Abstract
Theileria orientalis is a tick-borne intraerythrocytic protozoan parasite of cattle causing oriental or benign theileriosis. It has a worldwide distribution and is not considered a zoonotic agent, while the disease symptoms range from subclinical to severe. Theileria spp. are very common co-infecting agents with other tick borne pathogens such as Babesia spp., Anaplasma spp. including zoonotic Anaplasma phagocytophilum with rising prominence. This study is an exploratory investigation of the occurrence of Anaplasma spp. and other blood parasites in cattle from Gacko and Bileća, Herzegovina. Blood samples from 35 clinically healthy cattle were selected based on veterinary reports of tick infestation and examined microscopically, by PCR (Babesia/Theileria; Anaplasmataceae) and qPCR (A. phagocytophilum, A.platys, A.marginale). Microscopy revealed intraerythrocytic bacterial forms in multiple samples. A. phagocytophilum DNA was detected in one and Babesia/Theileria DNA in 34 samples. Based on 16S rRNA gene sequencing, the A. phagocytophilum isolate was grouped with isolates from Europe, suggesting regional pathogen circulation. AnkA gene analysis placed the isolate in European group I, but outside the zoonotic subcluster, indicating a ruminant-associated lineage maintained primarily in wildlife–Ixodes ricinus cycles. The 18S rRNA gene sequencing showed subtle regional clustering of T.orientalis isolates. This is the first molecular confirmation of A. phagocytophilum in cattle from Eastern Herzegovina and identification of possible enzootic foci of T. orientalis infection, deeming further investigation into tick vectors and reservoir hosts in the region.
Keywords: Anaplasma phagocytophilum, Bosnia and Herzegovina, cattle, Theileria orientalis, tick-borne pathogens
1. Introduction
Anaplasma phagocytophilum is a cosmopolitan, emerging tick-borne, Gram-negative intracellular bacterium of the Anaplasma genus, along with other species A. platys, A. centrale, A. bovis, A. marginale, A. ovis, and A. capra. It infects domestic animals and humans, leading to significant morbidity, especially in immunocompromised individuals (1–3). The geographic distribution of this bacterium is linked to its primary vector, the hard tick Ixodes ricinus (Acari: Ixodidae), and epidemiological studies have shown its presence in different host species across most European countries (3). Reservoir species of A. phagocytophilum include large game (such as deer, roe deer, and moose) and rodents (such as mice, shrews, and voles). Anaplasma phagocytophilum causes tick-borne fever (also known as pasture fever) in ruminants, characterized by different severity of clinical symptoms ranging from severe in lambs to mild or subclinical in cattle (4). Furthermore, it causes equine granulocytic anaplasmosis (EGA) in horses, and canine granulocytic anaplasmosis (CGA) in dogs. Anaplasma phagocytophilum is a zoonotic pathogen causing human granulocytic anaplasmosis (HGA) with flu-like symptoms, fever, chills, headache, myalgia, and leukopenia that can lead to severe complications (2, 5). HGA is often found in areas where Lyme disease is endemic and was by now confirmed in North America, Europe, and Asia, often in co-infections (6).
The distribution of Anaplasma species in domestic animals in Bosnia and Herzegovina is still not sufficiently documented, while there is more data available for the distribution of tick vector species in the region and other tick-borne pathogens such as causative agents of Lyme Boreliosis. Anaplasma phagocytophilum was detected in ticks (7) and dogs (8, 9) from Bosnia and Herzegovina, but to the best of our knowledge there is no data available for the detections of A. phagocytophilum in domestic ruminants. Significant genetic diversity of A. ovis was detected in sheep in the Herzegovina region (10), indicating the possible presence of other Anaplasma species in the area based on observations of symptoms similar to tick-borne fever by local veterinarians, animal holders, and epidemiological service. Several studies conducted so far indicated the circulation of A. phagocytophilum in animal hosts and ticks in the Balkan region. In a study from neighboring Serbia, Vasić et al. found a low seroprevalence of A. phagocytophilum in cattle (2.45%; 4/163; 95% CI: 0.96%−6.14%) and no bacterial DNA in the blood of seropositive animals (11). Wild carnivores are recognized reservoirs and potential transmitters of a variety of zoonotic pathogens (12). Another study from Serbia found A. phagocytophilum DNA in 0.9% (2/216; 95% CI: 0.26%−3.31%) of golden jackal (Canis aureus) spleen samples (13) indicating the role of wild carnivores in the A. phagocytophilum transmission cycle in nature. Furthermore, the presence of A. phagocytophilum was also detected in ticks, in the regions of Serbia near the border with neighboring Bosnia and Herzegovina (14). In Croatia, A. phagocytophilum was found in animal reservoirs and ticks and there have been reported cases of HGA (15, 16).
Oriental theileriosis of cattle is a protozoan disease caused only by the Theileria orientalis complex—group Theileria orientalis/buffeli/sergenti (17, 18). Depending on the species of Theileria, a number of hard ticks of the genera Amblyomma, Haemaphysalis, Hyalomma and Rhipicephalus can transmit these pathogens (19). Theileriosis caused by T. orientalis complex was considered benign and asymptomatic, but clinical cases had been reported from Australia, Japan and New Zeland (20–22). Currently, from 11 genotypes of T.orientalis only Ikeda and Chitose are concidered to be pathogenic (18). Cattle infected with high burdens of the Ikeda genotype often become anemic with clinical signs including tachypnoea, lethargy, ataxia, abortion in pregnant animals and mortality in up to 10% of cases. Stressors (e.g., parturition, lactation and transport of infected animals) exacerbate the onset of the disease. The geographical range of T. orientalis Ikeda has expanded significantly in the past two decades facilitated by the widespread occurrence of its principal tick vector, Haemaphysalis longicornis (23, 24).
Theileria orientalis was previously recorded with high prevalence in Sarajevo-Romanija region of Bosnia and Herzegovina (25). Research conducted in Europe also indicates its presence in Italy (26), Greece (27), Spain (28), Portugal (29), Romania (30), Hungary (17), United Kingdom (31), Serbia (11), Croatia (32), and Russia (33). Numerous studies indicate frequent co-infections with T. orientalis and A. marginale (32, 34, 35) as well as T.orientalis and Babesia spp. (35–37). According to some studies, single infection with T.orientalis or hemoplasmas in cattle is more common than co-infections because of the “interference phenomenon” (38).
Based on previous results on A. ovis detection in Eastern Herzegovina and high prevalence of bovine piroplasmosis in the small geographical area of the Sarajevo-Romanija region, this study aimed to determine the presence of Anaplasma species (namely A. phagocytophilum, A. platys, and A. marginale based on epidemiological data in Europe) and Babesia/Theileria in cattle in this enzootic area for tick-borne infections in a targeted approach based on previously conducted epidemiological investigation. Furthermore, this study aims to provide the first molecular confirmation and typing using the polymorphic ankA locus of A. phagocytophilum in cattle from Eastern Herzegovina (39). Sequencing of the ankA gene has become a key molecular tool for the phylogenetic grouping of A. phagocytophilum, as its high genetic variability enables discrimination of ecotypes and host-associated lineages that cannot be resolved by more conserved markers such as 16S rRNA (40). Our study aims to lay a base for further surveillance of Anaplasma species in livestock and their potential impact on animal health and disease transmission, as well as bovine piroplasms and their epidemiological and clinical significance in Bosnia and Herzegovina.
2. Materials and methods
2.1. Sampling and study area
A total of 35 blood samples from clinically healthy cattle were collected by veterinary practitioners from the municipalities of Gacko (coordinates 43.1667, 18.5333) (n = 30) and Bileća (coordinates 42.8765, 18.4297) (n = 5) at the beginning of June 2024. Geographically, these epizootiological units belong to the region of Eastern Herzegovina and are characterized by hilly relief and the intersection of Mediterranean and continental–mountainous climates. Sampling was carried out at four locations in the municipality of Gacko and one in the Bileća municipality (Figure 1).
Figure 1.
Geographical map of investigated areas of Gacko and Bileća—red circle indicates a positive location for Anaplasma phagocytophilum infection.
The studied population was selected based on reports by owners and local veterinarians concerning the presence of large numbers of ticks on cattle from February to May, aiming to gain better insight into the diversity of pathogenic species that ticks may transmit to cattle in the area. The study was conducted according to the Law on Animal Protection and Welfare of the Republic of Srpska (“Official Gazette of Republic of Srpska 111/08”). An ethical statement does not apply to this research, as samples were taken during formal examinations by local veterinarians.
Before venipuncture, information was collected on the geographic coordinates of the households, as well as the breed, sex, age, and other important characteristics of the animals, including observations of possible changes in their health status. This information was provided by veterinary practitioners and animal owners. All 35 animals that participated in the research were female, with ages ranging from 2 to 16 years. At the time of sampling (June), no ticks were observed on any of these animals. Blood samples were collected by puncture of the coccygeal vein into EDTA tubes, thin glass slide smears were made and the blood was transported the same day under a cold chain to the Veterinary Institute of the Republic of Srpska “Dr. Vaso Butozan” in Banja Luka, where they were stored at −20 °C until DNA extraction.
2.2. Microscopic examination
All collected blood samples were used to prepare thin glass slide smears, which were then air-dried, fixed with methanol, stained with Giemsa, and examined under a light microscope (magnification of 40x to examine the entire surface of the slide and 100x to describe the morphology of the observed structures). The shape of the observed formations was carefully described using terms such as round, pear-shaped, and oval, as well as their number and arrangement (peripheral, subcentral, central) within the blood cells.
2.3. DNA extraction
Total DNA nucleic acid extraction from 200 μL of EDTA blood was primarily performed using the commercial IndiSpin Pathogen Kit (Indical Bioscience, Leipzig, Germany) according to the manufacturer's instructions. The DNA extracts were adequately labeled, packed and, as previously described, sent to the Laboratory for Virology and Molecular diagnostics of the Veterinary Institute of the Republic of Srpska “Dr Vaso Butozan” in Banja Luka, after which they were stored at −20 °C until further analysis.
2.4. Molecular pathogen detections
The extracted DNA samples were tested by conventional PCR to prove the presence of the A. marginale genome, using the specific PCR protocol described by Torina et al., targeting the msp4 gene (344 bp) (41). The reaction was performed using Himedia Hi—Chrom PCR Master mix (HigenoMB, India) on a Nexus PCR Thermal cycler (Eppendorf, Hamburg, Germany). Electrophoresis of PCR products was performed on a 1.5% agarose gel (100 V, 60 min), pre—stained with Midori green and visualized under UV light.
Aliquots of the tested extracts (30 μl) were packed and sent under a cold chain to the Scientific Institute of Veterinary Medicine of Serbia, where they were stored at −20 °C until further use. First, a 345 bp fragment of the 16S rRNA gene specific to the Anaplasmataceae family was targeted according to Parola et al. (42). DNA of Babesia/Thelieria was detected by PCR using previously described primers (43). The reactions were carried out with PCR Master Mix 2x (Thermo Fisher Scientific, Waltham, Massachusetts, USA) using an MJ Mini Personal Thermal Cycler (Bio-Rad, Hercules, California, USA), followed by agarose gel electrophoresis stained with Simply Safe™ (EURX, Gdansk, Poland). The amplification products were visualized under UV light. A 6x Orange DNA Loading Dye (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was used for visual monitoring of DNA migration during electrophoresis.
Detection of specific DNA for A. platys and A. phagocytophilum was performed using primers and probes listed in Table 1. The molecular targets included the groEL gene for A. platys and the msp2 gene for A. phagocytophilum (44). The reaction was performed using Fast Gene Probe 2x qPCR Universal Mix (Nippon Genetics Europe, Düren, Germany) (primers 10 μM, probe 5 μM) on a MIC (Magnetic Induction Cycler) (Bio Molecular Systems, Brisbane, Australia). The sample identified as A. phagocytophilum was further tested using primers specific for the ankA gene and prepared for sequencing (45). Each PCR run included adequate positive and negative controls available in each laboratory, and primer sets are listed in Table 1.
Table 1.
List of pathogens, primers/probe sets and their sequences used in this study.
| Pathogen | Primer name | Sequence (55′-33′) | Reference |
|---|---|---|---|
| Babesia/Theileria | BJ BN2 | -GTCTTGTAATTGGAATGATGG- -AGTTTATGGTTAGGACTACG- | (42) |
| Anaplasmataceae | EHR 16SD EHR 16SR | -GGTACCYACAGAAGAAGTCC- -TAGCACTCATCGTTTACAGC- | (41) |
| Anaplasma marginale | AmargMSP4Fw AmargMSP4Rev | -CTGAAGGGGGAGTAATGGG- -GGTAATAGCTGCCAGAGATTCC- | (40) |
| Anaplasma platys | An_pla_groEL_F An_pla_groEL_R An_pla_groEL_P | -GCTATGGAAGGCAGTGTTGG- -GTCTTGAAGCGCTCGTAACC- -AATCTCAAGCTCAACCCTGGCACCAC- |
(43) |
| Anaplasma phagocytophilum | An_ph_msp2_F An_ph_msp2_R An_ph_msp2_P | -GCTATGGAAGGCAGTGTTGG- -GTCTTGAAGCGCTCGTAACC- -AATCTCAAGCTCAACCCTGGCACCAC- |
(43) |
| Anaplasma phagocytophilum | AnkaAP2074s AnkaAP2815a | -GGCAAATGAGGCAAGTAACC- -GCCACTACCCAAGGATGATAG- |
(44) |
2.5. Phylogenetic analysis
The positive PCR product for Anaplasmataceae and Babesia/Theileria was cut from the gel, purified using Agarose-out DNA purification kit (EURx, Gdansk, Poland) and sequenced in both directions with PCR primers. Consensus sequences were created using MEGA 12 software (46) and were further analyzed using BLAST (http://blast.ncbi.nlm.nih.gov). The pairwise distances were calculated using MEGA 12 software. The evolutionary history for A. phagocitophylum was inferred using the Maximum likelihood method and the evolutionary distances were computed using the Kimura 2-parameter method and bootstrap analysis with 1,000 reiterations. In total, 21 nucleotide sequences of Anaplasma spp. from GenBank were used for phylogenetic analysis (14 A. phagocytophilum strains, 2 A. marginale, 3 A. platys, and 2 A. bovis strains), while Rickettsia ricketsii was included as an outgroup. The obtained sequence was deposited to GenBank under the accession number PV056069.
The same sample was subsequently analyzed for the ankA gene using specific PCR primers. Maximum Likelihood phylogeny was reconstructed in MEGA 12 using the General Time Reversible substitution model. Rate heterogeneity was modeled with a discrete Gamma distribution (five categories, +G parameter = 2.9617) and a proportion of invariant sites (+I = 0.00%). Codon positions 1st, 2nd, 3rd, and non-coding sites were included, and positions containing gaps or missing data were eliminated using the complete-deletion option, yielding a final alignment of 494 positions. Ninety-one coding nucleotide sequences of A. phagocytophilum were compared and grouped according to Rar et al. (39) into 12 established clusters. The resulting ankA sequence was deposited in GenBank (accession no. PX255552).
The phylogeny for T. orientalis was inferred using the Maximum Likelihood method and Tamura (1992) model of nucleotide substitutions (47). The analytical procedure encompassed 35 nucleotide sequences with 436 positions in the final dataset and included analogous nucleotide T. orientalis sequences from GenBank as well as Babesia bovis sequence as an outgroup (48). The obtained sequences were deposited to GenBank under the accession numbers PV581906-PV581910.
3. Results
3.1. Microscopical observation
Microscopic examination of 35 blood samples revealed intracellular and extracellular bacteria in 31 samples. These bacteria were found in the erythrocytes, typically localized peripherally, and exhibited shapes ranging from spherical to coccobacillary (Figure 2). It was impossible to distinguish between the species within the genera Anaplasma and Mycoplasma. No bacterial structures were detected in white blood cells or platelets. Considering results, differential diagnostics were undertaken using molecular biology methods. Piroplasms were detected in 20 samples. The morphological analysis of merozoites and intraerythrocytic structures made it challenging to diagnose the species level accurately. However, the typical form of T. orientalis is presented in Figure 3.
Figure 2.
Spherical and coccobacillary bacterial forms in red blood cells (1,000x) (“Dr. Vaso Butozan”, 2024).
Figure 3.
Typical form of Theileria orientalis in red blood cell (1,000x) (“Dr. Vaso Butozan”, 2024).
3.2. Results of molecular detection of Anaplasma phagocytophilum
One sample from Gacko locality (Figure 1) tested positive for the presence of A. phagocytophilum using qPCR (Ct = 23.23). The positive dairy cow showed no signs of the disease and was 8 years old at the time of sampling.
3.3. Phylogenetic analysis of A. phagocytophilum sequence
Phylogenetic analysis of the partial 16S rRNA gene performed by aligning the 21 Anaplasma spp. sequences with the sequence obtained in this study confirmed its grouping with A. phagocytophilum representatives (Figure 4) with the highest similarity with isolates from Ghana OR241137.1 (100%) and Brasil KP642755.1 (100%) and Denmark 253AJ776165.1 (99%). Maximum-likelihood analysis of the partial ankA gene placed our cattle isolate (PX255552) within group I according to Rar et al. (39), clustering with European ruminant isolates (e.g., bison/cattle from Central and Northern Europe), and distinct from the zoonotic group I subcluster (marked “Z” in Figure 5). The Bosnian sequence formed a well-supported branch with neighboring European ruminant strains, indicating affiliation with a ruminant-associated lineage rather than the zoonotic ecotype. This ankA placement complements the 16S rRNA result and refines the molecular characterization of the detected A. phagocytophilum, consistent with the 12-cluster ankA scheme used in our study.
Figure 4.
The Maximum likelihood phylogenetic tree based on the nucleotide sequences of the partial 16S rRNA gene of Anaplasma spp. Rickettsia ricketsii was included as outgroup. The numbers represent the percentage of 1,000 bootstrap iterations supporting the nodes and only percentages >50% are shown. Nucleotide sequence of A. phagocytophilum from this study is marked with a black circle. GenBank accession numbers, isolate names, hosts, and the countries of origin are indicated for each sequence.
Figure 5.
The Maximum Likelihood phylogenetic tree based on the nucleotide sequences of the partial ankA gene of Anaplasma phagocytophilum. The numbers represent the percentage of 1,000 bootstrap iterations supporting the nodes and only percentages >50% are shown. Nucleotide sequence of A. phagocytophilum from this study is marked with a black triangle. GenBank accession numbers, isolate names, hosts, and the countries of origin are indicated for each sequence. Sequences are grouped according to Rar et al. (39) Zoonotic strains from group I are marked with “Z”.
3.4. Results of molecular detection of Babesia/Theileria
DNA fragment of Babesia/Theileria species was detected in 34 samples (29 from Gacko and 5 from Bileća). High quality amplicons (n = 5) were subjected to sequencing and phylogenetical analysis.
3.5. Phylogenetic analysis of T. orientalis sequences
The phylogenetic tree based on the partial 18S rRNA gene sequences of T. orientalis shows limited genetic variation, consistent with the highly conserved nature of ribosomal RNA genes (Figure 6). Despite this, regional clustering is noticeable, especially among Bosnian isolates (PV581906–PV581909 and ON148460–ON148462), which form a distinct monophyletic group. The presence of related sequences from other parts of the world reinforces the idea of a globally distributed and genetically stable T. orientalis population. Although the 18S rRNA gene offers limited resolution for intra-species variation, the tree reflects subtle geographic structuring. PV581910 is part of a regional T. orientalis population spanning southeastern Bosnia and Croatia, with minimal sequence divergence, consistent with the conserved 18S gene and denoting stable endemic transmission cycles in the area. Nucleotide sequence identities among the Bosnian isolates from this study ranged from 99.3 to 100% (pairwise distance up to 0.7%), while comparison with other previously reported sequences from Bosnia and Herzegovina showed 99.1% to 100% similarity (pairwise distance up to 0.9%). When compared to sequences from other countries, the similarity ranged from 98.8 to 100% (pairwise distance up to 1.1%).
Figure 6.
The Maximum Likelihood phylogenetic tree based on the nucleotide sequences of the partial 18S rRNA gene of Theileria orientalis. Babesia bovis was included as outgroup. The numbers represent the percentage of 1,000 bootstrap iterations supporting the nodes and only percentages >50% are shown. Nucleotide sequences of T. orientalis from this study are marked with black squares. GenBank accession numbers, isolate names, hosts, and the countries of origin are indicated for each sequence.
4. Discussion
Theileria orientalis is protozoan parasite that, in most cases, causes non-specific disease signs making it problematic to define its clinical significance. These piroplasms are genereally not eliminated from the circulation after infection, and parasitemia persists in infected animals for prolonged periods, even for life if carrier state is established (18). Our study confirms a high number of PCR positive piroplasmosis cases in grazing clinically healthy cattle from Romania region in Eastern Herzegovina. A case study from Croatia documented that T. orientalis contributed to the lethal outcome in three cows as a co-infection with A. marginale and A. bovis (32). Inconsistent with our findings, T. orientalis was recorded in asymptomatic cattle from neighboring Serbia with a very low prevalence (5/135, 3.70%) (11). It's increasingly detected in some European countries like Italy, Spain, Portugal, Greece, and the Balkans (25–28). Prevalence varies regionally but is rising due to climate change and animal movement. Although genotyping of the detected strains was not performed in this study, the molecular evidence confirms active circulation of T.orientalis in the region. The identification of the enzootic focus in southern part of the country suggests a stable transmission cycle likely supported by favorable environmental conditions and the presence of competent tick vectors, such as Haemaphysalis spp. and Rhipicephalus spp. (49). The high percentage of infected asymptomatic cattle in these areas could be an indicator of the endemic stability of oriental theileriosis, or the relatively low pathogenicity of the T. orientalis genotypes present (17, 25). This study was geographically limited to selected farms in the Herzegovina region, which may not fully represent the spatial extent of the enzootic focus and the lack of direct tick collection and identification limited our ability to confirm the local vector species responsible for transmission. Animal movement, pasture sharing and tick control practices are not considered, further limiting the ability to assess risk factors for enzootic establishment.
This study also documents the presence of A. phagocytophilum-specific DNA in a cattle blood sample, confirming its first detection in these animals in Eastern Herzegovina (Romanija region). These results align with previously published results of A. phagocytophilum presence in ticks (7) and dogs (8, 9) and extend current knowledge on its geographical prevalence within different hosts, also confirming its ongoing circulation.
The obtained nucleotide sequence clustered with sequences from Austria, Poland, Denmark, and Türkiye suggesting a potential epidemiological link and pathogen circulation across these regions. The observed clustering of 16S rRNA sequences suggests a stable A. phagocytophilum lineage circulating across Europe, extending through the Balkans, possibly maintained by wildlife reservoirs and tick migration rather than livestock trade. The detection of A. phagocytophilum in wild and domestic ruminants, especially sheep and goats, has been performed throughout Europe with varying reports concerning prevalence (4, 50–52). A recent study from Poland performed on several wild cervid species indicated the potential reservoir status of these animals (53). Interestingly, another Polish investigation (54) of free-grazing goats in forested regions found no evidence of A. phagocytophilum, suggesting this species has lower infection rates compared to other ruminants, consistent with the claims from a German study by Rubel et al. (55).
Available data from Germany indicates notable prevalence and confirms the circulation of numerous strains of A. phagocytophilum in dairy cattle, likely sustained by wildlife reservoirs (4). Also, in Türkiye, Aktas et al. (56) detected A. phagocytophilum in small ruminants, although a genetically distinct variant (A. phagocytophilum-like 1) was notably more prevalent, suggesting that the existence of these regionally adapted genetic variants warrants further examinations. Comparable to our examination, the msp2 gene was also used as a target for A. phagocytophilum detection in a study from Great Britain that confirmed its presence in cattle, sheep, deer, and I. ricinus ticks, and the potential role of cattle in the pathogen's epidemiological cycle was proposed (57).
The ankA-based phylogeny refines the molecular characterization of our strain. Rar et al. (39) showed that ankA diversity resolves A. phagocytophilum into 12 host/vector-associated clusters in agreement with MLST and groEL ecotypes. The sample from this study grouped within ankA cluster I. However it lies outside the zoonotic subcluster, paralleling reports of ruminant-restricted lineages circulating in Central and Northern Europe (4, 51). In Poland, cervids act as key reservoirs and harbor diverse groEL genotypes, and recent roe/red deer typing shows ankA/groEL combinations that include group I variants but also cervid-adapted lineages (53, 58). Wild boar have also carried human-pathogenic variants in Poland, underscoring a broader wildlife reservoir (59). Ecotype analyses from Great Britain further highlight that cattle infections often align with groEL Ecotype I, while Turkey has documented confirmed bovine cases without ankA typing (57, 60). Collectively, these data support our interpretation that the Bosnian isolate belongs to a ruminant-associated ankA group I lineage maintained primarily in a wildlife–I. ricinus cycle rather than through cattle-to-cattle transmission (61).
The diagnosis of anaplasmosis in cattle commonly relies on observation of clinical symptoms, followed by the examination of Giemsa-stained thin blood smears that enable the inclusion body identification. In our study, the spherical and coccobacillary bacteria were located within erythrocytes, primarily at the cell margins. However, the observation of Anaplasma and Mycoplasma species in microscopic examination seen in this study needed further evidence. Since there is a possibility that the observed changes in coloration in the blood smears might be due to stain residue or Howell-Jolly bodies, Heinz bodies, or Pappenheimer bodies. Our research did not confirm the presence of A. marginale, which had been reported in symptomatic cattle in neighboring Croatia (32). Additionally, microscopy yielded no bacterial structures in white blood cells or platelets. It is well known that A. phagocytophilum DNA is detectable days before visible inclusions can be identified through blood smear microscopy, and the proportion of infected leukocytes can vary considerably (2). The qPCR protocol (msp2 gene) that was used in this study was shown to have high specificity and sensitivity, so the detection of A. phagocytophilum DNA in one sample can be regarded within the limits of qPCR method.
Limitation of this study includes small sample size (n = 35) which might be reason for detection of only one positive sample for A. phagocytophilum. At this time, it was not possible to conduct large-scale research which is necessary to provide further insight into the spread and clinical significance of this tick-borne pathogen occurring in cattle. This study was conducted as an exploratory investigation rather than a prevalence study, with selective sampling guided by veterinary reports of tick infestations. Our results because of this limitation can not be extrapolated to the whole population of cattle of Bosnia and Herzegovina. Further limitation of the study is the lack of data on vector species ticks, which were not collected at this point because of the focus on animal health.
Different Anaplasma species have various transmission vectors: A. ovis and A. marginale are mainly spread by Rhipicephalus and Dermacentor ticks, A. phagocytophilum is transmitted via Ixodes species, while A. platys is spread by R. sanguineus, the brown dog tick (62–64). In Bosnia and Herzegovina, eleven species of hard ticks from the Ixodidae family have been documented, with I. ricinus being the most prevalent, followed by D. marginatus, R. bursa, Hyalomma marginatum, R. sanguineus sensu lato, Haemaphysalis punctata, I. canisuga, D. reticulatus, I. hexagonus, and H. concinna (65). A study by Stevanović et al. (66) validated that R. bursa dominates among the tick species parasitizing sheep in Eastern Herzegovina. However, this study failed to collect ticks parasitizing cattle, leading to uncertainty regarding the tick species composition, which can only be inferred from epidemiological data. Furthermore, the seasonal occurrence of the diseases caused by tick-borne pathogens was not considered since sampling was performed in a snapshot study in cattle based on epidemiological data.
Differently from certain Anaplasma species, there is no evidence that A. phagocytophilum undergoes transovarial transmission within Ixodes ticks. Accordingly, a reservoir host is essential for maintaining its endemic life cycle in the environment (1). Rubel et al. (55) reported a strong correlation between the presence of cats and dogs on livestock farms with exposure to Anaplasma spp. in farmed ruminants. However, their role as reservoir hosts of A. phagocytophilum is controversial and highly unlikely (67). High prevalence rates of A. phagocytophilum detected in wild ruminant populations in Italy highlight their importance as hosts in the infectious cycle of this pathogen (51). Additionally, wild ruminants not only aid in the development and reproduction of I. ricinus ticks but also harbor various A. phagocytophilum genetic variants. Nevertheless, at present, available information regarding reservoir hosts of A. phagocytophilum in Eastern Herzegovina remains insufficient, and the specific host species involved in its transmission cycle remains to be determined.
5. Conclusions
This study confirms the presence evidence suggestive of an enzootic focus of T. orientalis in Herzegovina, which is driven by subclinical infections. This suggests possible endemic stability and a dynamic equilibrium in the interactions among hosts, vectors and pathogens. The high number of Babesia/Theileria-positive cattle observed in this study supports the notion that oriental theileriosis is widespread in Bosnia and Herzegovina. Microscopic diagnostics appear to have limitations when compared to PCR diagnostics for detecting piroplasms in cattle.
In addition, we provide the first molecular evidence of A. phagocytophilum DNA in cattle from Herzegovina. The ankA gene analysis further indicates that this Bosnian isolate belongs to a ruminant-associated group I lineage distinct from the zoonotic subcluster, underscoring the need to monitor wildlife–I. ricinus cycles when assessing potential spillover to cattle and humans.
Funding Statement
The author(s) declared that financial support was received for this work and/or its publication. This study was funded by Ministry of Science, Technological development and Innovation of Republic of Serbia (Contract no. 451-03-136/2025-03/200030; 451-03-33/2026-03/200030 451-03-136/2025-03/200143; 451-03-34/2026-03/200143; 451-03-136/2025-03/200015; 451-03-33/2026-03/200015).
Footnotes
Edited by: Mihaela Kavran, University of Novi Sad, Serbia
Reviewed by: Paolo Tizzani, World Organization for Animal Health, France
Semiha Yalçin, Mugla University, Türkiye
Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.
Ethics statement
The requirement of ethical approval was waived by PI Veterinary Institute of the Republic of Srpska “Dr. Vaso Butozan” for the studies involving animals because the blood samples from cattle were taken in diagnostic procedure by authorized veterinary authority. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.
Author contributions
AV: Formal analysis, Writing – original draft, Project administration, Validation, Methodology, Investigation, Funding acquisition, Conceptualization, Resources. GV: Formal analysis, Software, Visualization, Investigation, Writing – review & editing. AR: Supervision, Conceptualization, Writing – review & editing, Data curation, Project administration. IP: Writing – review & editing, Validation, Data curation. JK: Methodology, Writing – review & editing. RS: Writing – review & editing, Formal analysis. ST: Methodology, Writing – review & editing. OS: Investigation, Conceptualization, Funding acquisition, Visualization, Validation, Writing – original draft, Project administration.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The handling editor MK declared a past co-authorship with the author AV and JK.
Generative AI statement
The author(s) declared that generative AI was not used in the creation of this manuscript.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.