Research Article

First Molecular Evidence of Anaplasma marginale Infection in Naturally Infected Cattle in Myanmar with Severe Hemolytic Anemia

Babi Kyi Soe1*, Toe Win Naing2, Su Lai Yee Mon1, Nay Chi Nway3, Hiroshi Sato4

1Livestock Upgrading Section, Livestock Breeding and Veterinary Department, Yangon 11021, Myanmar; 2Dairy Cattle Improvement Section, De Heus Myanmar, Yangon 11021, Myanmar; 3Veterinary Diagnostic laboratory, Livestock Breeding and Veterinary Department, Yangon 11021, Myanmar; 4Joint Graduate School of Veterinary Medicine, Yamaguchi University, Yamaguchi, 1677-1 Yoshida, Yamaguchi, 753-8515, Japan..

Received | October 19, 2023; Accepted | December 19, 2023; Published | February 12, 2024

*Correspondence | Babi Kyi Soe, Livestock Upgrading Section, Livestock Breeding and Veterinary Department, Yangon 11021, Myanmar; Email: [email protected]

Citation | Soe BK, Naing TW, Mon SLY, Nway NC, Sato H (2024). First molecular evidence of Anaplasma marginale infection in naturally infected cattle in Myanmar with severe hemolytic anemia. Adv. Anim. Vet. Sci., 12(3):559-565.

DOI | https://dx.doi.org/10.17582/journal.aavs/2024/12.3.559.565

ISSN (Online) | 2307-8316

Copyright: 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

The livestock population plays a major role in country economic development in terms of the fact that 10-12% of GDP of the Myanmar economy comes from the agricultural and livestock sectors (https://www.statista.com/statistics/1061469/myanmar-growth-rate-gdp-livestock-fishery-sector/). Even though the livestock industry serves as living insurance for farmers, the challenges of antimicrobial resistance, parasite diseases, and climatic disasters have increased as huge negative impacts. Nowadays, cattle are affected by various pathogens worldwide. Haemoprotozoan and rickettsial diseases are considered major threats to livestock health and productive performance in both tropical and subtropical regions (Das et al., 2022). Anaplasmosis is commonly known as “gall sickness,” which is caused by an obligate intraerythrocytic rickettsia microorganism (order Rickettsiales, family Anaplasmataceae). All ranges of ruminants can be infected by Anaplasma spp., whereas cattle seem to be more susceptible than others. Bovine anaplasmosis is an important tickborne disease that is caused by major species of A. marginale, A. bovis, A. centrale, A. phagocytophilum, A. capra, and A. platys (Selim et al., 2021), from which human cases have been reported by A. phagocytophilum, A. capra, and A. platys (Vanstreels et al., 2018). Parasite infestation results in a wide range of clinical symptoms, from asymptomatic to severe fatal hemolytic anemia, hepatosplenomegaly, decreased milk production, abortion, and susceptibility to other pathogens. Nowadays, hematobiochemical analyses have been extensively used to determine the underlined clinical status and to generate a more reliable diagnosis (Knowles et al., 2000). Once cattle have overcome the acute stage, they later develop long-lasting immunity and continue to harbor the infection for the rest of their lives, which serves as reservoir for susceptible animals (Chien et al., 2019). Of vector-borne transmission, 20 various ticks play a critical role as reservoirs for Anaplasma spp. infection, in which Boophilus microplus is known to be a major transmitting agent (El-Hamiani et al., 2021). Possibly mechanical transmission by means of biting flies or blood-contaminated fomites has been reported (Lankester et al., 2007). Until now, bovine anaplasmosis has been widespread globally and has been reported to be endemic in certain areas of Asia and Africa (Nasreldin et al., 2020; Ola-Fadunsin et al., 2018; Ybanez and Inokuma, 2016).

Giemsa-stained thin blood smear is a gold standard microscopic test for diagnosis because it is affordable and easy, despite recent advances in the diagnosis of Anaplasma spp. infection. However, either false positive or false negative results may come from staining artifacts or a low sensitivity percentage in pre-symptomatic and carrier animals (Noaman and Shayan, 2010). Therefore, molecular methods have been applied for conclusive diagnosis with high sensitivity and specificity results and definite species identification (Corona et al., 2014). Apart from that, hematobiochemical analyses provide valuable information relating to hemoparasites infection, which can lead to the interpretation of relevant diagnoses, drug efficacy, and disease progress (Abdullah et al., 2020). Once red blood cell damage occurs the intravascular/ extravascular, and immune-mediated severe hemolytic anemia have been reported in Anaplasma spp. infected cattle (Jalali et al., 2018). Among cattle hemoparasites, Babesia and Thelaria infections have been reported in certain areas of Myanmar (Bawm et al., 2014, 2016), whereas nothing has been informed about bovine anaplasmosis. The aim of this study was, therefore, to determine the presence of Anaplasma spp. infection using combination of techniques of both microscopy and molecular methods altogether with hematobiochemical alteration in clinically healthy cattle in order to promote specific management strategies.

Materials and Methods

In the present study, a total of 59 cattle blood samples were collected with convenience sampling. Briefly, about 5 ml of whole blood from the jugular vein of each cattle was collected into vials containing ethylenediaminetetraacetic acid (EDTA) anticoagulated tubes (Sigma-Aldrich Co. LLC, Saint Louis, Missouri, USA). The questionnaire survey was made based on the information regarding animal age (<1 year, 1–3 years, 3–5 years), breed (indigenous, crossbred, beef), gender (male, female), tick infestation (present, absent), management practice (poor, good, excellent), and acaricidal treatment (yes, no). Giemsa-stained (Sigma-Aldrich) thin blood smears were prepared immediately, as described by (Noaman and Shayan, 2010). In detail, blood smears were prepared and fixed in methanol for 5 mins. After that, the labeled smear was stained with a 5% Giemsa solution for 30 mins and air-dried. Finally, the labeled thin blood smears were observed under a light microscope (Olympus, Japan) at 1000x magnification using oil immersion for the presence of Anaplasma inclusion bodies. In order to perform molecular detection, the EDTA blood samples were kept at -20°C prior to DNA extraction. To analyze the hematobiochemical alteration of the PCR-positive sample, sera were isolated by centrifugation at 3000 x g for 5 mins at 4°C. Hematological parameters were determined by an automated hemoanalyzer (IDEXX VetAutoread Hematology Analyzer, USA), and the blood biochemical profile was determined using a VetScan VS2 chemical analyzer (Zoetis, UK).

Genomic DNA was extracted using the DNeasy blood and tissue kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions. The eluted DNA was stored at -20°C until use. The concentration and purity of the extracted DNA were measured by a nanodrop spectrophotometer (Thermofisher Scientific Inc., Waltham, USA). DNA samples with A260/A280 ratios between 1.7 and 2.2 were further analyzed. Polymerase chain reaction (PCR) was performed to amplify the 16S rRNA gene of expected size 429 bp shared by all Anaplasma spp. (F, 5ʹ-TACCTCTGTGTTGTAGCTAACGC-3ʹ; R, 5ʹ-CTTGCGACATT GCAACCTATTGT-3ʹ) as described by (Park et al., 2018). PCR was performed under the following cycling conditions: Initial denaturation at 96°C for 5 min, followed by 35 cycles of 10 s at 96°C of denaturation, annealing at 55°C for 35 s and final extension at 72°C for 5 min. After 1.5% agarose gel electrophoresis and ethidium bromide staining, PCR products were visualized under UV transillumination. Sequences were analyzed using the BioEdit version 7.2 sequence alignment software (www.mbio.ncsu.edu/BioEdit/bioedit.htm), and sequences alignment was performed using Clustal W software version 2.0 (Larkin et al., 2007). The resulting sequences were compared with the reference sequences in the GenBank database using the BLAST program of the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/). Phylogenetic trees were constructed using the neighbor-joining program in MEGA 11.0 software (Kumar et al., 2001).

 

 

Results and Discussion

In the present study, a total of 59 cattle blood samples were collected from Mingaladon township, Yangon region. Conventional microscopy revealed that three samples were positive (Figure 1), whereas molecular detection of 429 bp of the 16S rRNA gene shared by all Anaplasma spp., 2 out of 59 sampled cattle (3.38%) were found that positive for Anaplasma spp. infection of which two were positive from microscopy (Figure 2). Sequencing exhibited that the two sequenced samples were identified as A. marginale, with the sequence homology range between 98.8 and 100% compared with other sequences. The two sequences obtained in this study were deposited in the GenBank database under accession numbers OR654955 and OR654956. In addition, the two PCR-positive animals were in the age group of 1-3 years and are crossbred female dairy cattle, in which no ticks were infested during sample collection. Considering management practices, these positive cattle were from the same farm, whereas they were not raised under good hygienic measures, which might have led to breeding ground for ticks (Sajid et al., 2014) and were closely related to other livestock farms.

 

Phylogenetic analysis of the two partial 16S rRNA gene sequences (OR654955 and OR654956) was performed by aligning with selected Anaplasma spp. sequences in GenBank. As a result, the OR654955 and OR654956 sequences were closely related to A. marginale, followed by A. centrale, A. capra, A. platys, A. phagocytophilum, and A. bovis deposited in GenBank (Figure 3). The resulting Myanmar cattle isolates in the present study show 99.53% and 99.76% homology to sequences from A. marginale

strains originating from cattle in Pakistan (MK680805-MK680807), and 98.35% homology with sequences from A. centrale strains originating from cattle in Italy (EF520689 and EF520690). Interestingly, the two isolates found in this study were closely related to A. centrale, as they were in the same sister taxon as A. centrale isolates from cattle from Italy. However, in Myanmar, the information on A. centrale infection in livestock animals has not been updated yet. They were 97.64% sequence homology with A. capra isolated from water buffalo (ON763215-ON763217) from Turkey and 96.93% homology with A. platys sequence (LC545959) from dog from Myanmar. Previously, A. platys was reported from in dogs from Nay Pyi Taw, Myanmar, in 2020 with a low prevalence (0.25%). It was proposed that the Rhipicephalus sanguineus tick might be the main vector, as this is the dominant tick species found in Myanmar. Therefore, transboundary movement of animals could be a major transmission of sharing the same vector within the regions (Hmoon et al., 2021).

The two positive samples from both conventional microscopy and PCR were supposed to determine hematobiochemical alteration. By means of the hematology profile, the results indicated that the infected cattle have been shown to have severe hemolytic anemia. The hematological parameters of Hb, PCV, and total platelet count were remarkably lower, whereas the MCV and total WBC count were apparently higher in the infected cattle. In the present study, low hemoglobin levels and a remarkable increased of MCV in Anaplasma spp. infected cattle revealed evidence of hypochromic macrocytic anemia; hence, the type of anemia that occurred in Anaplasma spp. infection could depend the disease severity (Roland et al., 2014). Similar findings of hypochromic macrocytic anemia have been reported in A. marginale-infected cattle (Das et al., 2022). In addition to this, normocytic forms of anemia have been reported in acute anaplasmosis, whereas chronic cases with macrocytic progresses in Anaplasma spp. infected cattle Anton and Solcan (2022). Therefore, the infected cattle in this study seemed to have chronic cases of infection. In line with previous reports, Anaplasma spp. could reduce erythrocyte life span and favor erythrocyte destruction, which in turn leads to severe hemolytic anemia (Doyle et al., 2016). Contrast to Jurkovic et al. (2020), there was no clinical manifestation in Anaplasma spp.-infected cattle has been reported. In general, thrombocytopenia may occur relating to severe vasculitis, immune-mediated destruction, and pathogens of mainly blood sucking parasites (Rodriguez et al., 2018). However, other factors such as hormonal imbalance, nutritional deficiency, drugs intoxications, and environmental stress could not be ruled out in cases of erythrocyte destruction (Fazio et al., 2016).

Based on the serum biochemical profile, the infected cattle showed high levels of ALT, AST, ALP, total bilirubin, and BUN, whereas total protein and albumin were lower than

 

Table 1: Hematobiochemical parameters of two A. marginale infected cattle.

Clinical parameters	Diagnosed values	Elevated/ Decreased	Reference range (George et al., 2010; Kaneko et al., 2008)
Hb (g/dL)	5.31 - 5.84	Decreased	8.5 - 12.2
PCV (%)	19.02 - 20.21	Decreased	22 - 33
MCV (fL)	60.07 - 60.25	Elevated	38 - 50
MCH (pg)	16.71 - 16.34	Normal	14 - 18
MCHC (g/dL)	36.1 - 39.2	Normal	36 - 39
Granulocyte count (x 103 cells/µL)	9.9 - 10.4	Elevated	1.8 - 7.2
Lymphocyte count (x 103 cells/µL)	5.9 - 6.2	Elevated	1.6 - 5.6
Monocyte count (x 103 cells/µL)	1.57 - 2.12	Elevated	0 - 0.8
Total platelets count (x 103 cells/µL)	137 - 142	Decreased	193 - 637
Albumin (g/dL)	2.47 - 2.88	Decreased	3.03 - 3.55
Globulin (g/dL)	2.01 - 2.8	Decreased	3 - 3.48
ALT (IU/L)	62 - 65	Elevated	11 - 40
AST (IU/L)	148 - 150	Elevated	78 - 132
ALP (IU/L)	525 - 610	Elevated	0 - 488
Total bilirubin (mg/dL)	1.8 - 2	Elevated	0.01 - 0.5
BUN (mg/dL)	48 - 50	Elevated	14 - 37
Total protein (g/dL)	3.4 - 3.6	Decreased	6.7 - 8.8

 

normal, relating to excessive erythrocytes destruction, which later succumbed to hepatocellular damage (Anton and Solcan, 2021) (Table 1). These findings are in line with previous findings by Ashuma et al. (2013), from which impairment of the hepatocellular system and erythrocyte destruction in Anaplasma spp. infected cattle might result in elevated bilirubin levels because of retention in the biliary system and excessive destruction of erythrocytes in the reticuloendothelial system. High levels of BUN in infected animals could be due to renal ischemia, nephrosis, and dehydration, as these clinical symptoms have been reported in A. marginale infected cattle (Das et al., 2021). Resulting in decrease the capacity of protein synthesis due to hepatocellular damage, which can further lead to hypoalbuminemia, as previously described by Anton and Solcan (2022). Human infections have been reported by A. phagocytophilum, A. capra, and A. platys (Vanstreels et al., 2018) with mild to moderate clinical symptoms, from which high liver enzymes were important predictors of anaplasmosis. Even though bovine anaplasmosis has been shown to be clinical in most cases, the infected cattle from the study area shown to be infected sub-clinically. Despite the identified species in the study area was revealed as A. marginale, it is important to figure out the risk of zoonotic anaplasmosis in different geographical areas of Myanmar.

Even though, we could not calculate the significance due to the sample size limitation, management practices were shown to be important fact because cattle raised together with other livestock animals in poor hygienic practices were found to be more infected in the present study. The occurrence of A. marginale infection in the present study showed a relatively lower percentage compared to previous studies in other countries, even though the same analytic techniques were used: 72.6%, 43%, 23.2%, 11.2%, and 8.21% in Malaysia (Ola-Fadunsin et al., 2018), Philippines (Galay et al., 2021), Thailand (Saetiew et al., 2014), China (Yang et al., 2015), and Bangladesh (Mannan et al., 2021), respectively. Different geographical distribution and detection methods could produce different results hence, the main vector population could also be different (Das et al., 2021).

Conclusions and Recommendations

Overall, the very first molecular detection of A. marginale infection associated with hemolytic anemia in naturally infected cattle was revealed. Moreover, the remarkable changes in the hematobiochemical profile indicated that clinical bovine anaplasmosis might be a differential diagnosis in the presence of severe hemolytic anemia. However, our study still has limitations for interpretation of results since only one pathogen has been targeted; hence, the hematobiochemical alterations might also come out with other hemoparasite infections. Besides, the sampling area could not be representative of the whole Myanmar, and consequently, the assessment for bovine anaplasmosis still needs to be updated. Therefore, further studies with large sample sizes and an overall seasonal assessment of the vector population might investigating the association of potential risks with the presence of infection.

Acknowledgements

We are grateful to Dr. Khin Su Hlaing, Dr. Zin Hnin Thaw and Dr. Paik Htwe for their help in the fieldwork. The authors would also thank the farmers for their collaboration in sample collection. The authors acknowledged Dr. Arin Ngamniyom from Major Environment, Faculty of Environmental Culture and ECO tourist, Srinakharinwirot University, Thailand for providing molecular facilities throughout the study. As this is collaborative work, we don’t have any funding acquisition.

Novelty Statement

This study is the first molecular detection of bovine anaplasmosis associated with hemolytic anemia as it is considerable economic loss in livestock industry.

Author’s Contribution

BKS wrote the main manuscript text and prepared figures and table. BKS, SLYM, TWN and NN were responsible for conceptualization, investigation, draft revision, analyses, original draft editing and author proof revision. BKS and TWN were responsible for methodology. HS was responsible for supervision. All authors have reviewed the manuscript.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Abbreviations

16S rRNA: 16S of ribosomal RNA; A. marginale: Anaplasma marginale; A. capra: Anaplasma capra; A. phagocytophilum: Anaplasma phagocytophilum; A. centrale: Anaplasma centrale; A. bovis: Anaplasma bovis; A. platys: Anaplasma platys; PCR: polymerase chain reaction

Conflict of interest

The authors declare that the research was carried out without any commercial or financial affiliations that could be interpreted as a possible source of conflicting interests.

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