This systematic review and meta-analysis was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [18].

The literature search, study selection, data extraction, data analysis, and interpretation of the findings were conducted from January 2026 to April 2026 at Akkhraratchakumari Veterinary College, Walailak University, Nakhon Si Thammarat, Thailand.

The review protocol was registered with the Open Science Framework (OSF; DOI: 10.17605/OSF.IO/BFWQR). Relevant articles reporting the prevalence of hemoparasites in cattle and buffaloes (Anaplasma, Babesia, Theileria, Trypanosoma, and Mycoplasma) were retrieved from three bibliographic databases: PubMed, ScienceDirect, and Scopus. The search strategy included the following search terms: ("vector-borne diseases" OR "tick-borne diseases") AND ("cattle" OR "buffaloes"); ("Anaplasma" OR "Babesia" OR "Theileria" OR "Trypanosoma") AND ("cattle" OR "buffaloes"); and ("prevalence" OR "occurrence"). Articles published between 2015 and December 31, 2025, were eligible for inclusion. Three reviewers (NP, NS, and PP) independently screened titles and abstracts.

Studies were included in the systematic review if they met the following criteria: (i) cross-sectional study design; (ii) target population comprising cattle, buffaloes, or both; (iii) conducted in Southeast Asia; (iv) sample size ≥25 [19]; (v) use of PCR-based detection methods; and (vi) reporting the exact numbers of examined and positive animals or prevalence with a 95% confidence interval (CI). Studies were excluded if they (i) were not relevant to the prevalence of hemoparasites; (ii) investigated animal species other than cattle or buffaloes; (iii) used non-PCR-based detection methods; (iv) were case reports or review articles; (v) provided insufficient data for effect-size calculation; (vi) included fewer than 25 animals; (vii) were conducted outside Southeast Asia; or (viii) contained duplicate data published elsewhere. Full-text assessment and final study selection were independently performed by two reviewers (NP and NS).

Two reviewers (NS and TNT) independently extracted data, and discrepancies were resolved through discussion with a third reviewer (PP). The extracted information included (i) study characteristics (authors and publication year); (ii) methodological characteristics (sampling location, study period, and parasite detection method); (iii) host characteristics (host species, age, sex, total number of animals examined, and number of positive animals); and (iv) parasite characteristics (parasite species). The methodological quality of the included studies was independently assessed by two reviewers (NS and PP), with disagreements resolved through discussion. The quality assessment criteria were adapted from a previously published study [19]. Studies with quality scores of 0–8 were classified as low quality, whereas those with scores of 9–14 were classified as high quality.

Quantitative data, including the total numbers of cattle and buffaloes examined and the numbers infected with Anaplasma marginale, Anaplasma centrale, Anaplasma bovis, Anaplasma platys, Babesia bigemina, Babesia bovis, Babesia ovata, Theileria orientalis, Theileria annulata, Theileria sinensis, Theileria sp., Trypanosoma evansi, Mycoplasma wenyonii, and Candidatus Mycoplasma haemobos, were extracted and pooled for analysis. Pooled prevalence estimates were calculated for parasite groups represented by at least three eligible studies. Eight pooled prevalence analyses were performed, including overall estimates for Anaplasma spp., Babesia spp., and Theileria spp. infecting cattle and buffaloes. Species-specific pooled prevalence estimates were also calculated for A. marginale, B. bigemina, B. bovis, T. orientalis, and Tr. evansi. In addition to estimating pooled prevalence, this study systematically summarized the PCR gene targets used by laboratories across Southeast Asian countries for the molecular detection of major hemoparasites during 2015–2025.

A meta-analysis of proportions was performed using a random-effects model in R (version 4.5.3) [20] according to established methodological guidelines [21]. Between-study heterogeneity was assessed using Cochran's Q test and the I² statistic. Low heterogeneity was defined as p ≥ 0.10 or I² <50%, whereas high heterogeneity was defined as p ≤ 0.10 or I² >50% [22]. Forest plots were generated to visualize pooled prevalence estimates. Where substantial heterogeneity was detected, meta-regression analyses were performed to evaluate differences in the prevalence of A. marginale, B. bigemina, B. bovis, T. orientalis, and Tr. evansi according to geographical region (mainland vs. maritime Southeast Asian countries) and host species (cattle vs. buffaloes). Publication bias was assessed using funnel plots and Egger's regression test only for Babesia spp., because fewer than ten studies were available for the remaining parasite groups [23].

Database searching initially identified 4,803 articles (Figure 1). After removing 1,572 duplicate records, 3,157 articles were excluded during title and abstract screening because they did not meet the eligibility criteria. The full texts of 74 articles were subsequently assessed for eligibility, of which 51 were excluded for the following reasons: 12 were not relevant to the study objectives [24–35], seven provided insufficient data for effect-size calculation [36–42], ten used detection methods outside the scope of this review [43–52], one included a sample size of fewer than 25 animals [53], one was not a cross-sectional study [54], two were review articles, systematic reviews, or meta-analyses [55, 56], and 18 were conducted outside Southeast Asia [57–74].

A total of 22 cross-sectional studies were included in the qualitative and quantitative analyses, and their characteristics are summarized in Supplementary File 1. Among these studies, 14 investigated hemoparasites in both beef and dairy cattle [9, 75–87], three investigated buffaloes only [88–90], and five included both cattle and buffaloes [91–95]. Anaplasma spp. were reported in nine studies [9, 75, 76, 80–83, 85, 88], Babesia spp. in ten studies [77–81, 84, 87, 88, 90, 95], Theileria spp. in seven studies [9, 79–81, 88, 90, 92], Trypanosoma evansi in five studies [9, 86, 89, 91, 93], and Trypanosoma theileri in one study [91]. Two other pathogens, Candidatus Mycoplasma haemobos [9] and Mycoplasma wenyonii [94], were also reported.

Four Anaplasma species were identified, namely Anaplasma centrale [82], Anaplasma bovis [85], Anaplasma marginale [9, 75, 76, 80–83, 85, 88], and Anaplasma platys [9, 76, 85]. Three Babesia species were reported, including Babesia bigemina [77–81, 84, 87, 88, 90], Babesia bovis [77–81, 87, 88, 90, 95], and Babesia ovata [88]. Likewise, three Theileria species were identified, namely Theileria orientalis [9, 79–81, 88, 90], Theileria sinensis [9], and Theileria sp. [92]. These hemoparasites were reported from Indonesia [78, 86], Malaysia [9, 77, 82–84], the Philippines [88, 89, 91, 94], Thailand [75, 79–81, 85, 87, 90, 92, 93], and Vietnam [76, 95]. Quality assessment demonstrated that all 22 included studies were of high methodological quality (Figure 2).

Among the included studies, nine reported hemoparasite co-infections [9, 78–82, 88, 90, 91]. Three studies described co-infections in buffaloes, including dual infections [88, 90, 91] and triple infections [90]. The reported dual co-infection patterns included A. marginale with B. bigemina, A. marginale with B. bovis, B. bigemina with B. bovis, B. bigemina with T. orientalis, B. bovis with T. orientalis, and Tr. evansi with Tr. theileri. The only reported triple co-infection in buffaloes comprised B. bigemina, B. bovis, and T. orientalis.

Six studies reported co-infections in cattle, including dual [9, 78–82], triple [9, 79–81], and quadruple infections [81]. The dual co-infection patterns included A. centrale with A. marginale, A. marginale with B. bigemina, A. marginale with B. bovis, A. marginale with T. orientalis, A. platys with T. sinensis, B. bigemina with B. bovis, B. bigemina with T. orientalis, B. bovis with T. orientalis, T. orientalis with T. sinensis, and T. sinensis with Candidatus Mycoplasma haemobos. Triple co-infections included A. marginale, B. bigemina, and B. bovis; A. marginale, B. bigemina, and T. orientalis; A. marginale, B. bovis, and T. orientalis; A. marginale, T. sinensis, and Candidatus Mycoplasma haemobos; and B. bigemina, B. bovis, and T. orientalis. The only reported quadruple co-infection consisted of A. marginale, B. bigemina, B. bovis, and T. orientalis.

The 22 included studies investigated 5,789 cattle and 901 buffaloes. Hemoparasite infections included Anaplasma spp. (932 cattle and 29 buffaloes), Babesia spp. (781 cattle and 77 buffaloes), Theileria spp. (141 cattle and 40 buffaloes), Tr. evansi (128 cattle and 99 buffaloes), Candidatus M. haemobos (25 cattle), and M. wenyonii (32 cattle and 80 buffaloes).

Among the identified parasite species, A. centrale was detected in 23 cattle, A. marginale in 868 cattle and 29 buffaloes, and A. platys in 41 cattle. Among Babesia species, B. bigemina was detected in 472 cattle and 33 buffaloes, whereas B. bovis was detected in 309 cattle and 44 buffaloes. Among Theileria species, T. orientalis was detected in 73 cattle and 37 buffaloes, whereas T. sinensis was detected in 22 cattle.

The highest reported prevalence of each parasite group was 72.63% for Anaplasma spp. in Malaysia [83], 71.13% for Babesia spp. in Indonesia [78], 32.79% for Theileria spp. in Malaysia [9], 35.45% for Tr. evansi in Thailand [93], and 12.58% for Tr. theileri in the Philippines [91]. Candidatus M. haemobos was reported only in Malaysia, with a prevalence of 40.99% [9], whereas M. wenyonii was reported only in the Philippines, with a prevalence of 80.00% [94].

Anaplasma spp. infections in dairy cattle were reported in three studies [75, 81, 82], with the highest prevalence (31.40%) reported in Malaysia. In beef cattle, Anaplasma spp. infections were reported in two studies [9, 80], with the highest prevalence (26.23%) also reported in Malaysia.

Babesia spp. infections in dairy cattle were reported in two studies [81, 87], with the highest prevalence (8.39%) reported in Thailand. In beef cattle, Babesia spp. infections were reported in five studies [77, 79, 80, 84, 95], with the highest prevalence (37.14%) reported in Malaysia.

Theileria spp. infections in dairy cattle were reported in one study from Thailand [81], with a prevalence of 23.40%. In beef cattle, Theileria spp. infections were reported in five studies [9, 79, 80, 91, 92], with the highest prevalence (72.13%) reported in Malaysia.

Tr. evansi infection was reported exclusively in beef cattle in four studies [9, 86, 91, 93], with the highest prevalence (36.65%) reported in Thailand.

The PCR target genes identified for each hemoparasite are summarized as follows. A. centrale was detected using the 16S ribosomal ribonucleic acid (16S rRNA) gene [82]. A. marginale was detected using the 16S rRNA, groEL, and major surface protein 4 (MSP4) genes [9, 75, 76, 80–83, 85, 88]. A. platys was detected using the 16S rRNA gene [9].

For Babesia spp., B. bigemina was detected using the rhoptry-associated protein 1a (RAP-1a) and apical membrane antigen 1 (AMA-1) genes [77–81, 84, 87, 88, 90], whereas B. bovis was detected using spherical body protein 2 (SBP-2), spherical body protein 4 (SBP-4), and merozoite surface antigen (MSA) genes [77–81, 87, 88, 90, 95].

Among Theileria spp., T. orientalis and T. sinensis were detected using the major piroplasm surface protein (MPSP) gene [9, 79–81, 88, 90].

For Tr. evansi, the target genes included RoTat 1.2 variant surface glycoprotein (VSG) [9, 89], internal transcribed spacer (ITS) 1 [91], ITS2 [86], and satellite DNA [93]. Tr. theileri was detected using ITS1 [91]. Candidatus M. haemobos and M. wenyonii were detected using the 16S rRNA gene [9, 94].

This systematic review provides the first comprehensive, region-wide inventory of molecular diagnostic targets for major hemoparasites infecting cattle and buffaloes in Southeast Asia. The identified molecular targets included 16S rRNA, groEL, MSP4, RAP-1a, AMA-1, SBP-2, SBP-4, MSA, MPSP, RoTat 1.2 VSG, ITS1, ITS2, and satellite DNA. Collectively, these findings represent the first integrated regional synthesis of molecular diagnostic targets for these pathogens and the first comprehensive summary of molecular diagnostic evidence across multiple hemoparasite genera infecting both cattle and buffaloes.

The pooled prevalence estimates for the major hemoparasites are presented in Figure 3. Anaplasma spp. had a pooled prevalence of 12% (95% CI: 3%-38%; nine studies), with a 95% prediction interval (PI) of 0.37%-89.50% and substantial heterogeneity (I² = 98.6%). Babesia spp. had a pooled prevalence of 18% (95% CI: 11%-28%; ten studies), with a 95% PI of 3.16%-61.30% and substantial heterogeneity (I² = 97.0%). Theileria spp. had a pooled prevalence of 11% (95% CI: 3%-35%; seven studies), with a 95% PI of 0.43%-84.85% and substantial heterogeneity (I² = 94.8%).

Meta-analysis was not performed for Candidatus M. haemobos or M. wenyonii because fewer than three eligible studies were available. Species-specific analyses showed that Tr. evansi (Figure 3) had a pooled prevalence of 11% (95% CI: 4%-28%; five studies), with a 95% PI of 0.72%-70.64% and substantial heterogeneity (I² = 95.1%). A. marginale (Figure 4) had a pooled prevalence of 5% (95% CI: 1%-21%; nine studies), with a 95% PI of 0.29%-72.55% and substantial heterogeneity (I² = 94.7%). B. bigemina (Figure 4) had a pooled prevalence of 10% (95% CI: 5%-18%; nine studies), with a 95% PI of 0.88%-54.90% and substantial heterogeneity (I² = 94.6%). B. bovis (Figure 4) had a pooled prevalence of 9% (95% CI: 4%-17%; nine studies), with a 95% PI of 0.95%-52.07% and substantial heterogeneity (I² = 97.9%). T. orientalis (Figure 4) had a pooled prevalence of 10% (95% CI: 3%-31%; six studies), with a 95% PI of 0.77%-73.70% and substantial heterogeneity (I² = 90.2%).

The I² values for all pooled analyses exceeded 50% (Figures 3 and 4), indicating substantial between-study heterogeneity. Meta-regression analysis explored potential sources of heterogeneity and showed that the prevalence of A. marginale, B. bigemina, T. orientalis, and Tr. evansi was not significantly influenced by geographical region or host species (Table 1). In contrast, the prevalence of B. bovis differed significantly between mainland and maritime Southeast Asian countries (meta-regression, p = 0.0048), with a higher pooled prevalence observed in maritime countries.

Visual inspection of the funnel plot (Figure 5) suggested slight asymmetry, indicating possible publication bias. However, Egger's regression test did not detect statistically significant publication bias (p = 0.1293), suggesting no strong evidence of small-study effects. These findings should be interpreted with caution because of the limited number of included studies. Funnel plot assessment and Egger's regression test were performed only for Babesia spp., as fewer than ten studies were available for the remaining hemoparasite groups.

This systematic review advances the understanding of hemoparasite epidemiology in Southeast Asia in four important ways. First, it provides the first PCR-based pooled prevalence estimates of multiple hemoparasite genera infecting cattle and buffaloes across Southeast Asia during 2015–2025. Second, it presents the first comprehensive inventory of molecular diagnostic targets used in regional studies. Third, it identifies a significant difference in B. bovis prevalence between mainland and maritime Southeast Asian countries. Finally, it translates these findings into practical recommendations to strengthen regional veterinary diagnostic capacity, extending the evidence beyond previous single-country reviews.

The pooled prevalence estimates were 12% for Anaplasma spp., 18% for Babesia spp., 11% for Theileria spp., and 11% for Tr. evansi. These hemoparasites adversely affect livestock health and negatively impact the livelihoods of resource-poor farming communities in developing countries [96]. The tropical climate of Southeast Asian countries, characterized by high temperatures and humidity [97], provides favorable conditions for arthropod vector development [7]. In addition, the region harbors high arthropod vector diversity [8, 15], including 19, 61, 11, 48, 62, and 65 tick species reported from Cambodia, Indonesia, Laos, Malaysia, Thailand, and Vietnam, respectively [7]. Thailand alone has reported 128 tabanid species and 14 Stomoxyinae species, which serve as important vectors of hemoparasitic diseases [15]. Collectively, these environmental and ecological factors emphasize the continued risk of vector-borne diseases throughout Southeast Asian countries.

Meta-regression analysis further demonstrated a significant difference in B. bovis prevalence between mainland and maritime Southeast Asian countries (p = 0.0048). The higher prevalence observed in maritime countries may reflect ecological conditions that favor tick survival and B. bovis transmission. Differences in cattle movement and livestock trade may also contribute to the observed regional variation. However, the interpretation of this finding is limited by the availability of data on vector abundance, environmental conditions, livestock movement, and management practices across the two regions. These knowledge gaps highlight the importance of adopting a One Health approach, together with consideration of socioeconomic factors, to improve the understanding, surveillance, and control of hemoparasitic and other zoonotic diseases in Southeast Asian countries.

The synthesized evidence demonstrated diverse hemoparasite co-infection patterns in both cattle and buffaloes. Cattle exhibited dual, triple, and quadruple co-infections, whereas buffaloes exhibited only dual and triple co-infections. Co-infections involving multiple hemoparasites are likely to result in more severe clinical manifestations than single-species infections because of the combined pathogenic effects of different parasites. For example, A. marginale induces anemia through splenic clearance of infected erythrocytes, whereas Babesia spp. directly destroy erythrocytes during intracellular replication [10, 11]. Concurrent infection with these pathogens may therefore contribute to more severe disease.

Previous studies have reported substantial production losses associated with Anaplasma, Babesia, and Theileria infections, resulting in considerable economic losses due to reduced productivity and increased mortality [98]. In addition, A. platys and Tr. evansi have been reported in human infections, suggesting potential zoonotic importance, although their zoonotic roles remain to be fully established [14, 99]. The findings of the present study provide baseline information to support future epidemiological and molecular investigations of transmission pathways, zoonotic potential, vector control, acaricide resistance, and the effects of climate change on hemoparasite distribution. A One Health approach will be essential to strengthen surveillance systems and improve the prevention and control of these diseases in Southeast Asia.

The diagnosis of hemoparasite infections has traditionally relied on clinical signs together with laboratory detection of the causative agents [96]. PCR has been demonstrated to be a sensitive and reliable method for the molecular detection of hemoparasites in animals [100]. This systematic review provides the first comprehensive, region-wide synthesis of molecular diagnostic targets for the major hemoparasites infecting cattle and buffaloes in Southeast Asia by integrating evidence across multiple parasite genera. The compiled molecular targets may serve as a useful reference for veterinary diagnostic laboratories and help improve laboratory diagnostic capacity and technical competency.

However, PCR-based assays may not be readily available in all settings because of financial and technical constraints. In such circumstances, conventional methods, including blood smear and buffy coat smear examinations, remain practical and cost-effective diagnostic alternatives [101], particularly in rural areas where cattle and buffaloes are raised as sources of household income and financial security. The molecular and parasitological information summarized in this review may also help veterinary technicians and laboratory personnel improve their diagnostic competency for hemoparasitic diseases.

This systematic review and meta-analysis has several limitations. First, substantial between-study heterogeneity was observed, with I² values of 98.60%, 97.00%, 94.80%, and 95.10% for Anaplasma, Babesia, Theileria, and Trypanosoma, respectively. This heterogeneity may reflect differences in geographical location, study populations, host species, sample size, and study methodology. Although meta-regression identified geographical region and host species as significant contributors to heterogeneity for Babesia infections, a considerable proportion remained unexplained.

Second, although visual inspection of the funnel plot suggested possible publication bias for Babesia studies, Egger's regression test did not provide statistically significant evidence of publication bias. Moreover, these analyses could not be performed for the remaining hemoparasites because fewer than ten studies were available for each group, limiting the ability to assess potential publication bias and the possibility of underestimated pooled prevalence estimates.

Finally, the inclusion criteria restricted the analysis to PCR-based studies published in the English language and excluded studies using serological methods. These selection criteria may have introduced publication and language biases and should be considered when interpreting the findings of this review.