Impact of Equine and Camel Piroplasmosis in Egypt: How Much Do We Know about the Current Situation?

Piroplasmosis is a global tick-borne disease caused by hemoprotozoan parasites, which causes high morbidity and substantial economic losses in farm animals. Equine and camel piroplasmosis causes significant losses worldwide and in Egypt. The multifactorial effects and overall impact of equine and camel piroplasmosis in Egypt remain poorly characterized. However, several Babesia and Theileria spp. as well as potential tick vectors affecting these two species have been identified in the country. Equine and camel piroplasmosis has been reported by all governates in the country. Thus, in this work, we intend to provide a broad depiction of the current approaches used for diagnosis and control and the impact of piroplasmosis on the equine and camel industries in Egypt. We also identified current gaps in knowledge that might help develop future research efforts towards improved intervention and control of equine and camel piroplasmosis. It is important to develop specific diagnostic tools suitable for the early and chronic diagnosis of this disease. Altogether, the current situation warrants the development of large-scale epidemiological studies in order to obtain an accurate estimate for equine and camel piroplasmosis to secure the highly needed food resources in the country.

Cross-sectional analysis of Piroplasma species-infecting camel (Camelus dromedaries) in Egypt using a multipronged molecular diagnostic approach.

Camel piroplasmosis is a tick-borne disease (TBD) caused by hemoprotozoan parasites. Hereby, we describe a cross-sectional study aiming at identifying Piroplasma spp.-infecting camels in Egypt using a multipronged molecular diagnostic approach. A total of 531 blood samples from camels (Camelus dromedarius) were collected from slaughterhouses at different governorates in Egypt for analysis during the period from June 2018 to May 2019. Piroplasma spp. was identified using microscopical examination and several different and sequential polymerase chain reaction (PCR) assays targeting the 18S rRNA genes. The overall prevalence of Piroplasma spp. in microscopical and molecular analyses in the samples was 11% (58/531) and 38% (203/531), respectively. Further discriminative multiplex PCR analysis targeting the 18S rRNA gene applied on all Piroplasma spp.-positive samples allowed the detection of Theileria equi (41%), Babesia caballi (5.4%), Babesia bigemina (0.5%), and Babesia bovis (4%). Additionally, the blast analysis of nested (n) PCR, targeting the V4 region, amplicon sequences resulted in the identification of B. vulpes (22%), Babesia sp. (9%), and Theileria sp. (3%). Overall, the results of this study confirmed the high prevalence of TBDs caused by several types of piroplasm hemoparasites in camel and suggests the need for future interventions aimed at improving the control of these potentially debilitating diseases that may be t-hreatening important economic resources and food security in Egypt.

Molecular Epidemiological Investigation of Piroplasms and Anaplasmataceae Bacteria in Egyptian Domestic Animals and Associated Ticks.

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.

Fundamental Tick Vaccinomic Approach to Evade Host Autoimmune Reaction.

Ticks are obligate hematophagous ectoparasites that infect domestic animals, humans, and wildlife. Ticks can transmit a wide range of pathogens (viruses, rickettsia, bacteria, parasites, etc.), and some of those are of zoonotic importance. Tick-borne diseases have a negative economic impact in several tropical and subtropical countries. With climate change, tick distribution and tick-associated pathogens have increased. Currently, tick control procedures have more environmental drawbacks and there are pitfalls in vaccination process. Since vaccinations have helped to prevent several diseases and infections, several vaccination trials are ongoing to control ticks and tick-borne pathogens. However, autoimmune reactions to vaccinations are reported as an adverse reaction since vaccines were used to protect against disease in humans and animals. The antibodies against the vaccine antigen might harm similar antigen in the host. Therefore, in this chapter, we attempt to shed light on the importance of raising awareness of possible adverse events associated with vaccinations and the methods that should be used to address this problem. In silico and lab work should be performed ahead of the vaccination process to evaluate the vaccine candidates and avoid the vaccination opposing consequences.

Rapid Detection of Equine Piroplasms Using Multiplex PCR and First Genetic Characterization of Theileria haneyi in Egypt.

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.

Identification and antigenicity of the Babesia caballi spherical body protein 4 (SBP4).

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.

Camel hydatidosis diagnostic kit: optimization of turnip and horseradish peroxidase conjugates using glutaraldehyde method.

Echinococcosis/hydatidosis is one of the most important parasitic zoonotic diseases in the world. Cystic echinococcosis increases public health and socio-economic concern due to considerable morbidity rates that give rise to elevated economic losses both in the public health part and in the farm animal field. The enzyme linked immunosorbent assay (ELISA) is consider the more accurate tool for diagnosis of hydatidosis in camels. In the present study, affinity purified Echinococcus granulosus (E. granulosus) antigens (APA) were purified from crude hydatide E. granulosus germinal layer proteins for detection of E. granulosus antibodies in infected camels, using affinity matrix (camel IgGs coupled to CNBr-activated Sepharose). The electrophoretic profile of the APA showed that it was separated into two bands; one major band of 130 kDa and one minor band at 55 kDa. These antigens were used successfully as specific coating antigenic proteins in detection of echinococcosis in camel. In a trial to prepare an anti-camel IgGs peroxidase conjugate; peroxidase enzyme was purified from turnip roots (TPOD) using ammonium sulfate precipitation and affinity chromatography on phenyl Sepharose CL-4B. The purified TPOD showed a major band at 35 kDa. Rabbit anti-camel IgG antibodies (AC IgGs) were prepared then purified using affinity chromatography on Protein G-Sepharose. The TPOD, and commercial HRP for comparison, enzymes were conjugated to AC IgGs using 1%, 5% and 10% glutaraldehyde. The results revealed that the HRP was much better than TPOD in conjugation with AC-IgG antibodies and the 10% glutaraldehyde concentration was the most efficient concentration with ELISA titer 1:50.

Assessment of Theileria equi and Babesia caballi infections in equine populations in Egypt by molecular, serological and hematological approaches.

BACKGROUND: Equine piroplasmosis (EP) caused by Theileria equi, Babesia caballi, or both, contributes to significant economic loss in the equine industry and remains uncontrolled in Egypt. This study focuses on surveying T. equi and B. caballi infections and hematological disorders in equine populations in Egypt. METHODS: Theileria equi and B. caballi infections were assessed in blood from 88 horses and 51 donkeys in Egypt using light microscopy, indirect immunofluorescent antibody test (IFAT), nested PCR (nPCR), and competitive-ELISA (cELISA) assays. PCR products were examined for specificity by DNA sequencing. Hematological alterations were evaluated using a standard cell counter. RESULTS: Microscopic analysis revealed EP infection in 11.4% and 17.8% of horses and donkeys respectively. IFAT detected 23.9% and 17.0% infection of T. equi and B. caballi, respectively, in horses, and 31.4% of T. equi and B. caballi in donkeys. T. equi cELISA detected 14.8% and 23.5% positive horses and donkeys, respectively, but the B. caballi RAP-1-based cELISA failed to detect any positives, a result hypothesized to be caused by sequence polymorphism found in the rap-1 genes. Nested-PCR analysis identified 36.4% and 43.1% positive horses and donkeys, respectively for T. equi and it also identified 19.3% and 15.7% positive horses and donkeys, respectively for B. caballi. The overall EP incidence found in the population under study was relatively high and comparable regardless of the diagnostic method used (56.8% using nPCR and 48.9% using IFAT). Hematologic analysis revealed macrocytic hypochromic anemia and thrombocytopenia in all piroplasma-infected horses. CONCLUSIONS: The data confirm relatively high levels of EP, likely causing hematological abnormalities in equines in Egypt, and also suggest the need for an improved serological test to diagnose B. caballi infection in this region.

Morphological and molecular description of immature stages of Ornithodoros savignyi (Acari: Argasidae).

This study was designed to provide more details about larva, first nymph, and second nymph of Ornithodoros savignyi using a combination of light microscopy (LM), scanning electron microscopy (SEM), and partial sequence of mitochondrial 16S ribosomal ribonucleic acid (rRNA). The main characteristics of larva are wrinkled integument with many grooves, gnathosoma without camerostome cheeks, hypostome with a pair of large teeth apically, and tarsus without humps. The comparisons between the first and the second nymphs are different shape and distribution of dorsal grooves; a few spots without mammilla on the dorsal surface of the second nymph; 27 and 63-65 pairs of setae on the dorsal surface of the first and second nymphs, respectively; small holes on mammillae that are more dense in the second nymph; basis capitulum with two pairs of small setae in the second nymph; and one pair of sate in the first nymph, hypostome with dental formula 2/2 in the first nymph, and 3/3 in the second nymph. The partial 16S rRNA sequence of the second nymph that was determined as O. savignyi (450 bp) was deposited in GenBank under the accession number KU163242.

Serological and molecular diagnostic surveys combined with examining hematological profiles suggests increased levels of infection and hematological response of cattle to babesiosis infections compared to native buffaloes in Egypt.

BACKGROUND: Babesiosis threatens the development of the cattle and buffaloes industries in Egypt and improved control is needed. The main objectives of this study are surveying the presence of bovine babesiosis in distinct selected bovine and buffalo populations in Egypt using novel molecular and previously validated serological methods, while also comparing the occurrence of hematological alterations among Babesia infected cattle and buffalos. METHODS: A total of 253 and 81 blood samples from apparently healthy cattle and buffaloes, respectively, were randomly collected from diverse locations in Egypt. All samples were tested for Babesia bovis and B. bigemina infection using blood film examination, competitive ELISA (cELISA) and PCR. Novel semi-nested and nested PCR assays for the detection of B. bovis and B. bigemina respectively, were developed and used to analyze DNA extracted from bovine and buffalo samples. Hematological profiles were studied using a hematological analyzer. RESULTS: Blood films examination revealed 13.8% and 7.4% Babesia infection rates in cattle and buffaloes, respectively. However, in cattle, the cELISA detected 32.8%, 21.3% and 10.7% infection rates with B. bigemina, B. bovis and mixed infection, respectively. In addition, cELISA identified 22.2%, 22.2% and 6.2% infection rates with B. bigemina, B. bovis and mixed infection, respectively in buffaloes. The semi-nested PCR assay showed that 15% of the tested samples were positive for B. bovis in cattle, but just 3% in buffaloes. Infections with B. bigemina were also found in cattle (32.4%), but not in buffaloes upon nested PCR analysis. Sequencing analysis confirmed the identity of the PCR amplicons and showed that Egyptian genotypes of B. bigemina and B. bovis highly resemble sequences previously deposited in GenBank. Hemograms performed on the sampled animals revealed macrocytic hypochromic anemia associated with reduced platelet counts in infected cattle with babesiosis. In addition, marked increases in total leukocyte and granulocytic counts and decreases in lymphocytic counts were found in infected cattle. In contrast, no such hematological anomalies were found in presumably Babesia-infected buffaloes. CONCLUSIONS: Frequent occurrence of babesiosis among apparently healthy bovines in Egypt, suggests the need for appropriately designed prevalence studies in this country. Infected bovine, but not buffalo, populations often present hematological disorders compatible with intravascular hemolysis and thrombocytopenia.

First report of Toxoplasma gondii prevalence in Tibetan pigs in Tibet, China.

Toxoplasma gondii infection is widely prevalent in humans and animals, including pigs throughout the world. In this study, the seroprevalence of T. gondii infection in Tibetan pigs in China was investigated for the first time. A total of 427 serum samples were collected from Tibetan pigs in Nyingchi prefecture, Tibet, between April and December 2010, and were assayed for antibodies to T. gondii using the modified agglutination test (MAT). Ninety-seven (22.72%) pigs were found to be positive with MAT titers of 1:25 or higher. Slaughter pigs had the highest seroprevalence, compared with seroprevalence in fattening pigs, growing pigs, or piglets, although the difference was not statistically significant (p≥0.05). The results of the present survey indicate that T. gondii is highly prevalent in Tibetan pigs in Tibet, which poses a significant public health concern in this unique region of the world.

Evaluation of snail derived antigens of Fasciola gigantica for serodiagnosis of human fasciolosis.

Serum samples collected from a total number of twenty parasitologically confirmed cases of human fasciolosis were used to evaluate the diagnostic sensitivity and specificity of snail derived antigens; non infected snail (SA), infected snail (ISA), redia (RA), cercaria (CA) and encysted metacercaria (EMCA) of F. gigantica using the enzyme linked immunosorbent assay (ELISA) and enzyme linked immunotransfer blot (EITB). ELISA results showed that the highest level of sensitivity (100%) was with the CA as compared with ISA, RA and EMCA which displayed lower sensitivity levels of 30, 60 and 90%, respectively. All fasciolosis patients were seronegative with SA. Sodium dodecyle sulfate polyacrylamide gel electrophoresis and EITB (with rabbit antiserum raised against F. gigantica somatic antigen) of the snail derived antigens were carried out to characterize their protein profiles and detect cross-reactive polypeptide bands between them. The immunoblot profile of SA displayed cross-reactive bands at 61 and 30 kDa with ISA, RA, CA and EMCA; CA at 61, 34 and 30 kDa with ISA; 96, 61, 34, 30 and 23 kDa with RA and 61, 34, 23 and 19 kDa with EMCA. Cross reactive antigens may be important as possible candidates for vaccine and diagnosis of fasciolosis. Immunoblot of sera from fasciolosis patients using fractionated cercarial antigen on nitrocellulose strips showed that all sera recognized common reactive band at 32.5 kDa molecular weight from cercarial antigen. We suggest that the 32.5 kDa component of Fasciola cercarial antigen may be the most sensitive and specific for the diagnosis of human fasciolosis.