Molecular prevalence of Anaplasma spp. in cattle and assessment of associated risk factors in Northeast Thailand

Background and Aim: Anaplasma spp. are common rickettsia species described in ruminant hosts, including cattle. The clinical signs of anaplasmosis range from asymptomatic to mortality. However, there are insufficient studies on epidemiology surveys of this blood pathogen. This study aimed to estimate the prevalence and risk factors of anaplasmosis in beef and dairy cattle in Northeast, Thailand. Materials and Methods: A total of 187 blood samples of beef and dairy cattle were collected from five provinces in Northeast Thailand. Anaplasma spp. infections were screened by microscopic examination and polymerase chain reaction targeting specific genes (msp4 gene for Anaplasma marginale and 16S rRNA gene for Anaplasma platys and Anaplasma bovis). Moreover, the associated risk factors for the infections were evaluated. Results: Overall, blood samples from cattle revealed that 17.6% (33/187) were positive for Anaplasma spp. by microscopic examination and 20.8% (39/187) were positive by DNA amplification. Of these 20.8%, 17.6% were A. marginale and 3.2% were A. platys. However, A. bovis infection was not detected. Infection with Anaplasma spp. and A. marginale showed a significant association with breed and gender (p < 0.05) while age and packed cell volume levels showed no significant statistical relationship between Anaplasma spp. infected and uninfected animals. Conclusion: This study indicated that anaplasmosis is distributed in beef and dairy cattle in Thailand; therefore, prevention and control strategies for these pathogens should be improved. This information will benefit veterinarians and cowherds by avoiding vector exposure and eliminating tick breeding sites.


Introduction
Anaplasma is a Gram-negative intracellular bacterium in the family Anaplasmataceae which exists in the blood cells of a variety of mammals, including cattle and people [1].Moreover, this pathogen is also found exclusively within membrane-bound vacuoles in the invertebrate or tick host cytoplasm.Several types of Anaplasma: Anaplasma marginale, Anaplasma centrale, Anaplasma bovis, and Anaplasma platys can cause bovine anaplasmosis, which is a serious health problem in cattle [2][3][4][5].The clinical signs are hemoglobinuria, anemia, fever, jaundice, loss of appetite, weight loss, decreased milk production, abortion, and even death [2].To detect bovine anaplasmosis, microscopic examination is commonly used in combination with hematological values.Moreover, serological testing for antibody detection and polymerase chain reaction (PCR) for DNA detection have been developed to diagnose bovine anaplasmosis [4].
In Thailand, which is an agricultural state located in Southeast Asia, the livestock industry has been hampered by severe tick-borne hemoparasites.Beef and dairy cattle are predominant animals in Northeast Thailand and provide an important source of meat, horns, milk products, leather, land plowing, and transportation of people and crops [6].Bovine anaplasmosis in Thailand has been reported continually and its consequences impact economic losses in cattle production [7].Two species of Anaplasma that have been endemic in this region are A. marginale and A. platys.The previous studies by Saetiew et al. [8], Jirapattharasate et al. [9], and Nguyen et al. [10] reported that A. marginale is the most prevalent tickborne pathogen in North, Northeastern, and Western Thailand with a prevalence rate of approximately 8%-40%.However, there is less updated information on the epidemiology of Anaplasma spp.infection in cattle.
This study aimed to estimate the prevalence of Anaplasma spp.infections in beef and dairy cattle in Northeast, Thailand by microscopic examination and PCR.Moreover, the associated risk factors for Anaplasma spp.infections in naturally infected cattle were also determined.

Ethical approval
All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee, Mahasarakham University (IACUC-MSU-26/2022).

Study period and location
The cross-sectional study was conducted from June 2021 to May 2022.The blood samples were collected from cattle in smallholder farms in Khon Kaen, Maha Sarakham, Roi Et, Ubon Ratchathani, and Udon Thani provinces of Thailand (Figure -1).

Sample collection and study area
In total, 187 blood samples (106 samples from beef cattle and 81 samples from dairy cattle) were collected (approximately 3-5 mL from each) from the jugular vein or coccygeal vein in ethylenediaminetetraacetic acid anticoagulant tubes.The information on age, breed, gender, capillary refill time, and body condition score (BCS) were also recorded.Blood samples were transported on ice to the laboratory at the Faculty of Veterinary Sciences of Mahasarakham University.All blood samples were screened for Anaplasma infections using thin blood smear technique and measurement of the packed cell volume (PCV) levels which were performed on the same day of blood collection.The remaining blood was stored at -20°C until DNA extraction.

Microscopic examination using the blood smear technique
To perform this technique, approximately 10-20 μL blood was poured onto a slide and spread.The blood smear slides were then air-dried for 5-10 s, fixed with 100% methanol for 5 min, and stained with 10% Giemsa's solution for 15 min.Blood films were observed in the monolayer fields under a light microscope (Olympus, Japan) to determine the presence of parasites.

DNA extraction and PCR methods
DNA was extracted from whole blood (200 µL) following the GF-1 blood DNA extraction kit protocol (Vivatis, Malaysia).DNA of each sample was stored at −20°C for long-term preservation.Each extracted DNA sample was examined for blood parasitic infections by PCR or nested PCR.The first step of PCR used universal primers of the DNA belonging to Anaplasmataceae parasites for screening infections.In the next step, positive samples for Anaplasma infections were examined for species detection by specific primers (Table-1) [11][12][13][14].For the PCR reaction, approximately 10-50 ng of the extracted DNA was used as a template in a 25 µL reaction containing 1 µL of each primer (10 µmol/L), 1.5 mM MgSO 4 , 0.2 mM deoxynucleotide triphosphate, 1× PCR buffer, and 1 U of Taq Polymerase (Fermentas, USA).The reaction conditions comprised 35 cycles of denaturation for 45 s at 95°C, annealing for 45 s at 55°C-60°C, and extension for 1.5 min at 72°C using a PCR thermocycler (Biometra, Göttingen, Germany).The PCR products were electrophoresed running in 1% agarose gel stained with ViSafe Red Gel Stain (Vivantis, Malaysia) and visualized under ultraviolet light to check for positive amplifications.

Levels of PCV examination
Levels of PCV were evaluated as the height of the pack red cell column in a microhematocrit tube after centrifugation.The PCV was measured by filling blood directly into the microhematocrit tube, then centrifuged at 15,000× g for 3 min.The height of the total blood column and the height of the red cell layer was measured within 1 min after centrifugation was stopped.

Statistical analysis
The presence of blood parasites was determined and the percentages of infection were calculated.Confidence intervals (CI) were also used to compare the prevalence of parasitic infections.The association between blood parasite infections with other factors including gender, age, breed, and PCV levels was compared using Pearson's Chi-squared test.Statistical differences were considered when p < 0.05.
Under the light microscope, Anaplasma spp.infections were detected in erythrocytes (Figure -2).For microscope examination, the prevalence of Anaplasma spp. was 17.6 %.The occurrence of Anaplasmataceae was examined by PCR based on the 16S rRNA gene.Fragments of msp4 of A. marginale and 16S rRNA of A. platys were amplified to examine infections (Figure -3).For PCR, the overall prevalence of anaplasmosis in cattle in Northeast Thailand was 20.8% (95% CI: 15.3-27.4)based on 16S rRNA Anaplasmataceae primers.For specific primer detection, 5.3% and 3.2% of beef cattle were infected with A. marginale and A. platys.In addition, the molecular prevalence of A. marginale in dairy cattle was 12.3%, while no infection with A. platys was observed in this population.In addition, A. bovis infection was not discovered in this study.
Regarding breed, dairy cattle were more susceptible to Anaplasma infection (28.4%) than beef cattle  (15.1%).Male cattle (44.4%) were more likely to be Anaplasma infected than females (16.9%).For PCV values, the average PCV levels in both infected and uninfected groups were in the normal range (28.5% vs. 30%).Although the infected group had a lower trend of PCV, the results showed no statistical difference between infected and uninfected groups.Moreover, there were no clinical signs in any cattle infected with Anaplasma spp.In addition, statistical tests of the association between Anaplasma infections and other factors showed infection with Anaplasma spp.and A. marginale had an association with breed and gender (p < 0.05) while age and PCV levels showed no significant statistical relationship between Anaplasma spp.infected and uninfected groups.

Discussion
Anaplasmosis in cattle is a worldwide veterinary health problem, especially in tropical and subtropical regions.In this study, we screened Anaplasma spp.infection in beef and dairy cattle using both microscopic and molecular techniques.From this study, the overall prevalence of Anaplasma spp. in cattle was 20.8% based on PCR and 17.6% based on microscopic results.Although microscopic examination by direct blood smear technique is common, it is suitable for the detection of anaplasmosis during the acute phase of infection and requires an expert examiner.Polymerase chain reaction is an advantageous assay over microscopic examination because it has high sensitivity and specificity and is widely used to detect all phases of anaplasmosis infection in animals.The results indicated that the PCR method exhibited much higher sensitivity for the diagnosis of this blood parasite than the microscopic method, which is the routine method in the laboratory.
In Thailand, Anaplasma spp.infection in large ruminants is endemic with a higher infection rate reported in water buffalo (41%) [10] and beef cattle in the Western region (39.1%)[9].However, the prevalence in this study is higher than the previous studies by Junsiri et al. [15] in cattle in the northern and northeastern regions of Thailand in 2020 (10.30%) and water buffaloes in Northeast Thailand (8%) [8].The difference in the prevalence of anaplasmosis in cattle in Thailand could be explained by the climatic condition in each region which influences the spread of tick vectors [16], farm management, herd size, sampling period, sample size, antibiotic prevention [17], and diagnosis protocols [18].In addition, this study notices that good management practices on the farm have been observed to be the key factor in the infection rate.In other counties, the prevalence of anaplasmosis in cattle varies from 8.7% in Mongolia [19], 9% and 17% in Punjab (Pakistan) [20], 11.1% in Pakistan [21], 15.7% in India [22], 38.53% in Ohio [23], 49.1 % in Nigeria [24], and 68.3% in Egypt [25].The prevalence of bovine anaplasmosis in this study is reliable in range with previous epidemiological studies.
Apart from risk factor analysis, we found that the risk factors for Anaplasma spp.and A. marginale infections were significantly associated with breed and gender.Previous data also supported our finding that breed and gender had significant associations with Anaplasma spp.[26].For gender, the results showed that male cattle had a higher infection rate than female cattle according to the finding in cattle in China [27] and buffalo in Pakistan [28].For breed, the results revealed that dairy cattle are more susceptible to anaplasmosis than beef cattle.Although a previous study reported the age of the animals (below 1 year of age) showed a significant association with Anaplasma spp.infections [26], we found adverse results that age showed no significant relationship with infections.However, similar results in this study were also reported in water buffaloes from eight provinces of Thailand [10].In addition, the principal clinical sign of bovine anaplasmosis was considered anemia which can be directly measured by PCV levels; however, we found PCV levels showed no significant relationship with infections according to the report of PCV levels in infected cattle in Nigeria [24].This phenomenon may support the evidence that most cattle, especially animals that adapt well to a tropical climate show milder symptoms on infection.

Conclusion
From this study, Anaplasma infections in cattle in Thailand are common and several risk factors affect the rate of Anaplasma spp.infection, including host (age, gender, and breed) and environments (ecosystem, farm management, herd size, etc.).In addition, Anaplasma spp. is transmitted by ticks, the presence of this pathogen suggests that probable vectors may occur in ecological surroundings and requires further investigation.
Copyright: Seerintra, et al.Open Access.This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Table - 2
: Characteristics of cattle and risk factors analysis.