First Molecular Identification of Babesia, Theileria, and Anaplasma in Goats from the Philippines

Goats are key livestock animals and goat raising is an income-generating venture for smallholder farmers, supporting agricultural development in many parts of the world. However, goat production is often limited by various factors, such as tick-borne diseases. Goat piroplasmosis is a disease caused by apicomplexan parasites Babesia spp. and Theileria spp., while anaplasmosis is caused by bacterial Anaplasma spp. In the Philippines, the presence of Babesia, Theileria, and Anaplasma has not been reported in goats. In this study, DNA obtained from goats were molecularly screened for Babesia/Theileria and Anaplasma. Of 396, 77.02% (305/396) and 38.64% (153/396) were positive for piroplasma and Anaplasma using PCR assays targeting the 18S rRNA and 16S rRNA genes, respectively. Similarly, Babesia ovis was detected in six samples (1.52%). Representative Babesia/Theileria sequences shared 89.97–97.74% identity with each other and were most closely related to T. orientalis, T. annulata, and Theileria spp. Meanwhile, Anaplasma 16SrRNA sequences were related to A. odocoilei, A. platys, and A. phagocytophilum. This is the first molecular identification of B. ovis, Theileria spp., and Anaplasma spp. in goats from the Philippines.


Introduction
Babesiosis, theileriosis, and anaplasmosis are caused by tick-borne blood parasites of the genera Babesia, Theileria, and Anaplasma. These tick-borne diseases (TBDs) adversely affect livestock through direct and indirect losses in production. In small ruminants, the impact of TBDs has burdened farmers with losses linked to mortalities, less meat, milk, and wool produce, and increased costs for herd health management [1,2]. However, in endemic countries, TBDs are often overlooked despite being widespread in small ruminants, due to the lack of severe clinical manifestations during infection and strong tolerance through acquired natural immunity of infected hosts [3].

Sample Composition and Background Information
In this study, samples were collected from 396 randomly selected goats across six provinces in the Philippines (Figure 1), namely, Cavite (n = 42), Quezon (n = 20), Bohol (n = 35), Cebu (n = 74), Leyte (n = 26), and Davao del Sur (n = 199) ( Table 1). The goat population was comprised of the following: 60% adult (n = 237) and 40% young (n = 159); 87% female (n = 344) and 13% male (n = 52); 56% purebred (n = 222), 22% crossbred (n = 87), 15.4% Philippine native (n = 61), and unknown breed 6.6% (n = 26). The goats were raised in backyards by smallholder farmers, except those from Cebu and Bohol, which were domesticated in semicommercial and stock farms, respectively. The backyard goats were tethered and/or freely grazed, while goats from semicommercial farms were reared in semi-intensive and intensive systems. domesticated in semicommercial and stock farms, respectively. The backyard goats were tethered and/or freely grazed, while goats from semicommercial farms were reared in semi-intensive and intensive systems. where samples were collected from, with pinned sampling sites and GPS coordinates. The map was generated using the QGIS software [24]. Age-group where samples were collected from, with pinned sampling sites and GPS coordinates. The map was generated using the QGIS software [24].

Sequencing and Phylogenetic Analysis of Representative Sequences
We sequenced representative samples that showed strong bands for piroplasma (n = 7), Anaplasma sp. (n = 14), and B. ovis (n = 1) to determine their sequence identities and to analyze their phylogenetic relationships with previously published sequences in the Gen-Bank sequence database. The seven sequences (MW786647-MW786653) were confirmed as Theileria species and exhibited intersequence identities of 89.97-97.74%. As shown in Figure 2, three isolates (MW786649; MW786651; MW786653) were located in a subclade with other T. orientalis isolates from China, Pakistan, India, Bangladesh, and Malaysia. One Theileria sp. (MW786653) was most closely related to Theileria sp. Thung Song isolate from Thailand (99.30% identity) and formed a sister clade with the Chinese T. sinensis isolates, while MW786648 shared 99.53% identity with T. annulata isolates from India and Thailand ( Figure 2). MW786650 was similar to cattle isolate of T. orientalis from Pakistan, whereas MW786652 was located in a branch solitarily ( Figure 2). The Anaplasma sp. sequences (OP351259-OP351272) obtained in this study shared the following identities with each other: 99.68% (OP351261 and OP351271); 98.81-99.89% (OP351262, OP351263, OP351265-  (Figure 3). In addition, OP351261, OP351271, and OP351272 were phylogenetically related to various A. platys isolates. The B. ovis isolate from the current study (OP003548) was closely related and had 99.82% identity with sheep, goat, and horse B. ovis isolates from Turkey, Iran, Spain, and Portugal ( Figure 4).  Figure 3). In addition, OP35126 OP351271, and OP351272 were phylogenetically related to various A. platys isolates. T B. ovis isolate from the current study (OP003548) was closely related and had 99.82% ide tity with sheep, goat, and horse B. ovis isolates from Turkey, Iran, Spain, and Portug ( Figure 4).
Significant association between host parameters, including sex, age-group, breed, and location, and pathogen detection was noted in the present study. Piroplasma positivity in female goats was significantly higher than in male ones, while studies on small
Significant association between host parameters, including sex, age-group, breed, and location, and pathogen detection was noted in the present study. Piroplasma positivity in female goats was significantly higher than in male ones, while studies on small ruminants in Ethiopia [30], Turkey [39], and Tunisia [40] found goat sex to be negligible. On the other hand, Anaplasma spp. detection in young goats was significantly higher than in adults, which is parallel to the findings in Pakistan goats [35]. Moreover, detection of Anaplasma spp. and Babesia/Theileria were significantly associated with goat breed, wherein a higher number of purebred or exotic goats tested positive compared to upgraded and native goats. Earlier surveys observed a similar trend, where the indigenous goat breeds were described to have higher resistance to piroplasma [40,41] and Anaplasma [42]. Information available on goat breed resistance to TBDs is scarce, but one possible explanation may be the greater susceptibility of exotic goat breeds to tick bites compared to local breeds [43]. Piroplasma and Anaplasma detection rates significantly differed among the location of the goats. The same was observed in goat herds from China [29], Tunisia [40], and Oman [44]. Locationspecific factors in this study, such as management practices and macroclimatic conditions that affect tick proliferation, may be attributed to the different sampling sites characterized by diverse topography, terrain, microclimate, and fauna.
Sequencing and phylogenetic analysis confirmed that the representative Theileria sp. sequences obtained in this study were most closely related to T. orientalis, T. annulata, and Theileria sp. Thung Song isolate. Four Theileria isolates in the present study (MW786649; MW786651; MW786653) were similar to T. orientalis isolates from other locations. Members of the T. orientalis complex have been reported in cattle from the Philippines in previous studies [45][46][47]. More notably, we obtained an isolate highly similar to T. annulata. T. annulata is a species that can infect goats and causes a potentially fatal disease in cattle [48]. However, it should be confirmed whether the infected goats can be inapparent carriers of various T. orientalis genotypes and T. annulata and if they are able to spread the pathogens to bovids, as in sheep [48][49][50]. Theileria sp. Thung Song is an isolate from dairy cattle in Thailand and was genotypically divergent from other benign T. orientalis types (T. buffeli and T. sergenti) [51,52]. Its detection in goats in the current study may indicate host shifting of this parasite. We also obtained an isolate phylogenetically distinct from other analyzed sequences (MW786652; 98.75% identity with Theileria sequences in GenBank), which may be a new Theileria sp., although more studies are needed to verify this claim. On the other hand, T. luwenshuni, which is a common species in goats reported from nearby Southeast Asian countries, namely, Thailand [34,[53][54][55], Myanmar [56], and Vietnam [50,57,58], was not confirmed in the obtained sequences.
In this study, Anaplasma isolates (OP351259-OP351272) closely related to A. odocoilei, A. phagocytophilum, and A. platys were confirmed in goats from the Philippines. A. odocoilei is a species causing chronic Anaplasma infection in white-tailed deer discovered in the US [59]. A. odocoilei does not cause severe clinical disease in experimentally infected whitetailed deer, and natural infections have only been detected from North America and South America [60][61][62]. Additionally, we obtained isolates (OP351261 and OP351271) highly similar to A. platys (99.10% and 99.46% identity with other A. platys isolates, respectively) and OP351272, a potential novel A. platys-like isolate (97.30% highest identity with GenBank A. platys isolates). A novel A. platys-like species that can be vertically transmitted to the goat's offspring has been identified in China recently [63]. Furthermore, a previous study indicated that a couple of novel A. phagocytophilum-like and A. ovis-like variants are circulating in Philippine horses [64]. Thus, the isolates obtained from the current study warrant further probing on their genetic characteristics and clinical impact of the infections they inflict on goats.
Despite the high rate of positivity, clinical signs associated with TBDs were not observed. This may indicate the endemicity of these pathogens in goats in the Philippines. Likewise, this may correspond to persistent infections, a characteristic of natural infections in places where the disease is presumed endemic [7]. While B. ovis causes acute and severe disease in sheep, natural infection with B. ovis in goats is rarely clinical [8]. This was evident in the positive goats in the current study. In the case of Theileriaand Anaplasma-positive goats, the impact of subclinical infections should not be ignored because the pathogenicity of different species and genotypes vary depending on the host [15,65].
Some aspects were outside the scope of this study. For instance, few samples were subjected to sequencing analysis and species confirmation was performed by partial amplification of one fragment from one gene. Therefore, additional studies based on species-specific detection should be conducted to elucidate the species diversity of Babesia, Theileria, and Anaplasma in goats from the Philippines. In this study, the tick vectors were not determined. Since tethering and freely grazing systems are the more common production system in the Philippines [20], there are more opportunities for the ticks to feed on the host as goats are exposed to vegetation where questing ticks are abundant. The most likely vector of piroplasmas and Anaplasma is the ubiquitous R. microplus ticks, which was found infesting goats in Bulacan province [66]. The tick species R. microplus and Haemaphysalis bispinosa infesting goats from neighboring countries Thailand [34,67] and Malaysia [68] were also confirmed to be carrying T. luwenshuni. Similarly, R. sanguineus sensu lato (s.l.) ticks, the vector of canine A. platys in the Philippines [69,70], may also be implicated as the vector of A. platys-like variants detected in the present study. The three-host tick R. sanguineus s.l. commonly infests dogs, its main host, but humans and other animals may also be incidentally infested [71]. A majority of goats in this study were raised in backyards and regularly interacted with companion animals (dog and cats), which may have possibly exposed goats to the former's tick vectors. Moreover, the possibility of other transmission means, particularly, the role of mechanical transmission by insect vectors other than ticks, such as blood-sucking arthropods Tabanus, Stomoxys, and mosquitoes [17], should be further investigated.

Ethics Statements
Field sampling and animal handling protocols were conducted in accordance with the Philippine Animal Welfare Act (Republic Act 10631) and the guidelines set by the Institutional Animal Care and Use Committee of the University of the Philippines Cebu and Cavite State University. Experimental procedures and methodologies related to this study were permitted by Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan (permits 20-128 and 1723-4). The farmers and owners of the animals were oriented regarding the purpose of the study and provided verbal consent prior to the start of the sample collection.

Sample Collection and Sampling Sites
In this study, 396 whole-blood samples from randomly chosen goats were collected. The goats were randomly chosen irrespective of sex, age, and breed from March 2017 to March 2020. Sampling was done in backyards and farms selected by convenience in the provinces of Cavite (n = 42), Quezon (n = 20), Bohol (n = 35), Cebu (n = 74), Leyte (n = 26), and Davao del Sur (n = 199), Philippines. The specific sampling sites and their GPS coordinates are shown in Figure 1. Approximately 2 mL of blood was collected via venipuncture of the jugular vein of the goats into sterile EDTA tubes and kept cool until processing in the laboratory.

Genomic DNA Isolation
Genomic DNA was extracted using the QIAamp ® DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. About 200 µL of whole blood was used for the DNA extraction using the column-based blood kit. The DNA samples were transported to the National Research Center for Protozoan Diseases, Obihiro, Hokkaido, Japan and stored at −30 • C until use. Quality and the estimated concentration of the extracted DNA samples were checked using the NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) prior to screening the samples.

PCR Assays for Pathogen Detection
The PCR conditions performed in this study are referred to in Table 2. The samples were processed using a nested PCR assay targeting the hypervariable V4 region of the 18S rRNA gene of piroplasma [72,73] and 16S rRNA of Anaplasma spp. (A. phagocytophilum) [74]. In addition, a single specific primer set amplifying the B. ovis 18S rRNA gene was also used [75]. For the nested PCR assays, both first and final reactions were run to a final volume of 10 µL consisting of 1× ThermoPol ® buffer (New England Biolabs, Ipswich, MA, USA), 2 mM of dNTP mix (New England Biolabs) 2 µM of forward and reverse primers, 0.25 U of Taq DNA polymerase (New England Biolabs), and 2 µL of genomic DNA sample for the first assay or 1 µL of the PCR product for the nested assay. For the screening of B. ovis, the conventional assay was performed similarly to the aforementioned setup, except for the final concentration of primers (5 µM). The company-provided thermocycling conditions were followed, with the annealing temperature for each assay listed in Table 2. Positive (DNA samples confirmed positive for Theileria sp., B. ovis, and Anaplasma sp. [76]) and negative controls (UltraPure™ DNase/RNase-Free distilled water; Invitrogen, Waltham, MA, USA) were run alongside the samples in each assay. Visualization of amplicons after exposure to UV light was done after electrophoresis of PCR products in 1.5% agarose gel and staining with ethidium bromide solution.

Sequencing and Phylogenetic Analysis
Amplicons were purified using NucleoSpin ® Gel and PCR Clean-up kit (Macherey Nagel, Düren, Germany). Then, the purified amplicons were cloned by ligation into pGEM ® -T Easy Vector (Promega Corporation, Madison, WI, USA) and transformation in Escherichia coli DH5α strain calcium-competent cells. After overnight incubation of positive transformants, high-density bacterial cultures were lysed and plasmids were purified using NucleoSpin Plasmid QuickPure Kit (Macherey Nagel). Purified plasmids were sequenced by the Sanger sequencing method using BigDye ™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and ABI Prism 3100 Genetic Analyzer (Applied Biosystems).
Forward and reverse reads were manually trimmed and overlapped to assemble the sequences. Shared identities between presently obtained and previously deposited sequences were determined by NCBI BLASTn search, while the identity matrix generated from EMBL Clustal Omega multiple sequence alignment [77] determined the intersequence percentage identities. After nucleotide alignment by Clustal W and determination of the best DNA model, the maximum likelihood trees were constructed by phylogeny testing using the bootstrap method with 1000 replications. All analyses related to phylogeny were conducted using the Molecular Evolutionary Genetics Analysis (MEGA) X software [78]. The sequences obtained from this study were banked in the NCBI GenBank with accession numbers MW786647-MW786653 for Theileria sp. 18S rRNA (372-426 bp), OP003548 for B. ovis 18S rRNA (552 bp), and OP351259-OP351272 for Anaplasma sp. 16S rRNA (923-925 bp).

Statistical Analyses
Association between Babesia/Theileria sp. and Anaplasma sp. positivity (dependent variable) and animal parameters (categorical independent variables), namely, sex, age group, breed, and location, was evaluated. Background data of animal samples were available for all except for the breeds of goats from Leyte; thus, we excluded them in the analysis for the breed variable. Fisher's exact test was used to calculate the exact p values, whereas if not applicable, Pearson's chi-square test was employed to calculate the approximate p values. A p value of <0.05 was considered significant. All statistical analyses were performed using GraphPad Prism 8 (GraphPad software, San Diego, CA, USA). Institutional Review Board Statement: Field sampling and animal handling protocols were conducted in accordance with the Philippine Animal Welfare Act (Republic Act 10631) and the guidelines set by the Institutional Animal Care and Use Committee of the University of the Philippines Cebu and Cavite State University. Experimental procedures and methodologies related to this study were permitted by Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan (permits 20-128 and 1723-4). The farmers and owners of the animals were oriented regarding the purpose of the study and provided verbal consent prior to the start of the sample collection.

Informed Consent Statement: Not applicable.
Data Availability Statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.