases of Anaplasma phagocytophilum in North India. The results suggest the possibility of underdiagnosis of HGA in this geographical area. One of the HGA patients developed secondary invasive opportunistic Aspergillus fumigatus and Acinetobacter baumanii infection during the illness, which resulted in a fatal infection.

Host-pathogen associations inferred from bloodmeal analyses of Ixodes scapularis ticks in a low biodiversity setting.

Tick-borne pathogen emergence is dependent on the abundance and distribution of competent hosts in the environment. Ixodes scapularis ticks are generalist feeders, and their pathogen infection prevalence depends on their relative feeding on local competent and non-competent hosts. The ability to determine what host a larval life stage tick fed on can help predict infection prevalence, emergence, and spread of certain tick-borne pathogens and the risks posed to public health. Here, we use a newly developed genomic target-based technique to detect the source of larval bloodmeals by sampling questing nymphs from Block Island, RI, a small island with a depauperate mammalian community. We used previously designed specific assays to target all known hosts on this island and analyzed ticks for four human pathogenic tick-borne pathogens. We determined the highest proportion of larvae fed on avian species (42.34%), white-footed mice (36.94%), and white-tailed deer (20.72%) and occasionally fed on feral cats, rats, and voles, which are in low abundance on Block Island. Additionally, larvae that had fed on white-footed mice were significantly more likely to be infected with Borrelia burgdorferi and Babesia microti, while larvae that had fed on white-footed mice or white-tailed deer were significantly more likely to be infected with, respectively, mouse- and deer-associated genotypes of Anaplasma phagocytophilum. The ability to detect a nymph's larval host allows for a better understanding of tick feeding behavior, host distribution, pathogen prevalence, and zoonotic risks to humans, which can contribute to better tick management strategies. IMPORTANCE: Tick-borne diseases, such as Lyme disease, babesiosis, and anaplasmosis, pose significant public health burdens. Tick bloodmeal analysis provides a noninvasive sampling method to evaluate tick-host associations and combined with a zoonotic pathogen assay, can generate crucial insights into the epidemiology and transmission of tick-borne diseases by identifying potential key maintenance hosts. We investigated the bloodmeals of questing Ixodes scapularis nymphs. We found that avian hosts, white-footed mice, and white-tailed deer fed the majority of larval ticks and differentially contributed to the prevalence of multiple tick-borne pathogens and pathogen genotypes in a low biodiversity island setting. Unraveling the intricate network of host-vector-pathogen interactions will contribute to improving wildlife management and conservation efforts, to developing targeted surveillance, and vector and host control efforts, ultimately reducing the incidence of tick-borne diseases and improving public health.

Molecular epidemiology and associated risk factors of Anaplasma marginale in camels and possible co-infections.

Anaplasma spp. is an important pathogen that affects a wide range of animals, including camels. The current study aimed to assess the prevalence of six Anaplasma spp. in 400 camels from Ismailia, Suez, and Sharkia governorates in northern Egypt, as well as their associated risk factors and possible coinfections. Blood and fecal samples were examined using bacterial culture, the vitek2 system, and PCR. Genetic divergence among Anaplasma marginale (A. marginale) isolates was characterized using the msp4 gene. The overall prevalence of A. marginale was 19.5%. Sequencing analysis confirmed the PCR results, and a single A. marginale genotype was recognized by msp4 sequencing. The phylogenetic tree indicated that the study A. marginale isolates clustered together and were close to Egyptian A. marginale identified from buffalo (OP142725 and OP142726). Age, sex, housing type, tick infestation, body conditions, and tick control factors were significantly associated with camel anaplasmosis using a logistic regression model (odds ratio >1, P < 0.05). Multivariate logistic regression analysis revealed that the infection was 2.03, 1.9, 2.6, 1.9, and 1.8 times higher in females, semi-enclosed housing, ages >5 years, tick infestation, and emaciated camels. The risk of infection due to a tick control factor increased by 4.4 and 2.6 times when no control was applied or with irregular control, respectively. This is the first molecular report of A. marginale infection in camels in Ismailia, Suez, and Sharkia in northern Egypt, indicating a moderate prevalence of A. marginale and the involvement of multiple bacterial infections, mainly Escherichia coli and Salmonella spp. Thus, it is necessary to develop effective management and control for camel anaplasmosis.

Tick-borne encephalitis virus transmitted singly and in duo with Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum bacteria by ticks as pathogens modifying lipid metabolism in human blood.

BACKGROUND: Ticks are vectors of various pathogens, including tick-borne encephalitis virus causing TBE and bacteria such as Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum causing e.g. viral-bacterial co-infections (TBE + LB/HGA), which pose diagnostic and therapeutic problems. Since these infections are usually accompanied by inflammation and oxidative stress causing metabolic modifications, including phospholipids, the aim of the study was to assess the level of polyunsaturated fatty acids and their metabolism (ROS- and enzyme-dependent) products in the blood plasma of patients with TBE and TBE + LB/HGA before and after pharmacotherapy. METHODS: The total antioxidant status was determined using 2,20-azino-bis-3-ethylbenzothiazolin-6-sulfonic acid. The phospholipid and free fatty acids were analysed by gas chromatography. Lipid peroxidation was estimated by measuring small molecular weight reactive aldehyde, malondialdehyde and neuroprostanes. The reactive aldehyde was determined using gas chromatography coupled with mass spectrometry. The activity of enzymes was examined spectrophotometrically. An analysis of endocannabinoids and eicosanoids was performed using a Shimadzu UPLC system coupled with an electrospray ionization source to a Shimadzu 8060 Triple Quadrupole system. Receptor expression was measured using an enzyme-linked immunosorbent assay (ELISA). RESULTS: The reduced antioxidant status as a result of infection was accompanied by a decrease in the level of phospholipid arachidonic acid (AA) and docosahexaenoic acid (DHA) in TBE, an increase in DHA in co-infection and in free DHA in TBE with an increase in the level of lipid peroxidation products. The enhanced activity of enzymes metabolizing phospholipids and free PUFAs increased the level of endocannabinoids and eicosanoids, while decreased 15-PGJ2 and PGE2 was accompanied by activation of granulocyte receptors before pharmacotherapy and only tending to normalize after treatment. CONCLUSION: Since classical pharmacotherapy does not prevent disorders of phospholipid metabolism, the need to support treatment with antioxidants may be suggested.

A case of transfusion-transmission Anaplasma phagocytophilum from leukoreduced red blood cells.

BACKGROUND: Anaplasma phagocytophilum is a tick-borne bacterium and the cause of human granulocytic anaplasmosis (HGA). Here, we report a case of transfusion-transmitted (TT)-HGA involving a leukoreduced (LR) red blood cell (RBC) unit. CASE REPORT: A 64-year-old woman with gastric adenocarcinoma and multiple myeloma who received weekly blood transfusions developed persistent fevers, hypotension, and shortness of breath 1 week after receiving an RBC transfusion. Persistent fevers, new thrombocytopenia, and transaminitis suggested a tick-borne infection. RESULTS: The absence of blood parasites on thick and thin blood smears suggested that malaria and Babesia infection were not present, and the recipient tested negative for antibodies to Borrelia burgdorferi. Blood testing by polymerase chain reaction (PCR) for Ehrlichia and Anaplasma species identified A. phagocytophilum. Treatment with doxycycline resolved the infection; however, the recipient expired due to complications of her known malignancies. The recipient lived in a nursing home and did not have pets or spend time outdoors. The donor was a female in her 70s from Maine who was diagnosed with HGA 3 weeks after donating blood and whose LR-RBCs from the donation were transfused to the recipient 9 days following collection. CONCLUSION: This is a confirmed case of TT-HGA. Although rare, TT-HGA has been reported with LR-RBCs and platelets. In endemic areas, testing for tick-borne associated infections should be considered when investigating post-transfusion complications.

Dissecting the impact of Anaplasma phagocytophilum infection on functional networks and community stability of the tick microbiome.

CONTEXT: Pathogens can manipulate microbial interactions to ensure survival, potentially altering the functional patterns and microbiome assembly. The present study investigates how Anaplasma phagocytophilum infection affects the functional diversity, composition, and assembly of the Ixodes scapularis microbiome, with a focus on high central pathways-those characterized by elevated values in centrality metrics such as eigenvector, betweenness, and degree measures, in the microbial community. METHODS: Using previously published data from nymphs' gut V4 region's amplicons of bacterial 16S rRNA, we predicted the functional diversity and composition in control and A. phagocytophilum-infected ticks and inferred co-occurrence networks of taxa and ubiquitous pathways in each condition to associate the high central pathways to the microbial community assembly. RESULTS: Although no differences were observed concerning pathways richness and diversity, there was a significant impact on taxa and functional assembly when ubiquitous pathways in each condition were filtered. Moreover, a notable shift was observed in the microbiome's high central functions. Specifically, pathways related to the degradation of nucleosides and nucleotides emerged as the most central functions in response to A. phagocytophilum infection. This finding suggests a reconfiguration of functional relationships within the microbial community, potentially influenced by the pathogen's limited metabolic capacity. This limitation implies that the tick microbiome may provide additional metabolic resources to support the pathogen's functional needs. CONCLUSIONS: Understanding the metabolic interactions within the tick microbiome can enhance our knowledge of pathogen colonization mechanisms and uncover new disease control and prevention strategies. For example, certain pathways that were more abundant or highly central during infection may represent potential targets for microbiota-based vaccines.

Tick-Borne Bacterial Diseases in Europe: Threats to public health.

BACKGROUND: Tick-borne diseases, caused by bacterial pathogens, pose a growing threat to public health in Europe. This paper provides an overview of the historical context of the discovery of the most impactful pathogens transmitted by ticks, including Borrelia burgdorferi sensu lato, Rickettsia spp., Anaplasma spp., Francisella spp., Ehrlichia spp., and Neoehrlichia mikurensis. Understanding the historical context of their discovery provides insight into the evolution of our understanding of these pathogens. METHODS AND RESULTS: Systematic investigation of the prevalence and transmission dynamics of these bacterial pathogens is provided, highlighting the intricate relationships among ticks, host organisms, and the environment. Epidemiology is explored, providing an in-depth analysis of clinical features associated with infections. Diagnostic methodologies undergo critical examination, with a spotlight on technological advancements that enhance detection capabilities. Additionally, the paper discusses available treatment options, addressing existing therapeutic strategies and considering future aspects. CONCLUSIONS: By integrating various pieces of information on these bacterial species, the paper aims to provide a comprehensive resource for researchers and healthcare professionals addressing the impact of bacterial tick-borne diseases in Europe. This review underscores the importance of understanding the complex details influencing bacterial prevalence and transmission dynamics to better combat these emerging public health threats.

High genetic diversity of Anaplasma ovis in sheep from Bosnia and Herzegovina.

BACKGROUND: Ovine anaplasmosis (sensu stricto) is a rickettsial blood disease caused by the tick-borne species Anaplasma ovis. The disease is characterized by mild anemia, fever, and icterus. A more severe clinical presentation is possible in non-endemic areas. There is no existing data on the presence of Anaplasma ovis in Bosnia and Herzegovina. However, given the country's location within the Mediterranean Basin and the recent molecular detection of Babesia ovis, it is plausible that sheep in the region could naturally be infected with this tick-borne pathogen. METHODS AND RESULTS: Blood samples from 81 sheep in the Podrinje and Herzegovina areas were examined by PCR. PCR positivity was found in 38 (46.9%) cases indicating a high number of infected sheep. Mixed infections with Babesia ovis and A.ovis were observed in 63.3% of cases. A higher number of positive sheep was recorded in the area of Herzegovina. Phylogenetic analysis of the gltA, groEL, and msp4 genes of A. ovis revealed numerous genotypes and significant genetic variability. This diversity was not related to geographic origin, tick-borne infection status, or sheep breeding practices in Podrinje and Herzegovina. CONCLUSIONS: The data obtained in this study suggest that the emergence of new genotypes and the high genetic variability of A. ovis are driven by specific local and micro-environmental factors.

Molecular detection of Anaplasma bovis, Candidatus Anaplasma boleense and Rickettsia spp. in ticks infesting small ruminants.

Anaplasma spp. and Rickettsia spp. are intracellular vector-borne pathogens and harbored by a wide range of ticks and vertebrate hosts. Aim of this study was to molecularly characterize Anaplasma spp. and Rickettsia spp. in different ticks collected from livestock hosts in nine districts of Khyber Pakhtunkhwa (KP), Pakistan. In total, 862 ticks were collected from cattle, goats and sheep. Highest tick's infestation was observed on cattle 56.14% (32/57), followed by goats 45.45% (40/88), and sheep 42.05% (45/107). Rhipicephalus microplus (305/862, 35.38%) was predominant species, followed by Haemaphysalis sulcata (243/862, 28.19%), Hyalomma anatolicum (133/862, 15.42%), Haemaphysalis bispinosa (120/862, 13.92%), and Hyalomma kumari (61/862, 7.07%). A subset of 135 ticks were screened for Anaplasma spp. and Rickettsia spp. based on the amplification of partial 16 S rDNA and outer-membrane protein A (ompA) fragments, respectively. In total, 16 ticks (11.85%) were positive for Anaplasma spp. and Rickettsia spp. Obtained 16 S rDNA sequences for Anaplasma spp. detected in Ha. bispinosa and Ha. sulcata showed 99.98% identity with Anaplasma bovis, while other detected in Rh. microplus showed 99.84% identity with Candidatus Anaplasma boleense. Similarly, detected ompA sequence in Ha. sulcata showed 100% identity with Rickettsia sp. and 97.93% with Rickettsia slovaca, and another sequence detected in Rh. microplus showed 100% identity with Candidatus Rickettsia shennongii. In phylogenetic trees, these sequences clustered with corresponding species from Pakistan, China, Turkey, South Korea, South Africa, and Herzegovina. This is the first study reporting detection of A. bovis in Ha. bispinosa and Ha. sulcata, Ca. A. boleense in Rh. microplus collected from goats, and R. slovaca-like in Ha. sulcata. Our results enforce the need for regular surveillance of Rickettsiales in hard ticks infesting livestock in the region.

Molecular detection and characterization of Anaplasma ovis, Theileria ovis, and Theileria lestoquardi in sheep and goats in Luxor, Egypt.

BACKGROUND: Tick-borne diseases cause economically significant losses to animal production globally, and anaplasmosis and theileriosis are associated with the greatest losses. However, the spread of the relevant pathogens in flocks of domesticated animals in southern Egypt is little understood. Accordingly, in this study, we aimed to determine the prevalences of Anaplasma ovis, Theileria ovis, and Theileria lestoquardi in southern Egyptian sheep and goats through blood tests, and to make a molecular characterization of the A. ovis detected in sheep targeting a specific gene. RESULTS: We collected blood samples collected from 300 sheep and goats (n=150 /species) in Luxor Province in southern Egypt, and analyzed them for the presence of A. ovis, T. ovis and T. lestoquardi with screening by conventional and nested PCR targeting the msp4 and msp5, 18S rRNA, and merozoite surface protein genes. For A. ovis 140/300 samples (46.66%) were positive overall, with 90/150 (60%) and 50/150 (33.33%) positive samples in sheep and goats, respectively. Two major surface protein genes of A. ovis, msp4 and msp5, were sequenced using DNA extracted from sheep and goat blood samples, for phylogenetic analysis and genotyping. The msp4 gene sequence revealed no significant genetic diversity, to contrast to data on A. ovis strains from other countries. For T. lestoquardi, 8/150 (5.33%) samples were positive in sheep, but no samples were positive in goats (0%). For T. ovis, 32/150 (21.33%) samples were positive in sheep, but no samples were positive in goats (0%). Sequencing targeting the merozoite surface protein gene for T. lestoquardi and the small subunit ribosomal RNA gene for T. ovis revealed no significant genetic diversity in the study, another contrast to data on A. ovis strains from other countries. CONCLUSION: This study provides valuable data on phylogenetic and molecular classifications of A. ovis, T. ovis and T. lestoquardi found in southern Egyptian sheep and goats. It also represents the first report on detection and molecular characterization of T. lestoquardi in southern Egyptian sheep based on the specific merozoite surface protein gene, thus providing valuable data for molecular characterization of this pathogen in southern Egypt.

Molecular diagnosis and characterization of Anaplasma marginale and Ehrlichia ruminantium infecting beef cattle of Maputo Province, Mozambique.

BACKGROUND: Members of the Anaplasmataceae family, such as the Anaplasma and Ehrlichia species, cause economic losses and public health risks. However, the exact economic impact has not been comprehensively assessed in Mozambique due to limited data available on its basic epidemiology. Therefore, we investigated the molecular occurrence and identity of Anaplasma and Ehrlichia spp. infecting beef cattle in Maputo province, Mozambique. METHODS: A total of 200 whole blood samples were collected from apparently healthy beef cattle. Whole blood DNA was extracted and tested for presence of Anaplasma spp. and Ehrlichia ruminantium DNA through amplification of the 16S rRNA and map1 genes. Positive samples to Anaplasma spp. were subject to PCR assay targeting the A. marginale-msp5 gene. Amplicons obtained were purified, sequenced and subject to phylogenetic analyses. RESULTS: Anaplasma spp., A. marginale and E. ruminantium were detected in 153 (76.5%), 142 (71%) and 19 (9.5%) of all the samples analyzed, respectively. On this same sample group, 19 (9.5%) were co-infected with A. marginale and E. ruminantium. The 16S rRNA sequences of Anaplasma spp. obtained were phylogenetically related to A. marginale, A. centrale and A. platys. Phylogenetic analysis revealed that A. marginale-msp5 nucleotide sequences were grouped with sequences from Asia, Africa and Latin America, whereas E. ruminantium-map1 DNA nucleotide sequences were positioned in multiple clusters. CONCLUSION: Cattle in Maputo Province are reservoirs for multiple Anaplasma species. A high positivity rate of infection by A. marginale was observed, as well as high genetic diversity of E. ruminantium. Furthermore, five new genotypes of E. ruminantium-map1 were identified.

Phenology and habitat associations of the invasive Asian longhorned tick from Ohio, USA.

Geographically expanding and invading ticks are a global concern. The Asian longhorned tick (ALT, Haemaphysalis longicornis) was introduced to the mid-Atlantic US between 2010 and 2017 and recently invaded Ohio, an inland state. To date, ALTs in the US have been associated with livestock exsanguination and transmission of the agent of bovine theileriosis. To inform management, studies describing tick ecology and epidemiology of associated disease agents are critical. In this study, we described phenology, habitat and host associations, and tested for agents of medical and veterinary concern at the site of the first known established ALT population in Ohio, where pesticide treatment was applied in early fall 2021. In spring-fall 2022, we sampled wildlife (small mammals) and collected ticks from forest, edge, and grassland habitats. We also opportunistically sampled harvested white-tailed deer at nearby processing stations and fresh wildlife carcasses found near roads. Field-collected ALTs were tested for five agents using real-time PCR. We found that ALT nymphs emerged in June, followed by adults, and concluded with larvae in the fall. ALTs were detected in all habitats but not in wildlife. We also found a 4.88% (2/41) prevalence of Anaplasma phagocytophilum across ALT adults and nymphs. Host and habitat associations were similar to other studies in the eastern United States, but two potential differences in phenology were identified. Whether ALTs will acquire more endemic disease agents requires further investigations. Our findings provide the first evidence regarding ALT life history from the Midwest region of the United States and can inform exposure risk and guide integrated management.

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.

Tick-borne microorganisms in Amblyomma tigrinum (Acari: Ixodidae) from the Patagonian region of Argentina.

This study presents the results of the molecular detection of tick-borne microorganisms in Amblyomma tigrinum Koch collected near the city of Viedma, Río Negro, Argentina. Ticks were collected in their non-parasitic stage, on pet dogs and on Lycalopex gymnocercus (Pampa fox). Also, six tick samples from humans were analyzed. All ticks were morphologically identified to species level and genomic DNA was extracted. The DNA samples were examined by end point PCR assays to amplified DNA of Anaplasma sp., Babesia sp., Ehrlichia sp., Rickettsia sp. and Theileria sp. Although all tested DNA samples from the collected ticks resulted negative to the detection of Piroplasmida and Rickettsia spp., 16 samples (16.5%, including all hosts) were positive in the 16S rDNA gene PCR that detects bacteria from the Anaplasmataceae family. Phylogenetic analysis of seven obtained partial sequences resulted in the identification of three bacteria: two Ehrlichia spp. (related to Ehrlichia sp. strain Iberá and strain Viedma) and Candidatus Anaplasma boleense. The latter finding represents the first detection of this novel Candidatus species in A. tigrinum. Based on the results of this study, it must be assumed that the diversity of bacteria of the Anaplasmataceae family in Argentina is greater than previously thought, and that these bacteria can infect a wide range of domestic and wild animals.

Does parity order or cow antibody level influence the humoral immunity of calves for cattle anaplasmosis?

Anaplasmosis is a widely distributed vector-borne disease of cattle caused by the bacteria Anaplasma marginale, which may lead to severe losses in beef and dairy cattle production. Anecdotal information among farmers suggested that some cows may deliver calves more resilient to anaplasmosis. To investigate this, we tested two hypotheses: (i) whether the parity order or (ii) cow antibody levels may influence the humoral immunity of pre-weaning calves against cattle anaplasmosis. For this study, we sampled 170 cattle (Bos taurus taurus, Angus breed) on a farm in Southern Brazil, comprising 85 cows (50 multiparous and 35 primiparous) and their 85 calves (50 days old). Antibodies against A. marginale were investigated using iELISA. Eighty percent of the animals were considered seropositive (100% of the cows and 60% of the calves). There was no significant difference in seroprevalence between calves from primiparous and multiparous cows. However, calves from multiparous cows exhibited higher antibody levels. To address the second question, we classified cows based on their antibody levels to A. marginale (high or low). Calves from cows with high antibody levels also showed elevated antibody levels against A. marginale. Furthermore, calves from cows with high antibody levels had approximately four times greater odds of being seropositive for A. marginale at 50 days old than those born to mothers with low antibody levels. In conclusion, the calf's antibody level against A. marginale appears to be related to the cow's parity order and the mother's antibody level.

Exploring the relationship between flumethrin resistance and Anaplasma marginale infection in Rhipicephalus (Boophilus) microplus ticks of cattle.

The study explores the relationship between flumethrin resistance and Anaplasma marginale infection in Rhipicephalus (Boophilus) microplus of cattle in South Gujarat, India. Adult Immersion Test (AIT) was used to assess flumethrin resistance and polymerase chain reaction (PCR) to confirm A. marginale infection. Species-specific PCR resulted in the amplification of 576 bp of msp5 gene of A. marginale in 17.69% (49/277) groups of ticks, and subsequent digestion with EcoRI cleaved it into two distinct segments. Navsari district, noted level Ι resistance [resistance factors (RF) = 1.78-3.34], and A. marginale prevalence was 16.67, 15.38, 23.08, 15.38, and 11.76% in Navsari, Jalalpore, Gandevi, Chikhli, and Vansda sub-districts, respectively. Similarly, Vyara and Dolvan sub-districts of Tapi observed level Ι resistance (RF = 1-3.63), with A. marginale positivity of 21.43 and 22.22%, while Valod and Songhad demonstrated susceptibility, with 14.29 and 12.50% of A. marginale, respectively. Moving to Surat, the Mahuva, Bardoli, Mandvi, Palsana, and Kamrej sub-districts observed the level Ι resistance (RF = 1.94-2.89), coupled with 14.29, 17.65, 20, 20, and 21.43% of A. marginale, respectively. Lastly, in Valsad district, Dharampur, Kaparada, Valsad, and Umbergaon noted level Ι resistance (RF = 1.67-1.81), and corresponding A. marginale positivity rates of 18.18, 19.23, 15.00, and 20.00%. The scatter plot unveiled a significant moderate positive correlation between RF and A. marginale positivity% (p = 0.0362), characterized by a Pearson correlation coefficient (r) of 0.4963. The covariance (1.1814) highlighted fluctuations, while the coefficient of determination (r2) (0.2463) clarified that 24.63% of the variability in A. marginale positivity% could be attributed to the RF.

Insights into the involvement of male Hyalomma anatolicum ticks in transmitting Anaplasma marginale, lumpy skin disease virus and Theileria annulata.

Ticks can transmit viruses, bacteria, and parasites to humans, livestock, and pet animals causing tick-borne diseases (TBDs) mechanically or biologically in the world. Lumpy skin disease virus, Anaplasma marginale, and Theileria annulata inflict severe infections in cattle, resulting in significant economic losses worldwide. The study investigated the potential transmissions of LSDV, A. marginale, and T. annulata through male Hyalomma anatolicum ticks in cattle calves. Two 6-month-old Holstein crossbred calves designated as A and B were used. On day 1, 15 uninfected female ticks (IIa) and infected batch of 40 male ticks (I) were attached on calf A for 11 days. Filial transmission of the infections was observed in female ticks (IIb) collected from calf A, where 8 female ticks had been co-fed with infected male ticks. The blood sample of calf B was found positive through PCR for the infections. The larvae and egg pools obtained from the infected ticks were also tested positive in PCR. The study confirmed the presence of these mixed pathogens and potential intra-stadial and transovarial transmissions of A. marginale, T. annulata, and LSDV in male and female ticks of H. anatolicum and experimental calves to establish the feasibility of infections through an in vivo approach.

Current status of ruminant anaplasmosis in Algeria: a systematic review and meta-analysis.

Anaplasmosis is an emerging infectious disease that is being recognised all over the world, with impact on animal health.This systematic review and meta-analysis aimed to assess the rate infection of Anaplasma spp. infection in Algerian ruminants. Three databases were searched to identify eligible studies for the final systematic review and meta-analysis. The 'meta' package in the R software was used for the meta-analysis, and the random effects model was chosen to pool the data. Meta-analysis encompasses 14 research papers spanning 19 years (2004-2023), out of an initial pool of 737 articles retrieved from various databases. The study included a total of 1515 cattle, 190 sheep, and 310 goats, and the overall Anaplasma infection rate was estimated at 28% (95% CI, 17-41%). The analysis revealed varying infection rates among species, with cattle at 20%, sheep at 30%, and goats at 61%. Five classified species and two unclassified strains belonging to Anaplasma genus were identified in ruminants, which are A. marginale, A. centrale, A. bovis, A. ovis, A. phagocytophilum, A. phagocytophilum-like strains, and A. platys-like strains. Among these, A. marginale was prevalent in ten out of eleven cattle studies. The data also revealed regional variations, with Northeastern Algeria showing a higher infection rate (26%) compared to North-central Algeria (9%). In the subgroup analysis, clinically healthy cattle had a higher infection rate (28%) compared to suspected disease cattle (16%). Molecular biology screening methods yielded a significantly higher infection rate (33%) than microscopy (12%). Gender analysis suggested slightly higher infection rates among male cattle (19%) compared to females (16%). Age analysis indicated that Anaplasma infection was more common in cattle less than 12 months (14%) compared to those over 12 months (9%). This systematic review provides valuable insights, highlighting the need for continued surveillance and potential preventive strategies in different regions and among different animal populations in Algeria.