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Piroplasmosis and anaplasmosis are serious tick-borne diseases (TBDs) that are concerning for the public and animal health. This study aimed to detect the molecular prevalence and epidemiological risk factors of Piroplasma and Anaplasma species in animal hosts and their associated ticks in Egypt. A total of 234 blood samples and 95 adult ticks were collected from animal hosts (112 cattle, 38 sheep, 28 goats, 26 buffaloes, 22 donkeys, and 8 horses) from six provinces of Egypt (AL-Faiyum, AL-Giza, Beni-Suef, Al-Minufia, Al-Beheira, and Matruh). Blood and tick samples were investigated by polymerase chain reaction coupled with sequencing targeting 18S and 16S RNA genes for Piroplasma and anaplasmataceae, respectively. Statistical analysis was conducted on the potential epidemiological factors. Of the 234 animals examined, 54 (23.08%) were positive for pathogens DNA distributed among the six provinces, where 10 (4.27%) were positive for Piroplasma, 44 (18.80%) for anaplasmataceae, and 5 (2.14%) were co-infected. Co-infections were observed only in cattle as Theileria annulata and Anaplasma marginale plus Babesia bigemina, A. marginale plus B. bigemina, and T. annulata plus B. bigemina. Piroplasmosis was recorded in cattle, with significant differences between their prevalence in their tick infestation factors. Animal species, age, and tick infestation were the potential risk factors for anaplasmosis. All ticks were free from piroplasms, but they revealed high prevalence rates of 72.63% (69/95) with anaplasmataceae. We identified T. annulata, B. bigemina, and A. marginale in cattle; A. platys in buffaloes; A. marginale and A. ovis in sheep; for the first time, A. ovis in goats; and Ehrlichia sp. in Rhipicephalus annulatus ticks. Our findings confirm the significant prevalence of piroplasmosis and anaplasmosis among subclinical and carrier animals in Egypt, highlighting the importance of the government developing policies to improve animal and public health security.

Background The tick-borne intra-erythrocytic apicomplexan Babesia caballi is one of the etiological agents of equine babesiosis, an economically important disease of equids in most tropical and subtropical areas of the world. Discovering candidate antigens for improved diagnostic tools and vaccines remains needed for controlling equine babesiosis. This study describes the B. caballi sbp4 (Bcsbp4) gene and protein (BcSBP4) and analyzes its antigenicity in infected equids. Methods BLAST searches of an uncurated B. caballi assembly genome using the B. bovis SBP4 as a query were carried out, followed by PCR amplification and sequencing of a newly identified BcSBP4. Characterization of this novel gene and protein was performed by bioinformatics analysis, western blots, immunofluorescence (IFA) and an in vitro neutralization test using anti SBP4 peptide antibodies. Antigenicity of recombinant BcSBP4 (rBcSBP4) was tested with sera from field animals (n = 18) using an indirect ELISA (iELISA). Results Babesia caballi genome searches using B. bovis SBP4 as a query allowed identification of a novel gene termed Bcsbp4. The Bcsbp4 gene encodes for a protein of 30.58 kDa, which is fully conserved among B. caballi isolates from USA and Egypt. Bioinformatics analysis indicates that BcSBP4 contains a signal peptide and lacks additional transmembrane domains. Expression of BcSBP4 in blood stages of B. caballi was confirmed by western blot and IFA using antibodies against synthetic peptides representing putative B-cell epitopes of BcSBP4 predicted by in silico analysis. In vitro neutralization tests using anti-BcSBP4 peptide antibodies showed a marginal, but statistically significant inhibitory effect on the infectivity of B. caballi merozoites in horse red blood cells. Sera from eight B. caballi-infected equids, but none out of ten negative equid control sera, gave a positive signal in an rBcSBP4 based iELISA. Conclusions The Bcsbp4 gene is expressed in B. caballi blood stages. The BcSBP4 protein is a potential candidate for developing a novel serological test that could detect B. caballi infection in equids in tropical and subtropical countries worldwide.

Equine Piroplasmosis (EP) is an infectious disease caused by the hemoprotozoan parasites Theileria equi, Babesia caballi, and the recently identified species T. haneyi. Hereby, we used a multiplex PCR (mPCR) targeting the 18S rRNA gene of T. equi and B. caballi for the simultaneous detection of EP in Egyptian equids and examined the presence of T. haneyi infections in Egypt. Blood samples from 155 equids (79 horses and 76 donkeys) collected from different governorates of Egypt were examined by mPCR and PCR targeting T. hayeni. The mPCR method revealed a prevalence of T. equi of 20.3% in horses and of 13.1% in donkeys and a prevalence of B. caballi of 1.2% in horses. B. caballi was not detected in donkeys in the current study. The mPCR method also detected coinfections with both species (2.5% and 1.3% in horses and donkeys, respectively). Additionally, we report the presence of T. haneyi in Egypt for the first time in 53.1% of the horse and 38.1% of the donkey tested samples. Coinfection with T. haneyi and T. equi was found in 13.5% of the samples, while infection with the three EP species was found in 1.9% of the samples.