Analysis of management factors influencing Anaplasma marginale transmission.

The aim of this study was to identify factors that play a key role in the epidemiology of bovine anaplasmosis by adapting a model primarily developed for cattle babesiosis. A cross-sectional observational study was conducted to study the proportion of calf herds in endemic stability/instability for A. marginale in a semi-arid area of Argentina. The A. marginale inoculation rate (h) was calculated from age-specific seroprevalence using double-antigen sandwich ELISA in 58 herds of 4.5-8.5-month-old calves. Herds were considered to be in endemic instability (EI) at h < 0.005 and, therefore, at risk of anaplasmosis outbreaks. A generalized linear model was performed to explore husbandry practices associated with differences in A. marginale transmission. Additionally, spatial clustering of herds with the same immunological status was analyzed using spatial scan statistics (SatScan, Bernoulli model). Spearman's correlation was used to explore a possible association between A. marginale h and Babesia bovis and B. bigemina h (data obtained in previous works). Almost half (43 %) of the herds were in the EI zone for A. marginale. Calves raised under forage combinations had a greater risk of being in EI (OR = 5.41, CI95 %OR = 1.43-20.41) than those reared exclusively on permanent pastures, where cattle density is higher (P = 0.01). Moreover, calves from herds treated only with pyrethroids to control ticks had more chances of being in EI (OR = 4.16, CI95 %OR = 1.12-15.38) than calves from herds receiving different acaricide combinations (P = 0.03). Calves from herds subjected to more than two treatments against Haematobia irritans had higher odds for EI (OR = 5.69, CI95 %OR = 1.24-26.11) than those from herds using fewer than two treatments (P = 0.02). The spatial analysis revealed no spatial clustering of the immune status of the herds (P = 0.67 and P = 0.74 for low and high incidence rates, respectively). A significant variation between farms was observed in A. marginale h (CV = 90.38 %). The correlation analysis revealed a strong epidemiological link of A. marginale h with B. bovis h (Rho=0.794, P<0.001) and B. bigemina h (Rho=0.839, P<0.001). Given that R. microplus is the only vector of B. bovis and B. bigemina in the region, the results of this work strongly suggest an active and significant role of R. microplus in the transmission of A. marginale.

Tick fever agents in Holstein calves grazing in a tropical region: predisposing factors, impact on milk production, productivity, and role of Rhipicephalus microplus in epidemiology.

The present study aimed to evaluate under dairy farm conditions the predisposing factors, impact on milk production and productivity, and the role of Rhipicephalus microplus in the epidemiology of tick fever agents in Holstein calves grazing in a tropical region. A total of 4292 pure female Holsteins were evaluated at a commercial farm. Until April 2020, calves had contact with R. microplus for between 3 and 24 months, while after April 2020, no animal had further contact with ticks. Three times a week the rectal temperature (RT) of all animals was determined, and blood samples were collected for evaluation of tick fever (TF) agents from those that showed RT >39.3 °C. Specific treatment was performed against Anaplasma marginale, Babesia bigemina and Babesia bovis when these TF agents were diagnosed in the blood smears. The number of relapses and treatments for TF agents were sub-classified into scales (1, 2, 3, 4, 5, 6 or 7-10 treatments or relapses, and animals that received blood transfusions). Within each sub-class, the health data of calves during lactation along with productivity data were analyzed. Based in the results, whether an animal received colostrum enriched with powdered colostrum substitute, whether the animal was an embryo transfer calf, and the weight at which each calf was weaned were ascertained as factors leading to more recurrences or treatments against TF agents in post-weaned calves. On average, each recurrence of TF agents that a heifer presented between three and seven months decreased milk production by 213.5 liters in the first lactation. Calves that received a blood transfusion had lower milk production at first lactation; lower weight at first fixed-time artificial insemination (FTAI); older age at first FTAI; older age at first, second, and third calving; and delayed age at third calving by 140 days compared to the farm average. R. microplus was the main agent causing clinical cases of TF on the farm, and 10,770 treatments against TF agents were carried out when calves aged between three and seven months had contact with this tick species (2018 and 2019). When the animals no longer had contact with ticks (2022 and 2023), there were no recurrences or treatments against TF agents despite the presence on the farm of S. calcitrans, which can maintain the transmission of A. marginale to the herd.

An adjuvant formulation containing Toll-like Receptor 7 agonist stimulates protection against morbidity and mortality due to Anaplasma marginale in a highly endemic region of west Africa.

Efficient cattle production and provision of animal-sourced foods in much of Africa is constrained by vector-borne bacterial and protozoal diseases. Effective vaccines are not currently available for most of these infections resulting in a continuous disease burden that limits genetic improvement. We tested whether stimulation of innate immunity using the Toll-like Receptor (TLR) 7 agonist imiquimod, formulated with saponin and water-in-oil emulsion, would protect against morbidity and mortality due to Anaplasma marginale, a tick-borne pathogen of cattle highly endemic in west Africa. In Trial 1, haplotype matched Friesian x Sanga (F1) A. marginale negative calves were allocated to either the experimental group (n = 10) and injected with the synthetic TLR 7 agonist/saponin formulation or to an untreated control group (n = 10). TLR7 agonist/saponin injected calves responded with significantly elevated rectal temperature, enlarged regional lymph nodes, and elevated levels of IL-6 post-injection as compared to control group calves. All calves were then allowed to graze in pasture for natural exposure to tick transmission. All calves in both groups acquired A. marginale, consistent with the high transmission rate in the endemic region. The need for antibiotic treatment, using pre-existing criteria, was significantly lower in the experimental group (odds ratio for not requiring treatment was 9.3, p = 0.03) as compared to the control group. Despite treatment, 6/10 calves in the control group died, reflecting treatment failures that are typical of anaplasmosis in the acute phase, while mortality in the experimental group was 1/10 (odds ratio for survival was 13.5, p = 0.03). The trial was then repeated using 45 Friesian x Sanga calves per group. In Trial 2, the odds ratios for preventing the need for treatment and for mortality in the TLR7 agonist/saponin experimental group versus the control group were 5.6 (p = 0.0002) and 7.0 (p = 0.004), respectively, reproducing the findings of the initial trial. Together these findings demonstrate that innate immune stimulation using a TLR7 agonist formulated with saponin and water-in-oil emulsion provides significant protection against disease caused by tick borne A. marginale in highly susceptible cross-bred cattle, critically important for their potential to increase productivity for smallholder farmers in Africa.

Spatial patterns of Hyalomma marginatum-borne pathogens in the Occitanie region (France), a focus on the intriguing dynamics of Rickettsia aeschlimannii.

Hyalomma marginatum is an invasive tick species recently established in mainland southern France. This tick is known to host a diverse range of human and animal pathogens. While information about the dynamics of these pathogens is crucial to assess disease risk and develop effective monitoring strategies, few data on the spatial dynamics of these pathogens are currently available. We collected ticks in 27 sites in the Occitanie region to characterize spatial patterns of H. marginatum-borne pathogens. Several pathogens have been detected: Theileria equi (9.2%), Theileria orientalis (0.2%), Anaplasma phagocytophilum (1.6%), Anaplasma marginale (0.8%), and Rickettsia aeschlimannii (87.3%). Interestingly, we found a spatial clustered distribution for the pathogen R. aeschlimannii between two geographically isolated areas with infection rates and bacterial loads significantly lower in Hérault/Gard departments (infection rate 78.6% in average) compared to Aude/Pyrénées-Orientales departments (infection rate 92.3% in average). At a smaller scale, R. aeschlimannii infection rates varied from one site to another, ranging from 29% to 100%. Overall, such high infection rates (87.3% on average) and the effective maternal transmission of R. aeschlimannii might suggest a role as a tick symbiont in H. marginatum. Further studies are thus needed to understand both the status and the role of R. aeschlimannii in H. marginatum ticks.IMPORTANCETicks are obligatory hematophagous arthropods that transmit pathogens of medical and veterinary importance. Pathogen infections cause serious health issues in humans and considerable economic loss in domestic animals. Information about the presence of pathogens in ticks and their dynamics is crucial to assess disease risk for public and animal health. Analyzing tick-borne pathogens in ticks collected in 27 sites in the Occitanie region, our results highlight clear spatial patterns in the Hyalomma marginatum-borne pathogen distribution and strengthen the postulate that it is essential to develop effective monitoring strategies and consider the spatial scale to better characterize the circulation of tick-borne pathogens.

Molecular detection and characterization of Anaplasma marginale and Babesia canis vogeli infecting dogs in Luxor, Egypt.

Tick-borne diseases in animals are increasing rapidly worldwide, but there is insufficient information about tick-borne diseases infecting dogs in southern Egypt. Thus, in the current study, we detected the presence of Anaplasma marginale (A. marginale) and Babesia canis vogeli (B. canis vogeli) in the blood of dogs. The results revealed that 4/100 (4%) were positive, and a higher infection rate was found in males (75%), than females (25%). The phylogenetic analysis for the major surface protein 4 (msp4) gene in this study was compared with amplicons separate from other reported isolates with alignment by identity 100% with cattle and camels from Egypt, and the phylogenetic analysis for the B. canis vogeli small subunit ribosomal RNA (SSU rRNA) gene in this study identified identity by 99.89% with dogs from Egypt. This report is considered the first report in southern Egypt about A. marginale in dogs based on the sequence analysis of the msp4 gene, providing new data for the classification and identification of A. marginale in dogs compared to A. marginale isolated from other animals in southern Egypt.

Differential nested patterns of Anaplasma marginale and Coxiella-like endosymbiont across Rhipicephalus microplus ontogeny.

Understanding the intricate ecological interactions within the microbiome of arthropod vectors is crucial for elucidating disease transmission dynamics and developing effective control strategies. In this study, we investigated the ecological roles of Coxiella-like endosymbiont (CLE) and Anaplasma marginale across larval, nymphal, and adult stages of Rhipicephalus microplus. We hypothesized that CLE would show a stable, nested pattern reflecting co-evolution with the tick host, while A. marginale would exhibit a more dynamic, non-nested pattern influenced by environmental factors and host immune responses. Our findings revealed a stable, nested pattern characteristic of co-evolutionary mutualism for CLE, occurring in all developmental stages of the tick. Conversely, A. marginale exhibited variable occurrence but exerted significant influence on microbial community structure, challenging our initial hypotheses of its non-nested dynamics. Furthermore, in silico removal of both microbes from the co-occurrence networks altered network topology, underscoring their central roles in the R. microplus microbiome. Notably, competitive interactions between CLE and A. marginale were observed in nymphal network, potentially reflecting the impact of CLE on the pathogen transstadial-transmission. These findings shed light on the complex ecological dynamics within tick microbiomes and have implications for disease management strategies.

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.

Genetically modified live vaccine offers protective immunity against wild-type Anaplasma marginale tick-transmission challenge.

Anaplasma marginale is a tick-borne pathogen of cattle that causes bovine anaplasmosis in tropical and subtropical regions throughout the world. Killed vaccines derived from infected erythrocytes have been used for control of this disease with limited success. Recently, we described a targeted deletion mutation in the phage head-to-tail connector protein gene of A. marginale which caused bacterial attenuation in vivo and provided protection as a modified live vaccine (MLAV). Following intravenous injection of susceptible steers, the MLAV induced protective immunity against disease progression. In the current study, we demonstrated that the immunity resulting from MLAV in cattle prevents the disease progression resulting from virulent A. marginale intrastadial transmission from infected Dermacentor variabilis male ticks. The nonimmunized control steers receiving the infection from ticks developed fever, lethargy, and inappetence for several days post tick exposure with significant decreases in the packed cell volume and increases in bacteremia. In contrast, the MLAV immunized steers remained healthy after being challenged with infected ticks and this group of animals had a significant reduction in bacteremia as compared with the controls. This study demonstrated that the A. marginale MLAV provided protection against acute tick-transmitted anaplasmosis, in addition to protection documented in steers challenge-exposed with infected blood as reported previously.

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.

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.

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.

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.

Factors associated with perceived Anaplasma marginale infection and clinical anaplasmosis cases on beef operations in California.

Bovine anaplasmosis is a tick-borne disease caused by Anaplasma marginale in the United States. The objective of this study was to use a survey tool to generate information for beef operations in California on anaplasmosis prevention and control management, including to what extent management activities were informed by perceived herd-level exposure to A. marginale infection or occurrence of clinical anaplasmosis cases. We mailed 2,621 questionnaires with questions on Anaplasma status, herd demographics, anaplasmosis control and prevention measures, and environmental factors to beef ranchers in California in October 2020. Survey-weighted chi-square tests were used to compare management differences according to perceived Anaplasma infection status. Generalized estimating equations (GEEs) were used to analyze whether region of California, management practices, or environmental factors were associated with reported clinical cases of anaplasmosis in the previous five years. A total of 466 questionnaires describing 749 herds were obtained and used in this study. Use of management measures, including deliberate exposure of calves to ticks, vaccination for Anaplasma, infection control through antibiotics in feed, maintaining a completely closed herd, blood testing for Anaplasma on all herd additions, and taking no anaplasmosis control and prevention measures, were significantly different between herds with or without perceived A. marginale infection based on producers' self-declared status. The overall perceived prevalence for Anaplasma infection and reported clinical cases of anaplasmosis at the herd level was 26.0 % (95 % CI: 24.3-27.7 %) and 17.1 % (95 % CI: 15.6-18.6 %) respectively, with the highest perceived infection and case numbers reported in the Central Coast region. In the GEE model, higher odds of reporting clinical cases of anaplasmosis in the previous five years were observed in cattle located in the Central Coast region, cattle within a large herd, cattle that are treated with tick/fly control, cattle in a completely closed herd, and cattle receiving Anaplasma vaccine. Anaplasma infection and bovine anaplasmosis status may be underestimated in beef herds in California based on previous study results. Changing needles between cattle after injections and conducting blood testing for Anaplasma on herd additions are important Anaplasma management measures that are infrequently implemented in beef herds in California. The results show a need for producer education to improve producers' awareness of bovine anaplasmosis and implement proper measures for disease control and prevention.

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.

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.

Can herd seroprevalence be used as an indicator of enzootic stability for bovine anaplasmosis? Insights from a case-control field study in Brazil.

Bovine anaplasmosis presents a significant challenge to livestock production in tropical, subtropical, and temperate regions. For many years, the concept of enzootic stability/instability (initially established for babesiosis) and herd seroprevalence as an indicator of outbreak risks have been applied to anaplasmosis. However, this model has never been definitively validated for Anaplasma marginale. The objective of this study was to examine the relationship between herd immunity (seroprevalence) and the occurrence of anaplasmosis outbreaks in Southern Brazil. A case-control study was conducted, categorizing farms into two groups: cases (farms with a history of clinical anaplasmosis) and controls (those without anaplasmosis). Thirteen farms were identified as "cases", while 23 were identified as "controls". A substantial difference in seroprevalence distribution between the two groups was observed. The majority of "control" farms exhibited over 75% of animals with antibodies to A. marginale in both calves and heifers, whereas the majority of "case" farms had a seropositive cattle percentage below 75%. Additionally, twelve months after cattle serology tests, we conducted a prospective follow-up survey to identify any clinical cases of anaplasmosis. Statistical associations (P < 0.05) were found between both retrospective and prospective anaplasmosis outbreaks and the hypothetical threshold of herd seroprevalence (75%). We hypothesize that herd seroprevalence may be an indicator of the risk of occurrence of clinical anaplasmosis. It appears that the epidemiology of cattle anaplasmosis, at least in our conditions, aligns with the well-known model of enzootic stability/instability originally applied to bovine babesiosis.

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.

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.

Control of Rhipicephalus microplus tick larvae in the field based on distancing from the host - Lone tick system.

The aim of this study was to evaluate a host-tick distancing control system using Rhipicephalus microplus in cattle. Its impact on the profiles of the pathogens Anaplasma marginale and Babesia bigemina was also assessed using enzyme-linked immunosorbent and real-time polymerase chain reaction. Experiments were conducted in Mato Grosso do Sul, Brazil. A total of 37 Senepol animals with natural tick infestation were used in this study. The ticks on the animals were counted, and pasture rotation was performed at 28-day intervals. This approach was repeated until the end of the experiment to maintain a low number of ticks, resulting in a final average of 6.3 ticks/host. Cattle maintained an immune response to the cattle tick fever (CTF) agents. DNA copies of A. marginale (X̄ 1.1) and B. bigemina (X̄ 0.05) did not vary significantly with the decrease in tick count throughout the study period. Based on these results, we conclude that a distancing period of 84 days between ticks and cattle in conditions of the Cerrado biome can control ticks in animals and maintain enzootic stability. Thus, it is possible to create productive breeds with lower tick control costs.