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Molecular prevalence and associated infection risk factors of tick-borne protozoan and rickettsial blood pathogens in small ruminants

BMC Veterinary Research volume 19, Article number: 138 (2023) Cite this article

Abstract

Background

Tick-borne blood pathogens cause highly pathogenic diseases, which are associated with substantial economic losses in ruminants. Despite this, epidemiological research on these pathogens remains neglected in many countries. This study initiated a regional epidemiological survey that included the detection of molecular prevalence, associated risk factors, and gene sequencing, combined with phylogenetic analysis, targeting the two main tick-borne blood protozoan and rickettsial pathogens of Babesia, Theileria, and Anaplasma that infect small ruminants. One hundred blood samples were collected from 76 sheep and 24 goats.

Results

Microscopic examination of Giemsa-stained blood films revealed that 73% of the samples were infected with at least one species of the three blood pathogenic organisms. Molecular diagnosis based on the 18 S rRNA for Babesia and Theileria species and the major surface protein 4 (msp4) for Anaplasma species, revealed that 43% of the small ruminants were infected with at least one of these pathogens. The animal’s sex was the most significant associated risk factor, with 49.4% of female animals infected compared with only 4% of male animals (P < 0.05). The open breeding system recorded the highest infection rate for tick-borne blood pathogens. Homology-based and phylogenetic analyses indicated that the specific isolate species were Babesia ovis (B. ovis), Theileria ovis (T. ovis), and Anaplasma ovis (A. ovis), with sequences showing significant identities with isolates from sheep, goats, and other animal species, and geographically diverse countries in Africa, Asia, and Europe, in addition to Egypt.

Conclusion

This was the first molecular evidence of B. ovis, T. ovis, and A. ovis infections in sheep and goat populations in the North Coast region of Egypt. More extensive studies are required to develop an epidemiological map of blood pathogenic organisms, while more effective control strategies are required to reduce the burden of tick-borne pathogens on small ruminants.

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Background

Small ruminants play an important role in the survival, economy, and social livelihoods of people throughout the world. More significantly, in developing countries, keeping small ruminants is crucial to meeting people’s needs for food, specifically meat and milk. Their wool is also used in many industries that contribute to national economies [1].

Infections of small ruminants by the piroplasmid and rickettsial blood pathogens, including Anaplasma (A.) ovis, A. bovis, (A) phagocytophilum, Babesia (B.). motasi-like, (B) motasi-like Xinjiang, Theileria (T.). lestoquardi, T. uilenbergi, and T. luwenshuni have direct effects, including debilitating health conditions that are primarily attributable to hemolytic anemia and decreased feed conversion ratio. If not treated on time, progressive parasitic infections will ultimately result in morbidities and high mortality rates among infected animals [2, 3]. Parasitic and rickettsial blood infections are especially challenging due to their unknown prevalence rates, various modes of transmission, and the increasing resistance to commonly applied anti-parasitic chemotherapies [4].

Babesiosis in small ruminants is caused by various Babesia species, but the two most responsible species are Babesia motasi and Babesia ovis [5]. In contrast, ovine theileriosis is caused by several Theileria species, which vary in their infectivity and pathogenicity. Theileria lestoquardi is the most pathogenic, while Theileria ovis is the least pathogenic [6]. Anaplasmosis in small ruminants, on the other hand, is mostly caused by one of three species, Anaplasma ovis, Anaplasma phagocytophilum, and Anaplasma bovis [7, 8]. Worldwide, A. ovis is the most prevalent Anaplasma species in small ruminants [8] and is also the main causative agent of ovine anaplasmosis in tropical and subtropical regions [9].

Under field conditions, the diagnosis of tick-borne blood pathogens depends primarily on collecting case history data, identifying clinical symptoms in animals, and performing laboratory diagnoses [10, 11]. Laboratory diagnosis primarily includes microscopic examination of Giemsa-stained blood smears to detect erythrocytic stages of protozoan blood parasites and molecular detection of parasitic agents [10, 12]. Molecular techniques such as polymerase chain reaction (PCR), restriction fragment length polymorphism, and phylogenetic analysis are also applied to detect previously unidentified species of disease-causative protozoan parasites and are also used to differentiate between different species of the same genus in different hosts and different districts [10].

The current study was initiated to identify tick-borne blood pathogens infecting sheep and goats located in the northern coastal region of Egypt utilizing microscopic, molecular, and phylogenetic analysis techniques. During the identification process, risk factors and their potential associations with tick-borne blood pathogens infections were also analyzed.

Results

Demographic data of the study animals

The majority of the sampled animals were sheep (76%), compared with goats (24%), while female animals predominated in the sampling group over males (79% versus 21%) (Table 1). Additionally, most of the sampled animals were older (≥ 3 years), while 26% were medium-aged animals (1–3 years), and the remaining animals were younger than 1 year old (16%). When grouped according to grazing practices, 62% were free grazing compared with 38% that were maintained in semi-closed grazing systems. Most of the sampled animals generally had good body scores (70%). Among the sample animals, the mucous membrane was normal in 32%, pale in 22%, and congested in 46%. Only 10% of the animals were feverish at the time of sampling, while 33% and 67% were recorded with or without the presence of ticks on animals, respectively (Table 1).

Table 1 Demographic data of small ruminants enrolled in the current study
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Microscopic detection

Microscopic examination of the 100 Giemsa-stained blood smear samples (76 samples from sheep and 24 samples from goats) revealed that 73% of the samples were infected with at least one species of tick-borne pathogens including the piroplasmid protozoan parasites of genera Babesia and Theileria, and that of rickettsial tick-borne Anaplasma. When analyzed, the rates of infection with Babesia, Theileria, and Anaplasma were 17%, 30%, and 65%, respectively.

Molecular identification

PCR was successfully applied to amplify specific gene fragments from the three blood pathogens, with the expected molecular masses for the PCR products of B. ovis, T. ovis, and A. ovis being 549, 509, and 870 bp, respectively. Data obtained revealed that 43 out of 100 samples were positive for at least one of the three tick-borne blood pathogens in the four selected districts of the Matrouh Governorate (Table 2). Babesia ovis, T. ovis, and A. ovis were recorded in 16%, 17%, and 24% of the total examined samples, respectively. The molecular prevalence data were used in all analyses from now and henceforth.

Table 2 Molecular prevalence of Babesia, Theileria, and Anaplasma among the four selected districts (Elhammam, Marsa-Matrouh, Eldabaa, and Elalamein) in Matrouh Governorate
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Distribution of infection of B. ovis, T. ovis, and A. ovis among the four selected districts of matrouh governorate

Data showed that no significant differences (X2 = 4.738, P = 0.198) were detected between the four studied areas, concerning the distribution of infection by the three blood pathogens (Table 2). When analyzed as risk factors, Elhammam district was considered a higher risk factor when compared with the reference area (Matrouh district), with odds Logistic Regression 95% CI 1.050 (0.373–2.959). Moreover, Table (3) showed that the association between infection of B. ovis, T. ovis, and A. ovis species and animal species (sheep and goats) was insignificant, with only the more risk of getting an infection with Babesia and Anaplasma among sheep than goats, with the Odds Ratio 95% Confidence Interval (1.444 (0.375–5.566) and 1.267(0.416–3.857, respectively).

Table 3 The molecular prevalence of babesia, theileria, and anaplasma obtained among the different animal groups in matrouh governorate
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Analysis of infection prevalence and associated epidemiological risk factors

Infections with tick-borne pathogenic organisms were recorded in 42.1% of sheep and 45.8% of goats, with no significant species-dependent differential, as shown in (Table 4). The second and only significant factor observed was animal sex, with 49.4% of females infected, compared with only 4% of male animals (P < 0.05). A higher infection rate of 48% was observed in older animals (≥ 3 years) compared with medium-aged and young animals, in which 42.3% and 25% infection rates were observed, respectively (Table 4).

Table 4 Analysis of the epidemiological risk factors and its association with blood parasites
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The open grazing system was found to be more favorable to infection than the semi-closed one, with 45.2% and 39.5% infection rates, respectively. The remaining factors were found to have differential effects on infection rates, although with non-significant association patterns. Animals with high-score body conditions recorded lower infection rates (38.6%) than weak and emaciated animals (53.3%). Congested eye mucous membranes were associated with a 47.8% infection rate, while normal and pale membranes were associated with lower infection rates of 43.8% and 31.8%, respectively. When the animal body temperature was examined, 50% of animals were found to be infected, compared with 42.2% of animals with normal body temperatures. Finally, 54.5% of animals in which ticks were detected during sampling were found to be infected with blood pathogens, compared with a 37.3% infection rate in tick-free animals (Table 4).

DNA sequencing and analysis

Homology analysis performed using GenBank sequence data revealed highly significant scores of identities, similarities, query coverage, and E. values, with top hits for highly homologous sequences associated with each of the two identified blood protozoon and rickettsial pathogens. Homology-based data confirmed the identities of the current isolates as B. ovis, (A) ovis, and T. ovis, respectively. The nucleotide sequences of (B) ovis, T. ovis, and A. ovis isolates in the current study were recorded in GenBank under accession numbers MZ801782.1, MZ801783.1, and MZ889099, respectively.

The current B. ovis isolate was well-aligned and highly homologous with B. ovis isolates from sheep, goats, and horses, and countries as diverse as Uganda, Turkey, and Spain, with identities of > 94% (Table 5). Albeit with slightly lower identities, other Babesia species, such as Babesia orientalis, Babesia bigemina, and Babesia divergens from China, Iraq, and Ireland, respectively, were also highly homologous. Top homology hits of T. ovis included isolates of T. ovis from sheep and donkeys located in Iran, Pakistan, and Egypt, with > 92% identities (Table 5). Similarly, Theileria parva, Theileria lestoquardi, and Theileria orientalis, presented > 90% homology over their 18 S rRNA sequences. In contrast to Babesia and Theileria, all BLAST homologous outputs of Anaplasma 16 S rRNA were those of A. ovis isolates from Iran, Sudan, and Russia, with > 90% identities (Table 5).

Table 5 BLAST-Homology analysis of sequence output of Babesia ovis, Theileria ovis, and Anaplasma ovis
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Phylogenetic analysis

Phylogenetic trees of B. ovis and T. ovis were constructed based on 18 S rRNA gene sequences, and a phylogenetic tree of A. ovis was constructed based on msp4 gene sequences, which were generated in this study or downloaded from GenBank.

The neighbor-joining-based phylogenetic analysis of the 18 S rRNA nucleotide sequences of Babesia spp. revealed a highly conserved evolutionary relationship of current B. ovis as it has been grouped in the same clade with other related species from sheep, goats, horses, and ticks from various geographical locations (Fig. 1). The only exception was B. orientalis from ticks in China. Other Babesia species were systemically grouped into three specific clades, which were then grouped exclusively as B. bigemina, Babesia bovis, and Babesia centralis. These included isolates from different hosts and geographically diverse countries (Fig. 1).

Fig. 1
figure 1

The dendrogram was constructed using the neighbor joining (NJ) method based on 18 S rRNA partial sequences of Babesia ovis isolate. The distance of similarities is based on the molecular basis evolution of minisatellite sequence regions in Babesia ovis genomic DNA. The new sequence reported in the current study is indicated (squared)

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The phylogenetic analysis of partial 18 S rRNA nucleotide sequences of T. ovis resulted in two main clades, with T. ovis isolates being grouped into a large clade, and other species, such as T. lestoquardi, T. parva, and Theileria annulata, being grouped into another large clade (Fig. 2). The current isolates in the same clade as T. ovis are isolated from countries as diverse as Iran, Sudan, Serbia, China, Mongolia, Pakistan, and Russia (Fig. 2). The current isolate was primarily associated with T. ovis in sheep, goats, and donkeys in Egypt, Turkey, Algeria, India, and Tunisia. The evolutionary distant relationships among Anaplasma isolates were more homogenous as inferred from their NJ tree. A. ovis isolates were clustered into a single parental clade that was further subdivided into two branches, with one branch linking the current isolate with A. ovis from sheep, goats, and ticks from Russia, Iran, and Serbia, while the other branch grouped A. ovis from sheep, deer, ticks, and humans from Spain, Cyprus, China, and Turkey (Fig. 3). Species of Anaplasma, other than A. ovis, were remarkably grouped into unique clades with isolates of the same species, whether A. bovis, A. centrale, Anaplasma marginale, and A. phagocytophilum, and were clustered together regardless of their hosts or geographic origins (Fig. 3).

Fig. 2
figure 2

NJ-based phylogenetic analysis of 18 S rRNA partial sequences of the Theileria ovis isolate. The distance of similarities is based on the molecular basis evolution of minisatellite sequence regions in T. ovis genomic DNA. The new sequence has been similarly indicated (squared) on the tree branches

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Fig. 3
figure 3

NJ-based phylogenetic analysis of the msp4 partial sequences of the Anaplasma ovis isolate. A. ovis sequence of the study has also been indicated. The distance of similarities is based on the molecular basis evolution of minisatellite sequence regions in A. ovis genomic DNA.

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BLAST analysis of the 18 S rRNA identified the current isolate as T. ovis, and it was deposited in GenBank with accession number MZ801783.1. The T. ovis sequence of the current study revealed high homology-based identities of 94.5% and 92.87% with T. ovis genotypes from sheep from Iran and Pakistan (KC599236.1, MT498784.1), respectively), while a T. ovis isolate from donkeys (MN625903.1) also recorded a high identity of 92.87%. In contrast, considerable homologies were recorded for T. parva, T. lestoquardi, and T. orientalis, from buffalo, sheep, and cattle, respectively. A more evolutionary grouping of the phylogenetic dendrogram was identified for Theileria compared with Babesia and Anaplasma, as all T. ovis isolates were on the same tree node, leaving other Theileria species, such as T. parva, T. lestoquardi, and T. annulata, on a separate node. A more in-depth look at the dendrogram tree of Theileria species revealed that the current T. ovis was placed in very close proximity to four T. ovis isolates, three from sheep (MN625886.1/Egypt, AY260172.1/Turkey, and MH327772.1/Algeria), and the fourth A. ovis from donkeys (MN625903.1/Egypt), respectively. The T. ovis of this study was grouped well with other Theileria species isolated from other countries and hosts, such as T. lestoquardi isolated from goats in India (GenBank accession number MZ220437.1), T. parva from water buffalo in Kenya (accession number AF013418.1), and T. annulata from cattle in Iraq (accession number MW940543.1). Interestingly, Theileria species other than T. ovis were from animals other than sheep, including buffalo, cattle, and goats.

Discussion

Small ruminants are affected by several infectious diseases caused by bacterial, viral, and fungal pathogens, as well as by parasitic diseases caused by protozoan parasites, which have severe negative impacts on their health and welfare. Understanding the epidemiology of these tick-borne blood pathogens is crucial to their effective control and treatment. Thus, the current study involving sheep and goats in the northern coastal region of Egypt, specifically the Matrouh Governorate, had the aim of determining the prevalence of B. ovis, T. ovis, and A. ovis infections and identifying associated risk factors. Molecular detection by PCR using species-specific primers combined with sequencing, BLAST queries, and phylogenetic analysis was applied to detect implicated species.

Utilizing PCR, the total prevalence of the tick-borne blood pathogens B. ovis, T. ovis, and A. ovis was 43%. This was a slightly lower incidence rate than that recorded in Turkey [13], where the PCR indicated the prevalence of these parasites in small ruminants was 68.2%. Similarly, a higher molecular prevalence rate of 69.2% was recorded in sheep and goats in Iraq [14].

The present study detected that there was a significant association between animal sex and the prevalence of tick-borne blood pathogens. This finding concurs with [15], who reported that females were significantly associated with higher infection rates of protozoan blood parasites, and argued that female animals often experienced stress factor-associated immunosuppression, which was primarily associated with pregnancy and lactation, which their male counterparts did not experience. However, in larger ruminants, such as cattle, no significant association between sex and protozoan blood parasite infection has been reported [16].

In this study, the breeding system design was one of the most important risk factors identified. The open breeding system reported a significant association with higher infection rates of tick-borne blood pathogens. Similar evidence was reported in other studies, as [17, 18] demonstrated significant associations between high infection rates and semi-intensive outdoor breeding systems. This is primarily attributed to the higher likelihood of encountering vector-borne disease agents, which is reflected in higher blood parasite infection rates. In a related study, the open breeding system and outdoor grazing were shown to have increased chances of exposure to several vectors, including hard ticks, which are the primary vectors of protozoan blood parasite transmission [19]. Moreover, the open breeding system is associated with climatic changes (fluctuating weather conditions) that, through their effects, affect animal immunity.

The prevalence of A. ovis in sheep and goats revealed by PCR was 24%. Similarly, [20] in Iran, and [21] in Medina, Saudi Arabia, reported that the prevalence of A. ovis among small ruminants according to PCR was 27.5% and 25.3%, respectively. In contrast, a higher A. ovis prevalence rate of 60.89% according to PCR was reported in 12 Chinese provinces, which is nearly double the prevalence rate seen in the present study [22].

PCR amplification of the 16 S rRNA gene of Anaplasma species yielded an amplicon of ~ 870 bp. This is as expected for the amplified product specific to Anaplasma species, as reported by another study in the West Azerbaijan province, Iran, in which the targeting of the same gene produced a closely related size amplicon and sequence of 866 bp [23]. BLAST-homology queries unsurprisingly produced hits that were all identified as A. ovis homologues from different animal species, mostly sheep, but also from other animal hosts such as cattle, deer, and ticks. BLAST analysis results were further confirmed by phylogenetic analysis and its dendrogram tree as A. ovis in the current study. It was placed in the same clade with A. ovis isolated from goats from Iran, and A. ovis isolated from sheep from Serbia and Russia under accession numbers EU92511.1, GQ92510.1, and MT062868.1, respectively. These were in close evolutionary proximity to A. ovis from sheep, ticks, humans, and deer from Turkey, China, Cyprus, and Spain, respectively. Although not in the same clade (node of branching), the current A. ovis isolate relates to other species of Anaplasma, such as A. bovis isolated from deer in Japan (GenBank accession number AB211163.1), cattle in China (GenBank accession number MH255937.1), and ticks in China (GenBank accession number MN044717.1). It also relates to A. centrale isolated from cattle from Kyrgyzstan and South Africa with accession numbers MW672119.1, and GQ130291.1, respectively, as well as A. marginale isolated from cattle from China (GenBank accession number AJ63304.1), ticks from Pakistan (GenBank accession number ON52757.1), and A. phagocytophilum identified in rats and ticks from Canada and China, respectively.

The prevalence rate for Babesia infections according to PCR in this study is consistent with PCR-based molecular prevalence rates of Babesia infection recorded in two studies in Iran and Greece, which reported 16.7% and 15%, respectively [24, 25]. However, [26] in an independent study in the North Khorasan province of Iran, researchers reported a lower prevalence rate of 6.6% for B. ovis infection.

The amplification of the 18 S rRNA gene using Babesia-specific primers yielded a band at ~ 550 bp, which corresponds well with the same results of DNA amplification by the same primers as recorded by [27] in Elazig province, Turkey. Phylogenetic and BLAST analysis of the 18 S rRNA sequence of our Babesia isolate revealed that it was B. ovis, which was recorded in GenBank with accession number MZ801782.1.

These B. ovis isolates showed high identity (> 94%) with B. ovis isolates from other countries, including Uganda, Turkey, and Spain (GenBank accession numbers MT114713.1, MG569902.1, and AY150058.1, respectively), and were isolated from goats, horses, and goats, respectively. High homologous sequences from Babesia spp. other than B. ovis were retrieved and were highly identified (> 90%) with the current isolate. These included B. orientalis, B. bigemina, and B. divergens, from ticks (China), buffalo (Iraq), and cattle (Ireland), respectively.

In addition to B. orientalis, all B. ovis sequences were clustered together on the same node of the evolutionary dendrogram, including isolates from sheep, goats, horses, and ticks. For B. orientalis, this is not surprising, as until recently it had not been registered as a separate species due to the unusually high number of shared identities with other commonly known Babesia spp. [28].

More divergent Babesia species, such as B. bigemina, B. divergens, and B. bovis, are clustered on branches separate from B. ovis. When compared with Anaplasma, a more evolutionary conserved relationship was detected for various B. ovis isolates. The prevalence of Theileria according to PCR was 17% in the current study. Similar findings in Uganda were reported by [29], with an overall infection rate of 13.4%. In contrast, [30] recorded a higher prevalence of Theileria in different districts in China. Additionally, a high incidence rate of 66.25% was recorded for theileriosis in small ruminants in Kenya [31]. Lastly, PCR targeting the 18 S rRNA of Theileria species yielded an amplicon of 500 bp, an expected PCR product size as amplified by the same Theileria-specific primers on blood samples collected from small ruminants in China [32].

Conclusion

In our current study, the data revealed that the PCR, combined with sequencing and phylogenetic analysis was successfully applied in detecting two of the most important tick-borne protozoan and rickettsial pathogens that are circulating in populations of small ruminants on the North Coast of Egypt. Molecular phylogenetic analysis revealed significant identities with corresponding homologous isolates from Asia, Africa, and Europe. Analysis of risk factors showed that the grazing system is a crucial determinant of the propagation of vector-borne pathogens and that every effort should be directed toward changing the open grazing habits of small ruminant herds into a more closed system. The next study concerning the current topic should essentially be directed toward surveying small ruminants in a more extended geographical manner to develop an epidemiological map of tick-borne protozoon and rickettsial infective organisms in small ruminants in Egypt. Finally, more efforts are required to develop more effective measures to control blood parasites and, subsequently, optimize small animal health and productivity in Egypt.

Materials and methods

Study settings

This study was conducted in the northern coastal region of Egypt, with the main focus on Matrouh Governorate (31.352778°N, 27.236111°E), which included four districts: Elhammam, Marsa-Matrouh, Eldabaa, and El-Alamein. Herds of small ruminants are distributed throughout the four geographical regions. The study utilized a cross-sectional design. In this cross-sectional study, sheep and goats were sampled without regard to disease status and studied at a particular point in time. All methods were carried out following relevant guidelines and regulations. Ethical approval for this study was obtained from the scientific research ethical committee of the Faculty of Veterinary Medicine, Alexandria University (Approval #00012098).

Sample size

One hundred blood samples were collected from 76 sheep and 24 goats. The sample size was determined based on the total capital herd population of small ruminants in the designated four out of eight districts in Matrouh Governorate. The total was 178,408 heads, according to the latest data from the Matrouh Governorate Directorate of Veterinary Medicine (Access Link 1). According to a previous study of blood protozoan parasitic infection, which indicated a prevalence rate of 10% [33], the minimum sample size for this study was 98 heads, with a margin of error of 5% and a confidence level of 90%. Where the sample size was determined by using the Sample Size Calculator by Raosoft, Inc., http://www.raosoft.com/samplesize.html with a confidence level of 90%. The samples were collected from four districts. Within each district, by using the EPI method (bottle spinning) https://academic.oup.com/ije/article to determine a random direction to select random farms along the chosen direction pointing outwards from the centre of the district to its boundary, then within all farms selected, the total sheep and goats were required to be enrolled in the current study by a random systematic method. https://study.com/academy/lesson/systematic-random-samples.

Sampling technique

A multistage sampling technique was applied as follows: an unequal probability of selection with unequal-sized clusters was utilized during the first stage, and proportional allocation according to cluster size was used during the second stage to select 76 sheep and 24 goats from the four districts. From each of the selected animals, 2 ml of blood was collected from the jugular vein into anticoagulant EDTA (Ethylenediaminetetraacetic acid)-coated vacutainer tubes (Thermo Scientific™, Thermo Fisher Scientific Inc., Waltham, MA, USA). These samples were stored at − 20 ºC until further processed for microscopy and DNA extraction. In parallel to the sample collection, the body condition scoring was performed on the sampled animals following the technical body condition scoring protocol that was developed at Langston University (Access Link 2), which considered the status of the lumbar region, the rib cage, and the sternum, while examining the animal. Animals that scored ≥ 3 were considered high scores, while those ≤ 3 were recorded as having low body conditions. The animal temperature was also recorded, and the body temperature was considered normal or febrile (feverish) concerning the normal range of the body temperature of sheep and goats (sheep: 38.3–39.9 °C; goat: 38.5–39.7 °C; in the Merck Veterinary Manual 20th Edition).

Microscopic examination

For each sample, a thin blood smear was prepared on a glass slide, air-dried, and fixed for 5 min in methyl alcohol. The fixed blood smears were then stained with 5% Giemsa stain for 30 min. The Giemsa-stained blood smears were washed gently with tap water to remove the excess stain, and the stained films were examined using oil immersion lenses (1000x magnification) of a light microscope [34]. To set up a blindfolded examination strategy, three smears were prepared from each blood sample, and these were examined by two independent researchers, setting up the examination of up to six technical replicates from each sample, to conclude whether the sample was positive or negative.

DNA extraction and PCR-based amplification

Total genomic DNA was extracted from whole blood samples using the Gene JET Whole Blood Genomic DNA Purification Mini Kit (Thermo Fisher Scientific Baltics, UAB, Lithuania), as directed by the manufacturer. For the PCR, three sets of primer pairs were designed to amplify species-specific targets of Babesia ovis, Theileria ovis, and Anaplasma ovis (Table 6). For B. ovis and T. ovis, the primers were designed to specifically amplify targets from the 18 S rRNA sequences as previously specified [35], while for A. ovis, the primers were designed to specifically amplify targets from the major surface protein 4 (msp4) gene [36].

Table 6 Oligo primer sequences used in the current study
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DNA sequencing and analysis

To establish the molecular identities of the amplified PCR products, representative three samples that were positive (by microscopy and PCR) for the three pathogens (single sample per pathogen), and that also corresponded to the expected (annotated) molecular masses for each primer set, were selected for DNA sequencing. Selected PCR amplicons were sequenced in a single-sense stranded direction with the sample PCR sense primer, and by using the ABI 3500 Applied Biosystems genetic analyzer (Applied Biosystems, CA, USA). Assembled sequences (between 509 and 1345 bp) were manually screened and curated to remove non-allocated poly-N nucleotides (N). Curated clean sequences were queried in the standard nucleotide BLAST (BLASTn) search tool of the National Center for Biotechnology Information (NCBI, USA) (https://blast.ncbi.nlm.nih.gov/Blast.cgi) [37].

Throughout all the BLASTn queries, default settings were applied, except for the use of the “more divergent sequences” option instead of “closely related sequences” in the BLASTn search platform. The top sequence hits resulting from the BLASTn analysis showed significant similarities and identities with the sequences from the current study that were selected. Reverse-BLASTn using individual sequences was performed to confirm the molecular identities of the sequenced samples versus previously published sequence data deposited at GenBank (https://www.ncbi.nlm.nih.gov/genbank/) as reference sequences. Data corresponding to selected sequence hits, including names, accession numbers, animal hosts, countries, homology sequence identities (%), percentage query coverages (%), and E. values were extracted from BLAST and GenBank data and were correspondingly tabulated for each of the three blood pathogens. Finally, sequence data from highly homologous isolates was downloaded and used in the phylogenetic analyses.

Phylogenetic analysis

All extracted sequences, including the current ones, were subjected to sequence alignment, with two approaches applied. First, the BLAST sequence alignment for each of the three BLASTn outputs was downloaded, scanned, and curated manually to remove non-allocated gaps and to delete extensions at both ends of the aligned sequence data. In the second approach, homologous sequences were extracted from the GenBank followed by the use of the MEGA v.7 platforms for sequence alignment. The aligned sequences were then entered into the phylogenetic analysis within MEGA v.7 software [38]. Evolutionary relationships between different species of each of the three tick-borne blood pathogens were inferred from the analysis results by using the Saitou-Nei Neighbor-Joining (NJ) method with bootstrapping set at 1000 replications in MEGA v.7 [39].

Statistical analysis

Data were statistically analyzed using IBM SPSS Statistics for Windows, version 22 (IBM Corp., Armonk, NY, USA), and by applying Pearson’s Chi-squared test to determine the statistical significance and associations between prevalence rates and risk factors as independent variables. The confidence interval was set at level 95% and the odds ratio (OR) to predict the associations between infection rates and risk factors were considered significant at P < 0.05.

Data Availability

The datasets generated and/or analyzed during the current study are available in the GenBank of the National Center for Biotechnology Information (GenBank-NCBI) repository, which can be readily accessed by applying the following accession numbers: MZ801782.1, MZ801783.1, and MZ889099.

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Acknowledgements

We would like to offer great appreciation to all members of the Veterinary Authority and its administrative units at the Matrouh Governorate for their logistic support. Also, the authors would like to extend their sincere appreciation to The Science, Technology & Innovation Funding Authority (STDF) and The Egyptian Knowledge Bank (EKB) for their financial support and language editing proving support respectively.

Funding

No specific fund or financial aid for the research work has been received from funding agencies in the public, commercial, or not-for-profit sectors.

Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

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Authors and Affiliations

  1. Department of Parasitology, Faculty of Veterinary Medicine, Matrouh University, Matrouh, Egypt

    Mennat-Allah Abdelsalam & Wael Felefel

  2. Department of Biochemistry, Faculty of Veterinary Medicine, Matrouh University, Matrouh, Egypt

    Sabreen Fadl

  3. Department of Parasitology, Faculty of Veterinary Medicine, Alexandria University, Alexandria, Egypt

    Mohamed Bessat

  4. Faculty of Veterinary Medicine, King Salman International University KSIU, Ras Sudr, South Sinai, Egypt

    Mohamed Bessat

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Contributions

All authors were equally involved in setting up the research proposal, conducting the experiments, and drafting the manuscript. Investigation, Methodology, and Writing – original draft, Mennat-Allah Abdelsalam, Wael Felefel, and Mohamed Bessat; Supervision, Wael Felefel, Sabreen Fadl, and Mohamed Bessat; Revised and edited manuscript, Mennat-Allah Abdelsalam, Wael Felefel, Sabreen Fadl, and Mohamed Bessat; Writing – review & editing, Mennat-Allah Abdelsalam, Wael Felefel, Sabreen Fadl, and Mohamed Bessat.

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Correspondence to Mennat-Allah Abdelsalam.

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Handling of the animals was conducted ethically, with no excessive force or pain inflicted upon the animals. This followed the standard protocol approved by the Ethical Committee of the Faculty of Medicine, Alexandria University (Animal Ethical Handling Protocol #00012098).

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Oral informed conscience where obtained from the animal owners before samples collection from their animals.

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Abdelsalam, MA., Felefel, W., Fadl, S. et al. Molecular prevalence and associated infection risk factors of tick-borne protozoan and rickettsial blood pathogens in small ruminants. BMC Vet Res 19, 138 (2023). https://doi.org/10.1186/s12917-023-03702-4

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BMC Veterinary Research

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