Great diversity of Piroplasmida in Equidae in Africa and Europe, including potential new species

doi:10.1016/j.vprsr.2019.100332

Veterinary Parasitology: Regional Studies and Reports

Volume 18, December 2019, 100332

https://doi.org/10.1016/j.vprsr.2019.100332 Get rights and content

Highlights

•
New PCR assays were developed to potentially identify all piroplasms.
•
Epidemiological study was conducted on Equidae from sub-Saharan Africa and France.
•
Four species of piroplasms infecting horse, including two potentially new ones, were detected.
•
The newly identified species were characterized by amplifying three genes.

Abstract

Piroplasms are Apicomplexa tick-borne parasites distributed worldwide. They are responsible for piroplasmosis (theileriosis and babesiosis) in Vertebrata and are therefore of medical and economic importance.

Herein, we developed a new real time PCR assay targeting the 5.8S rRNA gene and three standard PCR assays, targeting 18S rRNA, 28S rRNA, and cox1 genes, for the detection of piroplasmids. These assays were first optimized and screened for specificity and sensitivity. Then, they were used to study a total of 548 blood samples and 97 ticks collected from Equidae in four sub-Saharan countries (Senegal, Democratic Republic of the Congo, Chad, and Djibouti) and France (Marseille and Corsica).

DNA of piroplasms was detected in 162 of 548 (29.5%) blood samples and in 9 of 97 (9.3%) ticks. The highest prevalence in blood samples was observed in Chad in 2016 with 72.9% positivity rate. Sequencing allowed the identification of four species of piroplasms, including two potentials new species. Theileria equi was mainly found. The highest prevalence was observed in Senegal (14 positive out of 23, 60.87%). Babesia caballi was detected in one horse in Senegal. Two new potential Theileria species were detected: Theileria sp. “Africa”, observed in all areas excepted in Marseille and Theileria sp. “Europa”, observed in Marseille and Corsica.

In conclusion, sensitive and specific PCR assays were developed for epidemiological studies of Piroplasmida. The circulation of multiple species of piroplasms, including two potentials new species, observed among Equidae from sub-Saharan Africa and France.

Introduction

Apicomplexa protists are stealth invaders, they can escape the immune response in host cells while using them as a source of nutrients (Striepen et al., 2007). Almost all apicomplexan are parasites, including multiple pathogenic species, both for humans and animals like malaria, toxoplasmosis, cryptosporidiosis, and piroplasmosis.

The parasites belonging to the apicomplexan order Piroplasmida include three genera namely: Babesia, Theileria, and Cytauxzoon (Schreeg et al., 2016). Equine piroplasmosis is an infectious tick-borne disease caused by the hemoprotozoan parasites Theileria equi and Babesia caballi (Wise et al., 2013). These piroplasms affect equid species, including horses, donkeys, mules and zebras(Wise et al., 2013). T. equi was initially named Piroplasma equi by Laveran in 1901, but after the discovery of schizogony in horse lymphocytes, which is known for Theileria but not for Babesia, this parasite was reclassified by Mehlhorn and Schein as T. equi (Uilenberg, 2006). In 1912, B. caballi, formerly named Piroplasma caballi, was first identified as another parasite infecting equids and different from T. equi and was assigned to the genus Babesia (Nuttall and Strickland, 1912).

Equine piroplasmosis caused by B. caballi and T. equi is endemic in tropical and subtropical zones; the latter is most prevalent (Friedhoff and Soulé, 1996). Southern Europe and Africa are highly endemic (Wise et al., 2014). Moreover, a high genetic diversity of these parasites has been observed in Tunisia(Ros-García et al., 2013) and Jordan (Qablan et al., 2013).

Equine piroplasmosis has serious impacts on horses' health. Agricultural production is strongly affected with high cost of control rules and impact on the carriage of merchandises and international commerce(International Office of Epizootics. Biological Standards Commission, 2012). In the United States, with a population of about 9.2 million horses, the direct economic impact of the equine industry is about 39 billion dollars per year. This industry also supports approximately 1.4 million full-time jobs (The American Horse Council, 2005). It took 25 years and $23 million for Southern California to eradicate equine piroplasmosis (USDA, 2010).

For a long time, T. equi and B. caballi were considered specific to their hosts. However, both parasites were recently identified in clinically healthy dromedaries by PCR, in Jordan (Qablan et al., 2012). Both were also detected in a dog in Croatia in 2009 (Beck et al., 2009). In France, in 2010, Fritz et al. analyzed 166 dogs; 31 were infected by T. equi and one by B. caballi (Fritz, 2010). Finally, Babesia canis, Babesia rossi and Babesia capreoli were recently reported to infect also horses (Fritz, 2010; Zanet et al., 2017).

Piroplasmosis is a typical zoonotic vector-borne infection (Schnittger et al., 2012). However, T. equi and B. caballi are not considered to infect humans (Maslin et al., 2004). Ticks and iatrogenic blood transfers are efficient modes of transmission in equids (Ueti et al., 2005). More than 21 tick species, mainly hard ticks, are associated with the transmission of these parasites. Co-infections are frequently reported in Equidae, often associated with co-infestation by tick species of the genera Dermacentor, Hyalomma and Rhipicephalus, (Tamzali, 2013). Babesia and Theileria exhibit different ecological relationships with their vectors and hosts. The reservoirs of Babesia include chronically infected animals and ticks (Yabsley and Shock, 2013). Infected ticks are able to transovarial and transstadial transmission of B. caballi from female ticks to its offspring. In contrast, Theileria are only transmitted transtadially (Ueti et al., 2005). Besides, Equidae are the primary reservoirs of Theileria.

Clinical signs of equine piroplasmosis are similar for both parasites (Tamzali, 2013).They occur after transmission within 10–30 days for B. caballi and 12–19 days for T. equi(de Waal, 1992). In endemic areas, most of infected horses are asymptomatic carriers with low level of parasitaemia. In case of concurrent disease or stress, they may develop clinical equine piroplasmosis (Allsopp et al., 2007). Acute forms of equine piroplasmosis can include fever (over 40 °C), sweating, congested mucous membranes, limb and supraorbital edema, icterus, anorexia, tachypnoea, tachycardia, anemia, and occasionally petechiae or ecchymoses (Tamzali, 2013). Death may occur in severe cases.

In this study, our aim was to develop sensitive and specific molecular tools able to potentially detect all piroplasm species and to analyze their phylogeny, in order to perform an epidemiological study on Equidae from sub-Saharan Africa and France.

Section snippets

Study area and samples collection

In total, 548 blood samples and 97 hard ticks were collected from Equidae (horses and donkeys). Blood samples were collected in four countries from sub-Saharan Africa (Chad, Senegal, Djibouti, and Democratic Republic of the Congo [DR Congo] and from France (Marseille and Corsica) (Fig. 1). Hard ticks have only been collected in Chad. All equines were apparently healthy at the time of sampling (Table S1).

In Chad, we had two periods of sampling of saddle horses belonging to the Chadian National

Sensitivity and specificity of PCR assays

The 5.8S real time PCR, as well as the standard 18S RNA-based PCR assay, allowed the amplification of the following piroplasmids: T. equi, B. caballi, Theileria ovis, Theileria annulata, Theileria buffeli, Babesia canis, and Babesia vogeli, all are already identified and sequenced in our laboratory (Table S2). All 18S amplicons from controls were sequenced. The obtained sequences allowed an accurate identification of all controls. None of the DNA extracts from negative controls were amplified.

Discussion

To the best of our knowledge, all published PCR-based assays are able to amplify either only one species or a group of piroplasms (Qurollo et al., 2017; Salem et al., 1999). Herein, we have developed a real time PCR assay targeting the 5.8S gene able to detect well-known equine pathogens (T. equi and B. caballi), well-known mammal pathogens as well as potential new species. The 5.8S gene is a conservative gene easy to amplify by PCR using one pair of universal primers (Gou et al., 2012). For

Conclusion

Overall, sensitive and specific PCR assays have been developed to potentially identify all piroplasm species and to study their phylogeny. Four horse-infecting piroplasms, including two potentially new species, were identified in Equidae from sub-Saharan Africa and France. However, few epidemiological investigations on equine piroplasmosis are conducted. Our study provides a better understanding of the situation. Further studies, covering larger geographical areas and larger number of samples,

Declaration of competing interests

None.

Ethical statement

1)
This material has not been published in whole or in part elsewhere;
2)
The manuscript is not currently being considered for publication in another journal;
3)
All authors have been personally and actively involved in substantive work leading to the manuscript, and will hold themselves jointly and individually responsible for its content.

Acknowledgements

This study was supported by the Institut Hospitalo-Universitaire (IHU) Méditerranée Infection, the National Research Agency under the program « Investissements d'avenir », reference ANR-10-IAHU-03, the Région Provence Alpes Côte d'Azur and European funding FEDER PRIMI.

The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.

We thank Masse Sambou, Maxence Aubadie (†), Raphaël Tine, Mamadou Diara, Pierre Verhaeghe, Bertrand

References (41)

M.T.E.P. Allsopp et al.
Molecular evidence for transplacental transmission of Theileria equi from carrier mares to their apparently healthy foals
Vet. Parasitol.
(2007)
L.E. Bartolomé Del Pino et al.
Babesia caballi and Theileria equi infections in horses in Central-Southern Italy: Sero-molecular survey and associated risk factors
Ticks Tick Borne Dis
(2016 Apr)
R. Beck et al.
Diversity of Babesia and Theileria species in symptomatic and asymptomatic dogs in Croatia
Int. J. Parasitol.
(2009)
R. Bhoora et al.
Development and evaluation of real-time PCR assays for the quantitative detection of Babesia caballi and Theileria equi infections in horses from South Africa
Vet. Parasitol.
(2010)
D.T. de Waal
Equine piroplasmosis: a review
Br. Vet. J.
(1992)
H. Gou et al.
A DNA barcode for piroplasmea
Acta Trop.
(2012)
D.P. Knowles et al.
Discovery of a novel species, Theileria haneyi n. sp., infective to equids, highlights exceptional genomic diversity within the genus Theileria: implications for apicomplexan parasite surveillance
Int. J. Parasitol.
(2018)
M.A. Qablan et al.
Quest for the piroplasms in camels: identification of Theileria equi and Babesia caballi in Jordanian dromedaries by PCR
Vet. Parasitol.
(2012)
A. Ros-García et al.
Prevalence and genetic diversity of piroplasm species in horses and ticks from Tunisia
Infect. Genet. Evol.
(2013)
G.H. Salem et al.
Development and evaluation of an extra chromosomal DNA-based PCR test for diagnosing bovine babesiosis
Mol. Cell. Probes
(1999)

B. Salim et al.

Current status of equine piroplasmosis in the Sudan

Infect Genet Evol

(2013 Jun)

L. Schnittger et al.

Babesia: a world emerging

Infect. Genet. Evol.

(2012)

G. Uilenberg

Babesia-A historical overview

Vet. Parasitol.

(2006)

L.N. Wise et al.

Equine piroplasmosis

Vet. Clin. North Am. Equine Pract.

(2014)

M.J. Yabsley et al.

Natural history of zoonotic Babesia: role of wildlife reservoirs

Int. J. Parasitol. Parasites Wildl.

(2013)

S. Zanet et al.

Horses infected by Piroplasms different from Babesia caballi and Theileria equi: species identification and risk factors analysis in Italy

Vet. Parasitol.

(2017)

S. Derycke et al.

Exploring the use of cytochrome oxidase c subunit 1 (COI) for DNA barcoding of free-living marine nematodes

PLoS One

(2010)

K.T. Friedhoff et al.

An account on equine babesioses

Rev. Sci. Tech.

(1996)

D. Fritz

A PCR study of piroplasms in 166 dogs and 111 horses in France (March 2006 to March 2008)

Parasitol. Res.

(2010)

E. Guven et al.

Balkaya İ, Abay U, Yavuz Ş, Akyüz M. Prevalence and molecular characterization of Theileria equi and Babesia caballi in jereed horses in Erzurum, Turkey

Acta Parasitol

(2017 Mar 1)

Cited by (31)

Molecular characterization of some equine vector-borne diseases and associated arthropods in Egypt
2022, Acta Tropica
Citation Excerpt :
The extracted DNA was stored at −20 °C until use in molecular methods. Quantitative PCR (qPCR) was performed in all extracted DNA samples (equines and associated arthropods) for multi-pathogen DNA screening using genus-specific primers and probes targeting the 5.8S rRNA gene of Piroplasmida (Dahmana et al., 2019), 23S rRNA gene of Anaplasmataceae (Dahmani et al., 2017), gltA gene Rickettsia sp. (Rolain et al., 2002), 16S rRNA gene Borrelia sp. (Bottieau et al., 2012), IS1111 intergenic spacer for C. burnetii (Mediannikov et al., 2010), and 16S-23S intergenic spacer for Bartonella sp. (Raoult et al., 2006) (Table 1). The qPCR was applied using the CFX96 Real-Time System (Bio-Rad, Marnes-La-Coquette, France).
Equine vector-borne diseases (EVBDs) are emerging and re-emerging diseases, and most of them are zoonotic. This study aimed to investigate EVBDs in equines and associated arthropods (ticks and flies) from Egypt using molecular analyses, in addition to a preliminary characterization of associated ticks and flies by the matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) and molecular techniques. In this study, 335 blood samples were obtained from equines that appeared to be in good health (320 horses and 15 donkeys) in Cairo and Beni Suef provinces, Egypt. From the same animals, 166 arthropods (105 sucking flies and 61 ticks) were collected. Ticks and flies were preliminary characterized by the MALDI-TOF and molecular tools. Quantitative PCR (qPCR) and standard PCR coupled with sequencing were performed on the DNA of equines, ticks, and flies to screen multiple pathogens. The MALDI-TOF and molecular characterization of arthropods revealed that louse fly (Hippobosca equina) and cattle tick (Rhipicephalus annulatus) infesting equines. Anaplasma platys-like (1.6%), Anaplasma marginale (1.6%), Candidatus Ehrlichia rustica (6.6%), a new Ehrlichia sp. (4.9%), and Borrelia theileri (3.3%) were identified in R. annulatus. Anaplasma sp. and Borrelia sp. DNAs were only detected in H. equina by qPCR. A. marginale, Anaplasma ovis, and Theileria ovis recorded the same low infection rate (0.6%) in donkeys, while horses were found to be infected with Theileria equi and a new Theileria sp. Africa with recorded prevalence rates of 1.2% and 2.7%, respectively. In conclusion, different pathogens were first detected such as A. platys-like, Candidatus E. rustica, and a new Ehrlichia sp. in R. annulatus; A. marginale, A. ovis, and T. ovis in donkeys; and a new Theileria sp. “Africa” in horses.
A report on tick burden and molecular detection of tick-borne pathogens in cattle blood samples collected from four regions in Saudi Arabia
2021, Ticks and Tick-borne Diseases
Citation Excerpt :
Total genomic DNA (gDNA) was isolated from the blood samples (100 μL) using a Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA) following the manufacturer’s instructions. Primers were designed to target a conserved region that encoded for ribosomal 18S rRNA (969 bp) and 23S rRNA (485 bp) to detect the DNA of piroplasms (Dahmana et al., 2019) and Anaplasmataceae (Dahmani et al., 2017), respectively (Supplementary Table 1). Polymerase chain reaction (PCR) assay was performed in an automated BIO-RAD Thermal Cycler (BIO-RAD, Singapore) using one PCR master mix (GeneDireX, Taiwan) following the instructions of the manufacturer.
Babesiosis, theileriosis and anaplasmosis are among the most commonly reported tick-borne diseases in cattle and are associated with significant economic losses. Through the present study the researchers aimed to report the presence of various pathogens that cause babesiosis, theileriosis and anaplasmosis in cattle collected from different provinces in Saudi Arabia and to report their phylogenetic relationship. A total of 362 blood samples of cattle along with ticks that were present on the cattle were collected from four regions (Riyadh, Al-Kharj, Al-Hasa and Al-Qassim) of Saudi Arabia. Blood samples were screened by polymerase chain reaction (PCR) for the presence of various Babesia, Theileria and Anaplasma species by amplification of their 18S rRNA and/or 23S rRNA genes. A total of 541 ticks were collected and identified from the cattle. These included Hyalomma anatolicum, Hyalomma dromedarii, Hyalomma impeltatum, Hyalomma excavatum, Rhipicephalus annulatus and Rhipicephalus turanicus. Regarding tick-borne pathogens, the overall prevalence was 1.9 % (7/362) for Theileria annulata, (2/362) 0.6 % for Theileria and (21/362) 5.8 % for Anaplasma ovis. Four of the cattle were found to be co-infected with more than one pathogen (1.1 %). We did not detect any Babesia species in the blood of the studied cattle. Prevalence of the Theileria and Anaplasma species was highest in cattle that resided in Riyadh, followed by cattle from Al-Hasa and Al-Qassim. Representative amplified partial-gene sequences of T. annulata (GenBank accession numbers MK826137-39) and A. ovis (GenBank acc. no. MK 880224) were submitted to GenBank. The presence of ticks on cattle was found to be associated with a high prevalence of Theileria spp. (P = 0.02) and Anaplasma ovis (P < 0.001). We report novel genotypes of T. annulata and A. ovis from cattle in Saudi Arabia and we recommend that molecular surveys are undertaken throughout the country to address the prevalence and geographical distribution of tick-borne infections for their effective diagnosis and treatment.
Infection dynamics of Theileria equi in carrier horses is associated with management and tick exposure
2020, Ticks and Tick-borne Diseases
Citation Excerpt :
A future study investigating co-infection in both horses and vectors should be conducted to support this hypothesis. It should be noted that the genotyping based on the 18S rRNA gene has recently been scrutinized with regard to its sensitivity (Dahmana et al., 2019; Knowles et al., 2018). Therefore, the differences between genotypes may be attributed to different Theileria species or sub-species, and a more comprehensive genomic study should be made to fully characterize and compare the two.
The tick-borne equine hemoparasite, Theileria equi, is endemic in many parts of the world where prevalence may be high, and most infected horses are apparently healthy but serve as life-long carriers. To determine the factors that affect T. equi dynamics, we followed parasitic loads in apparently healthy horses at four time points during one year. A total of 1094 blood samples were collected from 395 horses, along with ticks and demographic and clinical data. Infection and load of T. equi were tested by PCR and qPCR, and for the spring dataset, infection was also tested serologically by IFAT (n = 268). Theileria equi was molecularly detected in 64.8 % of the horses. The agreement between molecular and serological results was 79.8 % (K > 0.674) and positively correlated with parasitic load. Infection was associated with pale mucus membranes, lower packed cell volume and higher total solids (all P < 0.001), although these changes had only minor clinical importance. While parasitic loads in qPCR-positive samples (n = 561) were generally low (mean = 7.9−10⁻⁴% parasitized erythrocytes), younger horses showed higher loads, possibly suggesting development of immunity. Infection and parasitic load were associated with housing management and tick exposure, illustrating different patterns of exposure. Endemic stability is suggested in pasture farms with constant exposure to ticks, where parasite prevalence was high (96 %) and associated with T. equi 18S rRNA genotype D, low parasitemia and high antibody titers. Endemic instability can be suggested in case were horses are kept in paddocks (prevalence = 49 %) with intermittent exposure to ticks, where infection was associated with high parasitemia when ticks were present. A steady state is suggested in stabled horses (prevalence = 46 %), with no exposure to ticks; where infection was associated with genotype A, low parasitemia and low antibody titers. The ability to identify different risk groups within endemic areas may improve the administration of suitable treatment and control practices in an effort to reduce the risk of clinical disease.
Molecular and serological detection of animal and human vector-borne pathogens in the blood of dogs from Côte d'Ivoire
2020, Comparative Immunology, Microbiology and Infectious Diseases
In Côte d’Ivoire, limited information are available on vector-borne pathogens, their prevalence and distribution. Here, we assess the occurrence and diversity of canine vector-borne diseases (CVBDs) in Abidjan and Yamoussoukro cities. Blood from a total of 123 dogs were tested for Leishmania infantum and Ehrlichia canis antibodies and screened for Leishmania and Trypanosoma spp., Piroplasmida, Filariidae and Anaplasmataceae by PCR and sequencing. Among dogs, 39 % were positive for at least one pathogen. Seroprevalences were: 15.4 % and 12.2 % for L. infantum and E. canis, respectively. DNA of L. infantum and T. congolense (4.1 %), Baabesia vogeli (1.6 %), Filariidae (Dirofilaria immitis, D. repens and Acanthocheilonema reconditum) (10.6 %) has been detected. Anaplasmataceae were detected in (17.1 %) and E. canis was the only identified specie. Co-infections were observed in 13.8 % of dogs: E. canis-L. infantum co-infection was the most prevalent (4.9 %). Age, breed and sex of dogs do not seem to influence infections. Village dogs were more susceptible to CVBDs than kennel dogs (PV = 0.0000008). This study reports for the first time the presence of L. infantum, B. vogeli, A. reconditum, D. immitis and D. repens in dogs from Côte d’Ivoire and determines the prevalence and diversity of CVBD pathogens. The results indicate that human and animal pathogens are abundant in Ivoirian dogs which requires attention of veterinarians, physicians and authorities against these diseases, especially against major zoonosis such as visceral leishmaniasis (L. infantum).
Data release: targeted systematic literature search for tick and tick-borne pathogen distributions in six countries in sub-Saharan Africa from 1901 to 2020
2024, Parasites and Vectors
Tick-Borne Diseases of Humans and Animals in West Africa
2023, Pathogens

View all citing articles on Scopus

View full text

Original ArticleGreat diversity of Piroplasmida in Equidae in Africa and Europe, including potential new species

Highlights

Abstract

Introduction

Section snippets

Study area and samples collection

Sensitivity and specificity of PCR assays

Discussion

Conclusion

Declaration of competing interests

Ethical statement

Acknowledgements

Vet. Parasitol.

Ticks Tick Borne Dis

Int. J. Parasitol.

Vet. Parasitol.

Br. Vet. J.

Acta Trop.

Int. J. Parasitol.

Vet. Parasitol.

Infect. Genet. Evol.

Mol. Cell. Probes

Infect Genet Evol

Infect. Genet. Evol.

Vet. Parasitol.

Vet. Clin. North Am. Equine Pract.

Int. J. Parasitol. Parasites Wildl.

Vet. Parasitol.

Exploring the use of cytochrome oxidase c subunit 1 (COI) for DNA barcoding of free-living marine nematodes

PLoS One

An account on equine babesioses

Rev. Sci. Tech.

A PCR study of piroplasms in 166 dogs and 111 horses in France (March 2006 to March 2008)

Parasitol. Res.

Balkaya İ, Abay U, Yavuz Ş, Akyüz M. Prevalence and molecular characterization of Theileria equi and Babesia caballi in jereed horses in Erzurum, Turkey

Acta Parasitol

Original Article
Great diversity of Piroplasmida in Equidae in Africa and Europe, including potential new species