Incidence of hemoparasitic infections in cattle from central and northern Thailand

Sample and data collection

This study was conducted between June, 2020 and April, 2021. Dairy cattle farms and beef cattle farms with high population densities that are located in six provinces in northern and central Thailand were selected for this study. The sample-collection protocols were reviewed and approved by Animal Care and Use Committee at Faculty of Veterinary Medicine, Chiang Mai University (S26/2563). Farm owner permission letters were approved before samples were collected. The provinces included in this study were Chiang Mai (n = 143), Chiang Rai (n = 87), Lamphun (n = 557), Lampang (n = 76), Phayao (n = 122), and Nakhon Pathom (n = 81) (Fig. 1). A total of 1,066 blood samples were collected from randomly selected farms located in five provinces in northern Thailand and another province in central Thailand. The animals were restrained and blood was collected from their coccygeal or jugular veins and immediately transferred into EDTA-K2 lyophilized vacuum blood collection tubes (BD Vacutainer®, Franklin Lakes, NJ, USA). The blood sample tubes were kept in a cooled box equipped with ice packs during transport to the Faculty of Veterinary Medicine, Chiang Mai University and processed immediately. Data related to the characteristics of animals and farm management were obtained and recorded by the investigators. At each farm, farm owners or farm staff were interviewed with regard to specific individual animal characteristics, namely age, breed, and gender. Farm-based characteristics included location, history of hemoparasitic infection, treatment details, tick control programs, and farm management practices.

Microscopic analysis

A thin smear of blood was prepared from each blood sample. The blood smears were air-dried, fixed in methanol for 2 min, and stained by 10% Giemsa solution (Merck, Kenilworth, NJ, USA) in phosphate-buffered saline. The smears were examined at 1,000× magnification using an oil-immersion lens (CX31; Olympus, Shinjuku City, Tokyo, Japan). The identification process was carried out to decipher genus and species profiling to the greatest degree possible. A minimum of 1,000 red blood cells were counted and recorded. The percent of parasitemia was determined by counting the number of infected red blood cells (iRBCs) and by then dividing that number by the total number of red blood cells (RBCs): % parasitemia = (iRBCs/RBCs) × 100.

DNA extraction from blood

Genomic DNA was extracted from all blood samples using a PureLink^TM Genomic DNA mini kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturer’s instructions. The extracted DNA was eluted in 70 μL of the elution buffer. The quantity and quality of the DNA were determined by an UV/Vis spectrophotometer DU 730 (Beckman Coulter, Brea, CA, USA). The DNA was stored at −20 °C until it was used.

Packed cell volume determination using microhematocrit method

Blood samples were pipetted into capillary tubes and spun in a high-speed centrifuge. After 5 min of centrifugation, hematocrit results were estimated by calculating the ratio of the column of packed erythrocytes to the total length of the sample in the capillary tube and then measured with a hematocrit reader card. The reference value was determined to be between 24% and 46% (Miller et al., 1989).

PCR detection of cattle hemoparasites

All samples were analyzed by PCR parallel with microscopic analysis. For the purposes of PCR analysis, Babesia spp. and Theileria spp. parasites were screened for the presence of genetic differences among Babesia 18S rRNA (Hilpertshauser et al., 2006) and Theileria spp. 18S rRNA (Cao et al., 2013). Positive control samples were included for each specific screen. B. bovis was determined by B. bovis rhoptry-associated protein (rap-1) (Figueroa et al., 1993), B. bigemina was determined by B. bigemina apical membrane antigen 1 (ama-1) (Sivakumar et al., 2012). T. orientalis was determined by T. orientalis major piroplasm surface protein (mpsp) (Ota et al., 2009), and T. annulata was determined by T. annulata 30 kDa major merozoite surface antigen gene (tams-1) (Kirvar et al., 2000). In addition, the prevalence of A. marginale and A. phagocytophilum were determined by genetic variations of A. marginale major surface protein 4 (msp4) (M’Ghirbi et al., 2016) and A. phagocytophilum major surface protein 2 (msp2) (M’Ghirbi et al., 2016).

DNA from each sample was PCR amplified using the gene-specific primers for which the sequences is listed in Table 1. Babesia spp. and Theileria spp. parasites were further screened using nested PCR (nPCR). Positive samples were specifically screened, while B. bovis and B. bigemina were screened by nPCR. Meanwhile, T. orientalis, T. annulata, A. marginale, and A. phagocytophilum parasites were screened by primary PCR. The final volume of 30 μL was comprised of 5 μL of template DNA and 25 μL of the reaction mixture with 2X MyTaq HS Red Mix (Meridian Bioscience, Bioline, Memphis, TN, USA) and 10 μM of each sample, which was made up with deionized water to reach the final volume.

The conditions used for Babesia spp. and Theileria spp. amplification consisted of initial denaturing at 95 °C for 1 min, 35 cycles of a denaturing step at 95 °C for 15 s, an annealing step for Babesia spp. at 63 °C for 15 s and Theileria spp. at 55 °C for 30 s, an extension step at 72 °C for 15 s, and a final extension step at 72 °C for 30 s. The same concentration of MyTaq HS Red Mix was used for Theileria spp. amplification of 5 μL PCR product in the nPCR as has been described above. The nPCR condition included initial denaturation at 95 °C for 1 min and 35 cycles of a denaturing step at 95 °C for 15 s, annealing temperatures of 60 °C for 10 s for Babesia spp with an extension step at 72 °C for 10 s, and a final extension step at 72 °C for 30 s. Then, Babesia spp. positive samples were identified for B. bovis and B. bigemina. The same concentration of MyTaq HS Red Mix that was used for amplification consisted of an initial denaturing step at 95 °C for 1 min, 35 cycles of a denaturing step at 95°C for 15 s, an annealing step for B. bovis at 55 °C for 15 s and B. bigemina at 61 °C for 15 s, an extension step at 72 °C for 15 s, and a final extension step at 72 °C for 30 s. The same conditions used for B. bovis and B. bigemina amplification of 5 μL PCR product in the nPCR were used for the first PCR of each strain. While, Theileria spp. positive samples were identified for T. orientalis and T. annulata. Identification of T. orientalis and A. phagocytophilum was performed in a PCR thermal cycler consisting of initial denaturation at 94 °C for 3 min, 40 cycles of a second denaturation step at 94 °C for 1 min, an annealing step at 58 °C for 30 s, an extension step at 72 °C for 1 min, and a final extension step at 72 °C for 5 min. Similarly, in terms of the PCR specification of A. marginale and T. annulata., the initial denaturation step was set at 94 °C for 3 min, 40 cycles of a second denaturation step at 94 °C for 1 min, an annealing step at 60 °C for 30 s, an extension step at 72 °C for 1 min, and a final extension step at 72 °C for 5 min. All PCR products were separated by gel electrophoresis on 1% agarose in 1X TAE buffer and visualized using ethidium bromide under UV transilluminator.

DNA sequencing and phylogenetic tree analysis

B. bovis (n = 4), T. orientalis (n = 12), and A. marginale (n = 5) positive samples were randomly selected for DNA sequencing. PCR products were purified using PureLink^TM quick PCR purification kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). The purified PCR samples were sent to ATCC Co. Ltd. (Thailand Science Park, Khlong Nueng, Thailand) for identification of species by DNA sequencing. Nucleotide sequences were analyzed using the BLAST tool on the Clustal 2.1 software. The completed sequences were subjected to multiple sequence alignment with sequences previously available in the GenBank database. Phylogenetic trees in this study were analyzed using MEGA X program. Initial trees for the heuristic search were obtained automatically by applying Neighbor-Joining method and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura-Nei model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Rap-1 gene sequences of B. bovis (n = 4), mpsp gene sequences of T. orientalis (n = 12), and msp4 gene sequences of A. marginale (n = 5), while those reported from other regions were used to construct a subsequent phylogenetic tree. Bootstrap test with 2,000 replications was established as the confidence of the branching pattern of the trees. Finally, the phylogenetic relationship among the isolates identified in this study and those isolated from different countries were illustrated.

Statistical analysis

Statistical analysis of data categorized as positive or negative for B. bovis, T. orientalis, and A. marginale was accomplished based on PCR results and the packed cell volume. Variables were performed using the chi-square test. A P-value of <0.05 was considered to be statistically significant using GraphPad Prism version 8.4.

Microscopic examination of cattle hemoparasitic infections

According to light microscopic examinations, variable cattle hemoparasites, such as Babesia spp., Theileria spp., and Anaplasma spp., were detected in Giemsa-stained blood smears (Fig. 2). Paired-pyriform parasites within the erythrocyte were observed explicitly as a characteristic of Babesia spp. The pyriform shape of the Theileria parasites was clearly detected. A small spot located on the edge or center of the red blood cell was confirmed as a characteristic form of Anaplasma spp.

Hematological examination

Accordingly, 965 blood samples of total 1,066 samples were examined. The results indicated that 161 hemoparasite infected samples exhibited positive indications of anemia, as is shown in Table 2.

Molecular detection and identification of cattle hemoparasites

Using the specific primers, PCR products at 298, 776, and 420 bp were determined to represent B. bovis, T. orientalis, and A. marginale, respectively (Fig. 3). Meanwhile, B. bigemina, T. annulata, and A. phagocytophilum were not detected. The PCR results indicated that 1.22% with 95% CI [0.56–1.88] (13/1,066) of the blood samples were positive for B. bovis, T. orientalis, and A. marginale at 36.50% with 95% CI [33.60–39.38] (389/1,066) and 34.15% with 95% CI [31.30–36.99] (364/1,066), respectively, as is shown in Table 3. Furthermore, the multiple infections of two or more cattle hemoparasites appeared in 27.30% with 95% CI [ 24.62–29.97] (291/1,066) of the total blood samples (Fig. 4). By the presence of multiple infections, T. orientalis at 99.66% with 95% CI [98.98–100] (290/291) was found to be the most frequent hemoparasite.

DNA sequencing and phylogenetic analysis of cattle hemoparasites

The molecular characterizations of cattle hemoparasites were analyzed with respective gene targets of B. bovis rap -1, T. orientalis mpsp, and A. marginale msp4. The identity of the B. bovis specificity among isolates in this study ranged between 98.57–100%. Four isolates obtained from Lamphun (OK490920), Lampang (OK490921 and KO490922), and Nakhon Pathom (OK490919) provinces with a product size of 298 bp shared a degree of similarity with the isolates obtained from China (KT312809.1) and the Philippines (JX860283.1) (Fig. 5).

Further characterizations of T. orientalis and A. marginale were identified using phylogenetic analysis with PCR assay. The product size of 776 bp in this study was placed in four isolated genotypes by the percent identities ranging between 83.96–100% (Fig. 6). The phylogenetics of six isolates obtained from Lamphun (OK490929), Lampang (OK490926 and OK490928), Phayao (OK490927 and OK490930), and Nakhon Pathom (OK490931) provinces were related to T. orientalis type 5 and shared similarity with isolates obtained from several areas on the Asian continent. There were two isolates obtained from Phayao (OK490923) and Nakhon Pathom (OK490924) provinces that were related to the T. orientalis type 3 and shared similarity with isolates obtained from Sri-Lanka (AB701465) and Vietnam (AB560821). Moreover, another sequence result obtained from Lamphun (OK490925) province revealed the presence of T. orientalis type 4, which has been reported to be present in China (MH539832), Myanmar (AB871316), and Thailand (AB562561). T. orientalis type 7 was found lastly in this study, while three isolates from Chiang Mai (OK490933), Chiang Rai (OK490934), and Nakhon Pathom (OK490932) provinces exhibited similarity with the databases established from Japan (AB218430), China (MH539826), Indonesia (AF102500), and Vietnam (AB560823).

Finally, the PCR product size of 420 bp confirmed the presence of A. marginale. The phylogenetic findings of five isolates obtained from Lamphun (OK506074, OK506075, and OK506077), Chiang Rai (OK506076) and Nakhon Pathom (OK506073) provinces revealed 97.60–100% of sequence similarity when compared to isolates obtained from Brazil (JN022561), Colombia (MF771065), and South Africa (KF758944 and MT173811) (Fig. 7).