Molecular Detection and Identification of Babesia spp., and Trypanosoma spp. in one-humped camel (Camelus dromedarius) in Halayeb and Shalateen, Egypt

Addresses 1 Department of Biochemistry and Chemistry of Nutrition, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt. E-mail:abdo_dr_shimaa@yahoo.com (S.A.E.S.E.), drmohamedalymaher@hotmail.com (M.A.E.A.) 2 National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-Cho, Obihiro, Hokkaido, Japan 3 Department of Animal Genetics, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt. E-mail: mayar.othman132@yahoo.com 4 Department of Parasitology, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt. E-mail: alarabyma@yahoo.com Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 15 October 2020 doi:10.20944/preprints202010.0334.v1


Introduction
The world's event of desertification has highlighted the important role of camel in socio-economic aspects and the need of proper management system and appropriate disease control measures [1]. In several regions of the world, camels provide an important source of food and milk which is considered a high source of protein of high nutritional value. Moreover, the unique survival capability of camels and production in adverse environmental conditions permit the breeders for adopting semi-intensive farming systems for camels' production [2].
It was thought that camels are resistant to various pathogenic diseases [3]. Recently, different literatures described the susceptibility of camels to large number of pathogenic agents such as bacterial, fungal, parasitic and viral diseases [4].
In Egypt, vector transmitted diseases cause various clinical manifestations in farm animals [5]. Members of piroplasms are efficiently found in several species of equines, cattle and camel in Egypt, where specific DNA fragments were detected of those piroplasms in blood stream in apparently healthy camels of Babesia and Theileria species [6]. One of the famous piroplasms is Babesia spp. which is efficiently affected humans through ticks infestation [7]. Babesia microti has been identified in rodents and can efficiently affect human [8,9]. Camel babesiosis is an acute to chronic infectious disease that is distributed all over the world [2]. The disease is responsible for its deteriorative effects, high morbidity rate and higher economic losses, caused by the tick-borne hemo-parasitic protozoan [2,6].
Trypanosoma evansi is one of the famous parasite that affect animals and transmitted mechanically by tabanids species causing weight loss, anemia and abortion in several portions of the world such as South America, Asia and Africa [10]. In certain cases of trypanosomiasis, subclinical cases and other chronic infection can be found in hosts infected by low virulence strains [11].
Clinical signs of vector borne diseases are similar and is not sufficient for the ultimate diagnosis [12].
Moreover, the accurate diagnosis of piroplasms requires a wide range of laboratory techniques such as serological tests, blood smears and most definitely molecular diagnosis [13]. ELISA technique can be highly efficient in detecting both clinical and carrier animal, but lack specificity in determining recent and old infection [11]. Blood smear is considered a rapid field test for diagnosis of blood parasites definitely, but it has a low sensitivity against chronic and subclinical infections [14]. The molecular technique can offer a more accurate results in sensitivity and specificity in diagnosis of blood parasites and can be used frequently as a diagnostic approach for identifying active infections [5]. Therefore, the current study is aimed to use molecular diagnostic techniques for detecting blood parasitic infection (B. bovis, B. microti, and Trypanosoma spp.) in camels breeds in Halayeb and Shalateen that located at Lower Egypt and conducting phylogenetic analysis for all identified parasites.

Material and Methods
Ethical Statement: All experimental protocols in this study were approved by the Animal Care and Use Committee, Faculty of Veterinary Medicine, Mansoura University, Egypt (Approval No. 07-55). All Institutional and National Guidelines for the care and use of animals were followed according to the Egyptian Medical

Animals and blood sampling
A total of 142 blood samples were collected from one-humped camels during 2017. Blood samples were collected from camels reared in Halayeb and Shalateen, which located in Lower Egypt at the border with Sudan    [15][16][17]. The product was then chilled to 4 o C. After that gel electrophoresis of the PCR products was performed on a 1.5% agarose gel with TBE buffer and stained with ethidium bromide.

DNA extraction and PCR detections of hemoparasites
Then, the final PCR product was visualized under UV light.

Cloning and sequencing PCR products
Amplicons of PCR samples that exhibited high band intensities were extracted from agarose gels using a QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany), and cloned into a plasmid vector (PCR 2.1-TOPO, Invitrogen, Carlsbad, CA, USA). Sequencing was done for two colonies for each amplicon, using an AB1 PRISM 3100 genetic analyzer (Applied Biosystems, Foster City, CA, USA).

Phylogenic analysis
The evolutionary history was inferred using the Neighbor-Joining method [18]. The optimal tree with the sum of 3 branch length = 56.08480007 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [19] The evolutionary distances were computed using the Maximum Composite Likelihood method [20] and

Statistical analyses
The upper and lower limits of the confidence intervals of the positive rates were calculated for B. bovis, B. microti and T. evansi parasites using the Open Epi program (http://www.openepi.com/v37/Proportion/Proportion.htm).

Results
The genomic DNA sequences of the detected blood parasite in camel's blood were subjected to PCR with universal primers for all possible strains of Babesia and Trypanosoma species that was directed to amplify small ribosomal subunit in those parasites in order to conduct molecular diagnostic approach for blood parasite in camels.
Surprising, the highest infection rate in the present study was achieved by B. microti (  bovis. The phylogenetic analysis revealed the significant decrease in genetic distance between our obtained species and other species isolated from Argentine (AF030056), USA (AF030054) and Brazil (KC964615) (Fig. 2). Interspecies genetic distance analysis was conducted between our obtained RAP-1 gene sequences and other related taxa and a close genetic distance was detected between our isolated strain and other taxa from Argentine (AF030056.2) and USA (AF030054.1) in cattle ( Supplementary Fig. 2). For further confirmation, amino acids sequence was deduced from our obtained sequence and aligned with other related sequence of RAP-1 protein (Supplementary Fig. 2). The results of protein's phylogenetic analysis showed a close genetic relationship with other strain from Mexico (AAC27387.1) (Fig. 3). Pairwise genetic distance was performed between our obtained amino acid sequence and other related species of RAP-1 genes and revealed a close relationship with all aligned species except the isolated species from Brazilian bovine species (AFQ30755.1) (Supplementary Fig. 3).   under study. An accession number was deposited on GenBank for our obtained sequence (MF737081) (Fig. 5).
Phylogenetic tree was constructed between our isolated species of T. evansi and other related species where the higher similarity index species were aligned with our sequence and a lower genetic distance was observed between our isolated sequence and T. theileri that was isolated from cattle in Brazil. The phylogenetic analysis revealed the  Egypt in this study is boxed in black.

Discussion
A phylogenetic analysis was designed for our isolated sequence and others with high similarity index using neighbor joining method for statistical analysis of phylogenetic analysis with 500 replicates. A close genetic Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 15 October 2020 doi:10.20944/preprints202010.0334.v1 relationship was observed between our strain and other strains from Argentine, USA and Brazil. Similar result was investigated by a research work conducted by [21], they found that nested PCR technique conducted to amplify rhoptry-associated protein 1 (RAP-1) produced a close genetic relationship of South African strain of B. bovis strains with other strains from Uruguay, Argentine, Brazil and USA. However, the presence of polymorphism can adequately discriminate between a species and another [22]. Similarly, the conservation of Babesia spp. sequence between species was observed between Brazilian strains of B. bovis (98-100%) [23].
The amplification of small ribosomal subunit of B. microti was conducted which is considered a first record identification of B. microti in camels. B. microti was first discovered in the united states among human patients infecting mature erythrocytes [24]. In the current study, phylogenetic analysis of 18s rRNA gene of B. microti was revealed a close relationship between our isolated species and other species in Germany and France with a low genetic distance which was isolated from rodents. In Mongolia, a phylogenetic characterization of B. microti was executed and revealed a higher similarity index between isolated species of B. microti and other strains from united states, where the small ribosomal subunit gene was clustered with the United States -type of B. microti [25]. In fact, human babesiosis was detected in Egypt by El Bahnasawy and Morsy [26]. The symptoms, complete blood count, liver and kidney functions tests and all other serologic tests did not give concert diagnosis for B. microti in study performed by El Bahnasawy and Morsy [26]. Additionally, the patients were sero-negative for malaria infection. The patients were critically diagnosed by the demonstration of the typical ring forms of Babesia species in stained blood smears. These clinical cases were successfully treated with quinine and clindamycin, and were discharged from the hospital after the clinical and parasitological improvement. As this study demonstrated the infection of camels reared in Halayeb and Shalateen, in Lower Egypt by B. microti, future critical concerns must be taken to clarify the possible role of camels and its infested tick spp. in the zoonotic infection of babesiosis in Egypt. Importantly, the samples used in the present study were collected from apparently healthy camels, highlighting the fact that camels in Egypt might act as a silent reservoir for the infection of human by Babesia spp.
Internal transcript spacer 1 was used for simultaneous detection of three major trypanosome species (T. evansi, T. congolense and T. vivax) in circulating blood of camels. Phylogenetic analysis was conducted to identify the genetic relationship between our isolated species and other species of Trypanosma species. In the current study, T. evansi was identified by sequencing of the PCR products and was closely related to T. theileri that was detected from cattle in Brazil. Trypanosome is classified into several clades in a study in 1996 by Maslov et al., [27] and are as follows: mammalian, bird group, elasmobranch and are basically classified into vertebrate and in-vertebrates parasite.
Hughes and Piontkivska, [28] classified trypanosome species according to amino acid sequence of small ribosomal subunit into 42 protein families. They also classified Trypanosoma families according to host and location into American, African and bonoidae. In Egypt, internal transcript spacer 1 was used to investigate the phylogenetic relationship between trypanosome species and showed a genetic diversity among those species that can be used as a diagnostic tool for the identification of trypanosome species [12].
It should be noted that there are some limitations to the present study. Although this study report the first molecular evidence of Babesia microti in camel, the microscopy examination of the screened samples was not performed. Therefore, further investigation of Babesia microti in camels in Egypt by microscopy examination is needed. Also, to investigate the possible role of camels and its infesting tick as reservoir host for maintaining an enzootic cycle of Babesia transmission in Egypt, the simultaneous detection of B. microti prevalence in the tick species infesting camels, camels and human in the same area are urgently required. The small sample size used in this study may not allow obtaining a concrete conclusion about the incidence of different hemoparasites included in the study.
Moreover, this study was carried out in Upper Egypt only makes the need for further investigation in other areas in Egypt are needed.

Conclusions
This study provides the first evidence of B. microti in camel in Egypt and highlights the possible role of onehumped camels in maintaining the zoonotic cycle of Babesia transmission in Egypt. While, the prevalence of B.
microti infection in the most prevalent tick species infesting camels need to be further identified which may imply its possible role served as a reservoir host for maintaining an enzootic cycle of Babesia transmission in Egypt. These findings have economic significance and indicate the importance of introducing effective prevention and control strategies throughout Egypt to minimize the prevalence of infection by blood parasites in camels.
Obihiro, Hokkaido, Japan) for providing the authors by the positive control DNA samples. Also, the authors would like to thank owners and staff of the study farms in Egypt.

Author Contributions
Conceived

Funding
This research received no external funding