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.

Anaplasmosis outbreaks in taurine and zebu cattle in Brazil: causes, treatments, and Anaplasma marginale genetic diversity.

This study aimed to investigate seven outbreaks of A. marginale infection in two regions of Brazil, affecting taurine, zebu, and crossbred cattle. We assessed the possible causes, treatment measures, and genetic diversity of A. marginale. These outbreaks occurred in two states (Goiás: outbreaks 1-7; Mato Grosso do Sul: outbreak 3), breeds (Holstein, Nellore, and crossbreed), age groups (beef cattle: 18-25 days old and 7-8 months; dairy cattle: 18-25 days old, 13-14 months, and cow after the first birth) and rearing systems (feedlot, pasture, pen in a wood shaving bedding system and compost bedded-pack barns). Metaphylactic or prophylactic treatments varied according to outbreak (imidocarb dipropionate: outbreaks 1-4 and 6; enrofloxacin: outbreaks 5 and 7; diminazene diaceturate: outbreak 5). In outbreaks 6 and 7, the packed cell volume was monitored. In all outbreaks, the practice of needle/syringe sharing was discontinued. For outbreaks 1-3, clinical signs and mortality (range, 4.8-13.3%) occurred 36-45 days after entry into the feedlot. In outbreak 4, A. marginale was diagnosed in 66.2% of the calves (bacteremia, 0-4.5%), with a mortality of 8.6%. Among nursing calves aged 60 days during outbreak 5, 53.8% were infected with A. marginale, with average bacteremia of 2.7% (range, 0-21.3%), and a mortality of 13.8%. In dairy heifers aged 14 months, raised in paddocks lacking vegetation cover and infested with R. microplus, then transitioned to a rotational grazing system also infested with R. microplus, the A. marginale bacteremia ranged from 3.2 to 6.7%, with a mortality of 20%. Before monitoring during outbreak 7, the mortality was 17.9%, but no further deaths were observed after monitoring initiation. In conclusion, possible causes triggering the outbreaks included primary tick infestation, needle/syringe sharing, and stress factors which may have affected the immunological statues of animals in the feedlots. Control measures performed in all outbreaks were effective. The partial msp4 gene sequences of A. marginale generated herein belonged to two haplotypes, but further research would be needed to investigate if this finding has any clinical significance.

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.

Epidemiological assessment of Anaplasma marginale, Babesia bigemina, and Babesia bovis infections in Colombian creole cattle breeds: A molecular survey in northeastern Colombia.

Anaplasmosis and babesiosis are globally distributed arthropod-borne diseases known for causing substantial economic losses due to their high morbidity and mortality rates. This study aims to assess the frequency and epidemiological features associated with the infection of Anaplasma marginale, Babesia bigemina, and Babesia bovis in three Creole cattle breeds (Chino Santandereano (Chino), Casanareño (CAS), and Sanmartinero (SM)) in northeastern Colombia. Between June 2019 and March 2020, a total of 252 Creole cattle were sampled, with Chino, CAS, and SM accounting for 42.8%, 29.5%, and 29.5% of the samples, respectively. Blood samples were subjected to molecular analysis to detect the DNA of A. marginale, B. bigemina, and B. bovis, using species-specific primers. Additionally, Packed Cell Volume (PCV), total serum proteins, and body condition were evaluated. Molecular analyses revealed the presence of B. bigemina, A. marginale, and B. bovis in 83.7% (211/252; 95% CI = 79.1%-88.3%), 59.9% (151/252; 95% CI = 53.8%-66.1%), and 40.9% (103/252; 95% CI = 34.7%-46.9%) of the samples, respectively, with 69% (174/252; 95% CI = 57.8%-80.3%) exhibiting coinfections. Notably, in infected animals, no significant alterations in PCV, total serum proteins, or body condition were observed. Multivariate analyses indicated a statistically significant association between the frequency of A. marginale infection and the breed and season, with a higher frequency in SM during the rainy season (P < 0.05). To our knowledge, this is the first molecular survey that evaluates multiple arthropod-borne pathogens in Colombian Creole breeds. The results revel a high frequency of B. bigemina and A. marginale infections, coupled with a notable frequency of coinfections, all without significant alteration in the PCV, total serum proteins and body conditions. Our findings enhance the understanding of the epidemiological aspects of arthropod-borne pathogens in Colombian Creole breed and contribute to the improvement of sanitary programs for these animals.

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.

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.

Leading report of molecular prevalence of tick borne Anaplasma marginale and Theileria ovis in yaks (Bos grunniens) from Pakistan.

Domestic yak (Bos grunniens) is an economically important feature of the mountainous region of Gilgit-Baltistan in Pakistan where agriculture is restricted and yaks play multiple roles which includes being a source of milk, meat, hides, fuel and power. However little is known about the parasitic infections in Pakistani yaks. Aim of this research was to report the prevalence and genetic diversity of protozoa parasite (Theileria ovis, 18 S rDNA gene was targeted) and an obligate bacterium (Anaplasma marginale, msp-1 gene was amplified) in the blood that was sampled from 202 yaks collected from four districts in Gilgit-Baltistan during January 2023 till January 2024. Results revealed that 6/202 (3%) yaks were of Theileria ovis while 8/202 (4%) were Anaplasma marginale infected. Positive PCR products of both parasites were confirmed by DNA sequencing and their similarity with previously available pathogen sequences was determined by BLAST analysis. Phylogenetic tree indicated that isolates of both parasites displayed genetic. Anaplasma marginale infection varied with the sampling districts and Shigar district had the highest rate of bacterial infection. Cows were significantly more prone to Theileria ovis infection than bulls. Calf and hybrid yaks were more prone to Anaplasma marginale infection. In conclusion, this is the first report that yaks residing the Gilgit-Baltistan region in Pakistan are infected with Theileria ovis and Anaplasma marginale. Similar larger scales studies are recommended in various regions of Gilgit-Baltistan to document the infection rates of these parasites to formulate strategies that will lead to the effective control of these pathogens.

Molecular and serological detection of Anaplasma spp. in small ruminants in an area of Cerrado Biome in northeastern Brazil.

Anaplasmosis, caused by bacteria of the genus Anaplasma, is an important tick-borne disease that causes economic losses to livestock farms in many countries. Even though Anaplasma spp. have been detected in goats and sheep worldwide, few studies investigate the occurrence and genetic identity of these agents in small ruminants from Brazil. Thus, this work aimed to detect and determine the genetic identity of Anaplasma spp. in small ruminants from the Baixo Parnaíba region, state of Maranhão, northeastern Brazil. For this purpose, blood samples were collected from 161 animals (91 goats; 70 sheep) from 4 municipalities in the Baixo Parnaíba region. Sheep and goat serum samples were subjected to recombinant membrane surface protein (MSP5)-based iELISA. Whole blood samples were subject to DNA extraction and molecular diagnosis using PCR assays for Anaplasma spp. targeting msp1ß, msp1α, 16S rRNA and msp4 genes. Positive samples were sequenced and then subjected to Anaplasma marginale msp1α genetic diversity analysis and phylogenetic inferences based on the 16S rRNA and msp4 genes. The serological survey detected the presence of anti-A. marginale IgG antibodies in 18 animals (11.1%): 2.9% (2/70) sheep and 17.4% (16/91) goats. Anaplasma marginale DNA was detected in 2 goats (1.2%) using qPCR based on the msp1ß gene. Two distinct A. marginale msp1α strains, namely α ß and α ß ΓγΓγΓγΓγ were found in the infected goats, each one found in a different animal, both belonging to the H genotype. Phylogenetic analysis based on the 16S rRNA gene showed the sequences positioned in three different clades and grouped with sequences from 'Candidatus Anaplasma boleense', A. platys and A. marginale. Phylogenetic inferences based on the msp4 gene positioned the sequence variants in the A. marginale clade. The present work represents the first molecular detection of sequence variants phylogenetic associated to 'Candidatus Anaplasma boleense' and A. platys and α ß and α ß ΓγΓγΓγΓγ in goats from Brazil.

Molecular detection and associated risk factors of Anaplasma marginale, A. ovis and A. platys in sheep from Algeria with evidence of the absence of A. phagocytophilum.

Anaplasma species are obligate intracellular rickettsial pathogens that cause significant diseases in animals and humans. Despite their importance, limited information on Anaplasma infections in Algeria has been published thus far. This study aimed to assess the infection rate, characterize Anaplasma species, and identify associated risk factors in selected sheep farms across Oum El Bouaghi region in Algeria. In 2018, we collected 417 blood samples from sheep (Ovis aries) and performed molecular characterization of Anaplasma species infecting these animals. This characterization involved the use of 16S rRNA, msp2, rpoB, and msp5 genes, which were analyzed through nested PCR, qPCR, cPCR, DNA sequencing, and subsequent phylogenetic analysis. Our findings revealed infection rates of 12.7 % for Anaplasma species detected, with Anaplasma ovis at 10.8 %, Anaplasma marginale at 1.7 %, and Anaplasma platys at 0.2 %. Interestingly, all tested animals were found negative for Anaplasma phagocytophilum. Statistical analyses, including the Chi-square test and Fisher exact test, failed to establish any significant relationships (p > 0.05) between A. ovis and A. platys infections and variables such as age, sex, sampling season, and tick infestation level. However, A. marginale infection exhibited a significant association with age (p < 0.05), with a higher incidence observed in lambs (5.2 %) compared to other age groups. Remarkably, this study represents the first molecular detection of A. platys and A. marginale in Algerian sheep. These findings suggest that Algerian sheep may serve as potential reservoirs for these pathogens. This research contributes valuable insights into the prevalence and characteristics of Anaplasma infections in Algerian sheep populations, emphasizing the need for further investigation and enhanced surveillance to better understand and manage these diseases.

Systematic Review and Meta-Analysis on Piroplasma spp. Infection and Co-infection with Anaplasma marginale in Domestic Ruminants from Algeria.

BACKGROUND: Piroplasmosis and anaplasmosis stand out as the primary diseases affecting livestock during periods of tick activity. These vector-borne diseases continue to emerge worldwide, exerting a detrimental impact on both animal health and national economies. The purpose of this study is to assess the prevalence of Piroplasma spp. and its co-occurrence with Anaplasma marginale in domestic ruminants in Algeria. METHODS: Three databases were systematically reviewed to identify eligible studies for the final meta-analysis, following the PRISMA statement. The 'meta' package in the R software was employed for the meta-analysis with the random effects model chosen for data pooling. RESULTS: The meta-analysis encompasses 14 research papers spanning a 19-year period (2004-2023). Theileria spp. was identified in all studies, covering 1675 cattle, 190 sheep, and 128 goats, yielding an overall Theileria infection rate of 45% (95% CI 26-65%). Specifically, cattle had a 59% infection rate, while sheep and goats had rates of 18% and 20%, respectively. Babesia spp. was found in nine studies, involving 1183 cattle and 190 sheep, resulting in an overall Babesia infection rate of 7% (95% CI 4-15%), with cattle and sheep having rates of 10% and 3%, respectively. Notably, eight Piroplasma species T. annulata, T. orientalis, T. buffeli, T. equi, Theileria sp., B. bovis, B. bigemina, and B. occultans were detected in cattle, with T. annulata being the most prevalent at 54%. Regional disparities and host factors also impacted infection rates, with higher rates in Northeastern Algeria and among suspected disease cattle. Additionally, gender, age, and breed influenced cattle susceptibility to Theileria infection. Furthermore, six distinct co-infections between Piroplasma spp. and A. marginale were observed, with T. annulata/A. marginale identified in six studies, demonstrating an 8.3% co-infection rate. CONCLUSION: This analysis offers crucial insights into the current status of Piroplasmosis and its co-infection with A. marginale in Algerian domestic ruminants, providing valuable data for surveillance and prevention strategies.

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.

Molecular characterization and phylogeny of Anaplasma marginale, A. phagocytophilum and A. bovis in livestock of Bangladesh.

The emergence of Tick-borne Anaplasma spp. poses a significant threat to humans and animals worldwide. Traditional surveys based on examining blood smears overlook the existence of emerging pathogens. This study aimed to screen Anaplasma spp. in livestock species from diverse geographies with molecular tools. We collected 276 blood samples from cattle (Bos indicus), gayals (Bos frontalis) and goats (Capra hircus) in Jhenaidah, Bogura, Sirajganj and Bandarban districts, and Naikhongchari sub-district from June 2021 to March 2022. After that, a molecular screening was conducted through polymerase chain reaction (PCR) and sequencing was done to confirm the PCR results. The PCR assays were performed based on the analyses of groEL (Anaplasma marginale) and 16S rRNA (A. phagocytophilum and A. bovis). The Anaplasma spp. detected in this study were A. marginale (10.51%), A. phagocytophilum (0.72%), and A. bovis (63.77%). However, A. platys was not detected in this study. Among the screened pathogens, the detection of A. bovis (82.86%) was significantly high in the Bandarban district, while A. marginale was found only in cattle in this location. Regarding animal species, the occurrence of A. bovis was significantly higher in cattle. Moreover, the detection rate of A. marginale was significantly higher in adult cattle (≥2 years). The phylogenetic analyses revealed that the groEL sequences of A. marginale and 16S rRNA sequences of A. bovis and A. phagocytophilum were included in a single clade in the respective phylograms, showing a single genotype of each species circulating in Bangladesh. This study reports the existence of A. phagocytophilum in Bangladesh for the first time.

Simultaneous molecular detection of Anaplasma marginale and Theileria annulata in cattle blood samples collected from Pakistan-Afghanistan boarder region.

Theileria annulata (T. annulata) and Anaplasma marginale (A. marginale) are among the most extensively reported tick borne pathogens and are associated with huge economic losses worldwide. A total of 298 cattle blood samples were screened to report the presence of these two pathogens. The samples were collected from apparently healthy cattle (Achai, n = 155, Jersy, n = 88 and crossbred, n = 55) in Bajaur district of Khyber Pakhtunkhwa (KPK) during June and July of 2022. A total of 31 out of 298 cattle (10.4%) were found infected with T. annulata as PCR amplified a 156 base pair fragment from Tams-1 gene of T. annulata from their blood. While 16/298 animals (5.4%) were found infected with A. marginale as they amplified a 382 base pair fragment specific for msp5 gene of this bacterium. Three animals (1%) were found co infected. Cattle susceptibility to T. annulata infection was significantly higher than A. marginale infection (P < 0.001). Phylogenetic analysis revealed that Pakistani isolates of both detected pathogen clustered together and were closely related isolates from worldwide countries. Prevalence of T. annulata varied significantly among the sampling sites (P = 0.05) while no such association was observed for A. marginale among the tested cattle. Epidemiological data analysis revealed that none of the studied risk factors was found associated either with the prevalence of T. annulata or A. marginale (P > 0.05) among enrolled cattle. In conclusion, our study has revealed a relatively higher prevalence of T. annulata than A. marginale in cattle from the Bajaur district in KPK. This information is important for improving the productivity of the livestock sector, which is one of the main sources of income in the country. It is recommended that this data be taken into account for the development and implementation of effective tick control programs, as well as for the improvement of livestock management practices to prevent and manage TBDs in Pakistan.

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.

Molecular prevalence of Anaplasma marginale in ruminants and Rhipicephalus ticks in northern Pakistan.

Anaplasma marginale is the most prevalent tick-borne haemoparasite of cattle and causes huge economic losses to the dairy industry worldwide. This study aimed to determine the occurrence of A. marginale infection in blood and tick samples collected from livestock animals in the districts located in Khyber Pakhtunkhwa (KPK), Pakistan. A total of 184 blood and 370 tick samples were included in this study. It has never been reported that sheep, goats, and cattle in Tank, Ghulam Khan, Birmil and Miran Shah areas were infected with A. marginale. All samples of blood and ticks were collected through random sampling from March 2021 to January 2022 from cattle, sheep and goats and screened through PCR for anaplasmosis by using primer pairs of Anaplasma spp. Three hundred and seventy ticks were collected from infested hosts (120/184, 64.21%). Among the four morphologically identified tick species, the highest occurrence was recorded for Rhipicephalus sanguineus (n=138, 37.29%), followed by Rhipicephalus microplus (n=131, 35.4%), Rhipicephalus annulatus (n=40, 10.81%), Hyalomma anatolicum (n=31, 8.37%), and Hyalomma marginatum (n=30, 8.1%). The occurrence of female tick was highest (n=160, 43.24%), followed by nymphs (n=140, 37.38%) and males ticks (n=70, 18.9%). Among these ticks, A. marginale was detected in female ticks of R. microplus, and R. sanguineus. Molecular identification of A. marginale was confirmed in 120 out of 184 blood samples and 6 out of 74 tick samples. Overall, occurrence of A. marginale in blood and tick samples was found to be 65.21% and 8.1% respectively. Species-wise occurrence in blood samples of goats were 71.11% followed by sheep 68.31% and cattle 50%. Specie-wise occurrence of A. marginale in tick samples of cattle were 12.5% followed by goats 6.89%. The obtained sequence showed similarity with A. marginale reported from Kenya and USA. We report the first PCR based detection of A. marginale infection in blood samples and in R. sanguineus ticks of goats simultaneously.

Loop-mediated isothermal amplification assay for the rapid detection of Anaplasma marginale in cattle based on major surface protein 5 gene.

BACKGROUND & OBJECTIVES: Timely intervention is needed to minimize the economic losses of vector-borne bovine anaplasmosis which can be possible by the isothermal amplification assay. METHODS: Anaplasma marginale in the cattle of south Gujarat, India was detected in the PCR and LAMP by amplifying the fragment of msp5 gene. The PCR product was digested with EcoRI, and sequenced to confirm its pathogen specific detection. RESULTS: Species specific PCR observed a band of 457 bp of msp5 DNA following 1% agarose gel electrophoresis. Positive LAMP reaction turned into yellow colour while negative sample depicted original pink colour. A detection limit of PCR and LAMP was up to 10-6 and 10-8 of the original genomic DNA of A. marginale, respectively. A single cut site of EcoRI was observed in the PCR product. Current msp5 DNA sequences of A. marginale (MW538962 and MW538961) showed 100% homology with the published sequences. Monophyletic lineage type relationship was observed with high bootstrap proportion among the msp5 DNA sequences of A. marginale in the phylogram. Prevalence rate of A. marginale was significantly higher (p<0.05) in the PCR [43/280 (15.36%)] and LAMP [62/280 (22.14%)] than the microscopic technique [17/280 (6.07%)]. Diagnostic sensitivity, specificity, positive and negative predictive values at 95% CI for LAMP assay with respect to PCR were 93.02%, 90.72%, 64.52% and 98.62%, respectively. INTERPRETATION & CONCLUSION: Thus LAMP can be a practical alternative to the PCR for the diagnosis of A. marginale infection in the cattle even in field condition.

The Use of the Antigenically Variable Major Surface Protein 2 in the Establishment of Superinfection during Natural Tick Transmission of Anaplasma marginale in Southern Ghana.

Many vector-borne pathogens, including Anaplasma spp., Borrelia spp., Trypanosoma spp., and Plasmodium spp., establish persistent infection in the mammalian host by using antigenic variation. These pathogens are also able to establish strain superinfection, defined as infection of an infected host with additional strains of the same pathogen despite an adaptive immune response. The ability to establish superinfection results in a population of susceptible hosts even with high pathogen prevalence. It is likely that antigenic variation, responsible for persistent infection, also plays a role in the establishment of superinfection. Anaplasma marginale, an antigenically variable, obligate intracellular, tickborne bacterial pathogen of cattle, is well suited for the study of the role of antigenically variant surface proteins in the establishment of superinfection. Anaplasma marginale establishes persistent infection by variation in major surface protein 2 (msp2), which is encoded by approximately six donor alleles that recombine into a single expression site to produce immune escape variants. Nearly all cattle in regions of high prevalence are superinfected. By tracking the acquisition of strains in calves through time, the complement of donor alleles, and how those donor alleles are expressed, we determined that simple variants derived from a single donor allele, rather than multiple donor alleles, were predominant. Additionally, superinfection is associated with the introduction of new donor alleles, but these new donor alleles are not predominantly used to establish superinfection. These findings highlight the potential for competition among multiple strains of a pathogen for resources within the host and the balance between pathogen fitness and antigenic variation.

Molecular characterization and prevalence of bovine hemoprotozoan and rickettsial organism from Bhubaneswar, Eastern India.

The blood parasites adversely affect the bovine population and are one of the major hindrances to productivity and health of livestock, globally. This study aimed to study the prevalence of some bovine hemoparasites, their molecular characterization, and associated risk factors in and around Bhubaneswar, a place in Eastern India. Microscopy (Giemsa stained thin blood smear) and polymerase chain reaction (PCR)-based prevalence studies were undertaken for a total of 106bovine blood samples belonging to different breed, age, and sex presented to Veterinary Clinical Complex. DNA was extracted by conventional method and primer targeting msp5 gene (382 bp) for Anaplasma marginale, Tams1 gene (572 bp) for Theileria annulata and VSG RoTat 1.2 gene (110 bp) for Trypanosoma evansi, were used. Sequencing and phylogenetic analysis were also carried out for representative PCR products. The risk factors were correlated with prevalence by logistic regression analysis. Blood smear examination revealed a prevalence an overall of 48.11% (51/106) out of which the prevalence of T. annulata, A. marginale and T. evansi, and concurrent infection were reported to be 26.42%, 10.38%, 3.77%, and 7.55% respectively. All the samples found positive in microscopy were also found positive in PCR. PCR assay revealed an overall prevalence of 73.58% (T. annulata: 37.74%, A. marginale:17.92%, T. evansi: 5.66% and concurrent infection:12.26%). A higher prevalence was observed in the age group of 2-4 years (37.74%), females (61.32%) and cross-bred Jersey (50%). Representative PCR products were sequenced and assigned by GenBank (OL550058: A. marginale; OL604429: T. annulata and OL550059: T. evansi). There is a paucity of disease mapping of these parasites in most parts of the country. The study is probably the first report of molecular characterization of T. evansi and A. marginale from the region which gives some information of possible genetic diversity of the isolates circulating in the region. Future research should include large-scale epidemiological surveys as well as concurrent diagnosis of these hemoparasites for better treatment and control strategies.

Molecular of Anaplasma marginale Theiler (Rickettsiales: Anaplasmataceae) in horseflies (Diptera: Tabanidae) in Uruguay.

Anaplasma marginale is transmitted biologically by infected ticks or mechanically by biting flies and contaminated fomites. In tick-free areas, such as southern Uruguay, horseflies could be the principal vectors of this pathogen for bovines, causing anaplasmosis. The objective of this work was to detect the presence of A. marginale by MSP-5 PCR and Sanger sequencing in the most prevalent species of horseflies obtained using different collection methods in Colonia, Tacuarembó and Paysandú, Uruguay. Eight horsefly species were tested (Dasybasis missionum, Poeciloderas lindneri, Tabanus campestris, T. claripennis, T. fuscofasciatus, T. platensis, T. tacuaremboensis and T. triangulum); four species were found to be positive for A. marginale, with D. missionum and P. lindneri having the most frequent infections, while only one individual each of T. fuscofasciatus and T. tacuaremboensis was positive. Both D. missionum and P. lindneri were positive for A. marginale in tick-free areas, and the implications are discussed in this report.

Anaplasma marginale: Molecular discrimination, recombinant expression and characterization of major surface protein 2.

A. marginale's major surface protein 2 (MSP2) is an immunodominant protein that is encoded by a multigene family. Phylogenetic analysis revealed that the msp2 sequence Thailand strain was clustered in third clade, with similarity values between 90.4 and 100%. The haplotype diversity showed 10 haplotypes of the msp2 genes. The entropy analysis of the nucleic and amino sequences revealed 289 and 117 high entropy peaks, respectively. Interestingly, one predicted allele belonging to MHC-II represented the hypervariable region (HVR) of MSP2. A. marginale's recombinant MSP2 (rAmMSP2), which has a molecular weight of 42 kDa, was examined in SDS-PAGE. Antigenicity of rAmMSP2 (42 kDa) and AmMSP2 (36 kDa) showed the conserved epitopes. The distribution of AmMSP2 on infected erythrocytes' membrane and outside was demonstrated by immunofluorescence detection. Therefore, the rMSP2 could be utilized in the establishment of immunodiagnostic tools and vaccine approaches for the monitoring of anaplasmosis.