Molecular detection and characterization of Anaplasma marginale and Babesia canis vogeli infecting dogs in Luxor, Egypt

Tick-borne diseases in animals are increasing rapidly worldwide, but there is insufficient information about tick-borne diseases infecting dogs in southern Egypt. Thus, in the current study, we detected the presence of Anaplasma marginale (A. marginale) and Babesia canis vogeli (B. canis vogeli) in the blood of dogs. The results revealed that 4/100 (4%) were positive, and a higher infection rate was found in males (75%), than females (25%). The phylogenetic analysis for the major surface protein 4 (msp4) gene in this study was compared with amplicons separate from other reported isolates with alignment by identity 100% with cattle and camels from Egypt, and the phylogenetic analysis for the B. canis vogeli small subunit ribosomal RNA (SSU rRNA) gene in this study identified identity by 99.89% with dogs from Egypt. This report is considered the first report in southern Egypt about A. marginale in dogs based on the sequence analysis of the msp4 gene, providing new data for the classification and identification of A. marginale in dogs compared to A. marginale isolated from other animals in southern Egypt.

The protozoan morphology (piroplasm merozoites) within the red blood cell has traditionally been used as the primary taxonomic determinant based on a microscopic examination of a blood smear, this determination can categorize these protozoa as large size Babesia species (B.canis) or small size Babesia species (B.gibsoni), following that, molecular techniques identified several Babesia species that can infect dogs 12 .
The life cycle of Babesia protozoans spends part of it in the tick vector, but the merozoites circulating in the blood of infected animals can be transmitted directly to a healthy host via blood transfusion 13 .The first clinical evidence of possible vertical transmission of B. canis 14 and B. microti-like spp.has also been documented 15 .Babesia-infected dogs may be asymptomatic 16 or exhibit various clinical signs ranging from mild to severe and fatal.Clinical manifestations include anorexia, lethargy, fever, pale mucous membrane, jaundice, and renal disease depending on the parasite and the host's age, sex, and breed [17][18][19] .Subclinical signs of B. vogeli infection have been observed in both natural and experimental in vivo infections.The pathogen causes severe acute infection in immunocompromised animals, such as splenectomized dogs.The clinical signs can be accompanied by fever, anorexia, malaise, regenerative anemia, thrombocytopenia, and an increased white blood cell count 20 .
Anaplasma marginale is a frequently mechanically transmitted pathogen to susceptible cattle by blood-contaminated mouthparts of the blood sucking Diptera (Tabanus and Stomoxys) or via fomites 21 .The only site of replication in cattle is within the erythrocytes, where it forms membrane-bound vacuoles containing 4-8 A. marginale 22 .The prepatent period ranges from 7 to 60 days (depending on the infective dose), then up to 70% of erythrocytes may become infected during acute infections and with clinical signs 23 .Bovines older than one year are the most vulnerable to developing clinical diseases, severe anemia, icterus (without hemoglobinuria), fever, weight loss, lethargy, depression, and abortion were the main clinical signs observed 24 .The postmortem findings included icterus, splenomegaly, hepatomegaly, and petechial hemorrhage on the heart and pericardium.The blood was thin and watery, and all tissues were pale 25 , cattle that survived acute infections may remain carriers for the rest of their lives 3 .This study is part of many studies aimed to examine the anaplasmataceae and piroplasmosis in the different animal species including camel, cattle, buffalo, sheep, and goat in southern Egypt, the varied in breed, age, and gender, with most of animals showing no signs of severe disease 26 .The current study aimed to learn more about canine tick-borne diseases and provides the first sequencing data for A. marginale in dogs based on specific genes in southern Egypt, which suggests that dogs can become infected with A. marginale and become a source of infection.Governorate.This study occurred from April 2021 to January 2022 (Fig. 1).A total of 100 animals were used in this study, of which 55 were male and 45 were female.The animals ranged in age from one to three years and six months, with the numbers for each age group (1-2-years, 2-3 years, and 3-3.5 years) being 28, 37, and 35, respectively.Using clean, sterile vacutainer tubes with heparin as a target for PCR amplification, whole blood

Tick collection and pulverization
The tick samples were carefully cleaned by passing them through a fine mesh and a gentle stream of tap water to get rid of any surface dirt or debris.The ticks were then rinsed twice in sterile Milli-Q water and 70% ethanol for two minutes before being dried to show their taxonomic features.By using the appropriate standard identification keys 28,29 .After that, each tick was carefully put into a 2 ml tube that had a stainless-steel bead affixed to it to make crushing the tick easier after it was frozen for 12 h at − 80 °C.After that, each tick was crushed using an Automill crusher (Tokken.Inc., Chiba, Japan) for three cycles of 30 s each at 2000 rpm.Next, 200 μl of 1 M Tris-HCl (pH 7.5) was added to each sample, and the tubes were centrifuged at 13,000 rpm for 5 minutes at 4 °C.From the resulting mixture, 200 μl of tick homogenate was carefully collected for conventional PCR assay for mt-rrs gene detection for tick species identification 30 .Tick 16S rRNA forward and reverse primers used were respectively (5ʹCTG CTC AAT GAT TTT TTA AAT TGC TGTGG3ʹ) and (5ʹCCG GTC TGA ACT CAGA TCA AGT A3ʹ) for tick identification, and the PCR conditions are (95 °C)/(5 min) → [(98 °C)/(30 s)-(56 °C)/(30 s)-(72 °C)/ (30 s)]30 × → (72 °C)/(5 min).

DNA extraction, detection of control genes and pathogens by PCR
The DNA of one hundred blood samples from dogs was extracted using a (Wizard Genomic DNA Purification Kit, USA) and subjected to conventional Polymerase Chain Reaction (PCR), with the forward and reverse primers used were respectively, (5ʹ GTG AAC CTT ATC ACT TAA AGG 3') and (5ʹ CAA CTC CTC CAC GCA ATC G 3ʹ) for the detection of small subunit ribosomal RNA (SSU rRNA) gene of B. canis vogeli 31 and (5ʹ GGG AGC TCC TAT GAA TTA CAG AGA ATT GTT 3ʹ), and (5ʹ CCG GAT CCT TAG CTG AAC AGG AAT CTTGC 3ʹ) for the detection of major surface protein 4 (msp4) gene of A. marginale 32  The PCR reaction was done in a total volume of 10 µl using of 5 µl of 2 × Gflex PCR buffer and 0.5 µl of Tks Gflex DNA polymerase (TaKaRa, Shiga, Japan), and 0.5 µl of each of the forward and reverse primers with a concentration of 10 µM, 3 µl of nuclease-free water, and 0.5 µl of the template (DNA template with a concentration ranging from 10 to 30 ng/µl).Negative controls containing nuclease-free water were used as negative samples.Electrophoresis of PCR products was done in 1.5% agarose gel in a 1 × Tris-acetate-EDTA (TAE) buffer with an electrophoresis device (Mupid Co., Ltd., Tokyo, Japan).Bands were visualized through a gel documentation system UV device WUV-M20 (ATTO Co., Ltd., Tokyo, Japan) after being stained with 5 μg/ml ethidium bromide in 1 × TAE.

Sequence and data analysis
Sequence analysis for A. marginale, msp4 genes, and SSU rRNA gene of B. canis vogeli were subjected to PCR at 50 μl mixtures.The amplicons were purified using NucleoSpin Gel and a PCR Clean-Up Kit (Macherey-Nagel, Leicestershire, Germany) according to the manufacturer's protocol.Sequence readings were compared to the sequences of reported isolates using GenBank.A maximum likelihood phylogenetic tree was constructed using molecular evolutionary genetics analysis, version X (MEGA X software, https:// www.megas oftwa re.net/ citat ions) 33 with bootstrap values estimated using 1000 replicates based on Kimura model 34 .The 95% confidence intervals were calculated with www.vassa rstats.net 35  and pinpoint the cause of these abnormalities (Supplementary Fig. S1).The results of microscopic examination revealed the presence of two common species of ticks, brown dog ticks Rhipicephalus sanguineus, and Rhipicephalus annulatus (Supplementary Fig. S2).
The microscopic results were confirmed by sequencing and phylogenetic analysis, with our isolate of Rhipicephalus annulatus (PP837398.1)exhibiting 99.76% identity alignment when compared to amplicons that are distinct from previously published isolates in GenBank MK737647.1,and Rhipicephalus sanguineus PP832578.1 were identified with previously published isolates in GenBank MW560390.1 by 99.75% (Fig. 2).
Molecular identification for the tick species and A. marginale msp4 gene and B. canis vogeli SSU rRNA gene by PCR reaction was shown in (Supplementary Figs.S3 and S4).The results of infection rates for A. marginale and B. canis vogeli and hematological parameters are shown in Tables 1 and 2. Eight out of a total of one hundred animals (8%) tested positive for A. marginale and B. canis vogeli.Additionally, male animals (75%) had a greater  www.nature.com/scientificreports/infection rate than female animals (25%), with most of the infections occurring in the northern part of the governorate (75%).The total number of white blood cells and eosinophils increased slightly, and total red blood cells and hemoglobin decreased more than the reference parameter, with all other hematological parameters remaining within the normal range (Table 1).The results showed that A. marginale and B. canis vogeli had equal infection rates (4%) (Table 2).In addition to the location, the highest infection rate of A. marginale was found in the northern (50%), middle (25%), and southern areas (25%) of Luxor governorate, respectively.Regarding age, the highest infection rate (75%) was found in older animals (older than one year) than in younger (less than one year) animals (25%).
The phylogenetic analysis for the B. canis vogeli SSU rRNA gene in this study OP604258.1 and OP604259.1 compared with amplicons separate from other reported isolates with alignment by identity by 99.89% MN625891.1 with dog from Egypt, and by 99.78% HQ662635.1 with dog from Romania, and by 99.China and by 97.03% LC602472.1 with dog from Myanmar (Fig. 3).
The A. marginale (msp4) gene was sequenced for phylogenetic analysis and genotyping of A. marginale in dogs from different locations in the Luxor Governorate in southern Egypt.All sequences were also submitted to GenBank, and the following accession numbers can be used to access them for the msp4 gene OP244836.1,OP244837.1,OP244838.1,and OP244839.1.The phylogenetic analysis for the msp4 gene in this study was   4).

Discussion
In Egypt, dogs have recently received much attention from scientists, particularly in the southern region, and many findings have begun to be published.A simple method that is commonly used in clinical diagnosis is the microscopic examination of stained thin blood smears 36 .However, microscopic examination lacks sensitivity and makes it challenging to distinguish between species with mixed infections and low parasitemia.In addition to personal experience in evaluating blood smears, the diagnosis is frequently inaccurate 37 .The polymerase chain reaction assay, a variable amplification method, has exceptionally high sensitivity and is often used for research purposes 38 .For this reason, in our study, we focused on molecular techniques for the diagnosis and characterization of tick-borne disease.
The clinical and hematological examination of dogs in this study revealed no specific clinical signs for babesiosis and only a slight increase in total white blood cell counts and eosinophils, which could be related to tick infestation.These findings are consistent with the available literature, which regards B. vogeli as the lowest  www.nature.com/scientificreports/pathogenic strain, causing a relatively mild disease, usually without evidence of clinical signs, and can be found in clinically healthy dogs 39 .Microscopic examination of ticks revealed the presence of two types of ticks in dogs, Rhipicephalus sanguineus, which primarily infects dogs, and Rhipicephalus annulatus, which primarily infects cattle as well as some domestic animals like horses and dogs 40 .The phylogenetic analysis of our isolate, Rhipicephalus annulatus (PP837398.1),exhibited 99.76% identity alignment compared to other amplicons in GenBank MK737647.1,and Rhipicephalus sanguineus (PP832578.1)was identified with MW560390.1 by 99.75%.The mitochondrial 16S rRNA gene serves as a valuable marker for identifying ticks 41 .
This study is focused on tick-borne diseases in dogs, which include Babesia and Anaplasma.Babesia canis vogeli is the most widely distributed Babesia subspecies, occurring in Africa 42,43 , Europe 44,45 , Asia 46,47 , and Australia 48,49 .Babesia canis vogeli has been reported in Colombia 50 , Venezuela 51 , Brazil 52 , and Argentina 53 .In this study, the infection rate of B. canis vogeli in dogs from southern Egypt was 4%, which was lower than the result in dogs from northern Egypt, where the occurrence of B. canis vogeli infection was 5.1% 54 .We found that our reported rate of B. canis vogeli was lower than that in Iraq (5.1%) 55 , Brazil (4.8% and 15.6%) 56 , Iran (9.3%), Bosnia (85%), and Nigeria (10.8%) [57][58][59] .In this study, all dogs were local breeds, and babesiosis is more common and severe in imported dogs than in native species.However, not all dog breeds are equally susceptible to babesiosis 60 .
The current findings related to sex confirm that males have a higher probability of being infected with B. canis vogeli (75%) than females, only one animal was positive (25%) of being infected with B. canis vogeli.This result agreed with previous reports that males were more susceptible than females to Babesia infection 61 .The results of phylogenetic analysis for B. canis vogeli were obtained by comparing the B. canis vogeli sequences of Egypt isolates and other registered sequences in the gene bank.The phylogenetic analysis for the SSU rRNA gene was compared with amplicons isolated from other reported isolates, with alignment by identity ranging from 99.89% with accession number MN625891.1 to 97.03% with accession number LC602472.1.Two B. canis vogeli vouchers were clustered in distinct lineages and shared a well-supported subclade with parasites of the same host (Fig. 3).
Our study is the second report on A. marginale in dog blood in Egypt and the first report in southern Egypt in Luxor governorate.The first report was based on 16S ribosomal RNA sequences of Anaplasmataceae 62,63 .Major surface proteins have been designated as 1a, 1b, 2, 3, 4, and 5 64 , these proteins are recognized by neutralizing antibodies and have a strong intermolecular interaction in the membrane of the initial bodies, where they perform important functions 65 .Anaplasma species' biogeography and evolution have been studied by phylogenetic analysis using the msp4 gene 66 .Our report is considered the first report in Egypt for A. marginale in dogs based on the msp4 gene, which was sequenced for phylogenetic analysis.
All sequences were submitted to GenBank with accession numbers OP244836.1,OP244837.1,OP244838.1,and OP244839.1.Based on previous GenBank data, it was aligned by identity 100% with cattle and camels from Egypt, and by identity 99.72% with Canis lupus familiaris from Hungary, cattle from Russia, cattle from Tunisia, and by identity 99.57% with cattle from Tunisia and Sudan, and by identity 99.43% with cattle from Italy.It showed our sequences of A. marginale clustered together and shared a well-supported subclade with parasites of the same species (Fig. 4).
Based on the sequence data, the construction of the phylogenetic tree demonstrated the splitting of the tree into two major clades, and the phylogenetic position of the nucleotide sequences of A. marginale identified here was clustered with other A. marginale infecting other animals.The similarity between the sequences isolated from dogs and those isolated from cattle and camels in southern Egypt may be reflected in the close contact between dogs and these animals in rural areas.In an unpublished report, A. marginale was found in dogs in Hungary and was registered in GenBank based on the 16S ribosomal RNA gene under accession number MH020201.This finding supports the idea that some tick-borne pathogens accidentally parasitize other hosts 67 .
Based on the obtained data in the current study, future studies will be conducted including a higher number of samples from different Egyptian regions and more factors will be tested to identify the risk of infections.In this regard, different approaches will be additionally implemented such as serological testing and tick examinations.

Conclusions
The findings of this study revealed the widespread and considerable prevalence of A. marginale and B. canis vogeli in dogs in Luxor, Egypt.The identity of A. marginale was confirmed by amplifying the specific msp4 gene, which provided new data on A. marginale in dogs in southern Egypt.The phylogenetic analysis for the SSU rRNA gene of B. canis vogeli did not show 100% identity with any previously deposited sequence in GenBank (the maximum identity was 99.89%).The molecular characterization of A. marginale and B. canis vogeli in dogs is considered the first report in this area and provides significant data to the veterinary and public health sectors.
Dogs were used in the animal farm for guarding purposes and some dogs were in close contact with the animals especially in the transported animals and during outdoor feeding of the animals.All experiments were performed in accordance with relevant guidelines and regulation rules of the Ethical Research Committee of South Valley University, Faculty of Veterinary Medicine under letter number (No.19/11.08.2021).The current study focuses on the infection of local dog breeds with A. marginale and B. canis vogeli at various age and sex in the Luxor

Figure 1 .
Figure 1.Map of Egypt indicating the study areas where samples were collected from four different regions in Luxor.

Figure 2 .
Figure 2. Phylogenetic relations of tick species, obtained via the maximum-likelihood method 606 and the Tamura 3-parameter model based on 16S rRNA gene sequences.The percentage trees in which the related taxa clustered together is displayed next to the branches.Branch 608 lengths are expressed in terms of the number of substitutions per site, and the tree is drawn to scale.The circles represent the tick species infesting dogs in the present study.
74% MH100708.1 and OM069368.1 with Canis lupus familiaris from Paraguay and cat from China and by 99.66% LC331058.1 with Canis lupus familiaris from Zambia and by 99.61% KT323934.1 with dog from Brazil, and by 99.55% MK881089.1 and KT333456.1 with dog from China and Canis lupus familiaris from Brazil, and by 99.33% KY290979.1 with dog from Argentina and by 99.32% MH143394.1 with dog from China and by 99.30% MK495837.1 and MK495836.1 with dog from Cote d Ivoire and by 98.99% HQ148663.1 and Spain and by 98.88% KY805844.1 with dog from China and by 98.92% AY150061.1 with dog from Spain and by 98.32% LC602470.1 with dog from Myanmar, and by 97.96% AB083376.1 with Citellus erythrogenys from China and by 97.43% KX218439.1 with Panthera leo (African Lion) from Botswana, and by 97.39% MK571835.1 with dog from China, and by 97.32% MK256974.1 with canine form China, and by 97.28% MH143390.1 with dog from

Figure 3 .
Figure 3. Phylogenetic relations of Babesia canis vogeli in dogs, obtained via the maximum-likelihood method and the Kimura two-parameter model based on small subunit ribosomal RNA gene sequences.The percentage of trees in which the related taxa clustered together is displayed next to the branches.Branch lengths are expressed in terms of the number of substitutions per site, and the tree is drawn to scale.The circles represent Babesia canis vogeli in dogs obtained in the present study.

Figure 4 .
Figure 4. Phylogenetic relations of Anaplasma marginale obtained via the maximum-likelihood method and the Kimura two-parameter model based on major surface protein 4 (msp4) gene sequences.The percentage of trees in which the related taxa clustered together is displayed next to the branches.Branch lengths are expressed in terms of the number of substitutions per site, and the tree is drawn to scale.The circles represent Anaplasma marginale obtained in the present study.

Table 1 .
Hematological parameters of dogs.

Table 2 .
Anaplasma marginale and Babesia canis vogeli in dogs based on PCR detection in blood samples.95%CI calculated according to the method described at http:// vassa rstats.net/.CI confidence interval.Parasite Number