The “expanding universe” of piroplasms

doi:10.1016/j.vetpar.2003.11.015

Veterinary Parasitology

Volume 119, Issue 4, 6 February 2004, Pages 337-345

https://doi.org/10.1016/j.vetpar.2003.11.015 Get rights and content

Abstract

The present paper is the continuation of our previous studies dealing with the genetic characterization of piroplasmid species found in southern Europe. We report in this work new data concerning sequences of the 18s rRNA gene in Spanish piroplasms not studied (or not totally sequenced) in our former surveys.

Molecular data analysis indicated that

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Spanish Cytauxzoon felis (cat isolate) has 98% identity with Cytauxzoon sp. from Mongolia and 95% identity compared to African C. felis.
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There are at least two main genetic variants of Babesia caballi in Spain:
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  The first variety (isolate Spain 1) shows a relatively low homology with the African genotype (97% identity).
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  The second variety (represented by two isolates, Spain 2 and Spain 3, differing by a single base) shows high genetic similarity with the African genotype (99.7–100% identity).
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There are also two genetic variants of Babesia equi (isolates Spain 1 and Spain 2, differing by four bases) in Spain, sharing 99% identity with the African genotype. At least one of them (Spain 1) can infect dogs.
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All of the phylogenetic analysis procedures employed indicated that Spanish isolates of C. felis, B. caballi (Spain 1) and B. equi (Spain 1 and Spain 2) are genetically different from their African relatives, all those dichotomies showing very high bootstrap support. Nonetheless, the lack of information on their morphology and the fact that the sequences were obtained in a single isolate preclude any conclusion about their definitive taxonomic status.

Introduction

In the last years, some new species like Theileria annae (Zahler et al., 2000), Babesia leo (Penzhorn et al., 2001) and Babesia venatorum (Herwaldt et al., 2003) have been discovered within the piroplasms, a group of haematic protozoa that causes severe disease in mammals. The level of genetic divergence detected in other instances has led to some researchers to suggest that new subspecies (or even cryptic species), might be occurring within piroplasmids, although no specific names have been proposed for them (Kjemtrup et al., 2000, Gubbels et al., 2002, Criado-Fornelio et al., 2003d). The use of molecular diagnosis procedures, as well as gene sequencing, has prompted not only the discovery of a striking level of genetic diversity in those parasitic protozoa, but also the finding of unexpected new hosts for piroplasms like Babesia canis, T. annae and Babesia equi (Criado-Fornelio et al., 2003a, Criado-Fornelio et al., 2003b). Taking into account that sequencing of piroplasmid isolates is a procedure not used for routine diagnosis, we can affirm (talking in a figurative sense) that “the universe of piroplasms is still expanding”, both at genetic and host range levels. The issue of genetic divergence is not trivial, since some authors have indicated that it may have a great influence both in chemotherapeutic and vaccine trial failures (Bock et al., 1995, Hatcher et al., 2001, Criado-Fornelio et al., 2003b).

Due to the lack of morphological features that can help to identify genetic variants or even cryptic species of parasites, the best strategy for definitive diagnosis is the use of molecular methods (Herwaldt et al., 2003, Criado-Fornelio et al., 2003b). Such an approach was employed by us in a previous piroplasm survey in Spain, sequencing the 18s rRNA gene in some of the most frequent species (Criado-Fornelio et al., 2003a, Criado-Fornelio et al., 2003b, Criado-Fornelio et al., 2003d). In the present paper we report new sequence data of the 18s rRNA gene in Spanish isolates of Babesia caballi, B. equi and Cytauxzoon felis. In addition, these new sequences were used for phylogenetic analysis.

Section snippets

Clinical/biological samples

Veterinary practitioners from Spain sent suspect samples (symptomatic) with diagnostic purposes to our laboratory. Blood samples were collected from client-owned animals, placed into EDTA and transported with a cold pack. A total of 400 samples were screened, comprising 100 cats, 150 dogs and 150 horses. All these samples were negative for piroplasmosis by microscopic examination of blood smears. The cat samples were not symptomatic for piroplasmosis but for other pathologies like feline

Molecular epizootiology

Seminested PCR detected piroplasmid infection in 21% of dogs, 33% of horses and 5% of cats. As mentioned above, only isolates from species not studied before were sequenced. We show the homology found by BLASTN search between Spanish isolates and the closest GenBank™ entries in Table 2. DNA sequence data can be summarized as follows.

A domestic cat was positive to Theileria/Cytauxzoon infection by seminested PCR. After sequencing the complete 18s RNA gene, the isolate showed 98% identity with

Molecular epizootiology

Piroplasmosis levels detected in the present work are lower than in our former survey in Spain (Criado-Fornelio et al., 2003b). Such a difference can be explained by the fact that in our first trial we employed a very small and biased sample (some of the animals selected presented a severe symptomatology or were even positive by microscopic examination of blood smears).

Concerning the nature of the pathogens diagnosed, we must point out the importance of the finding of a C. felis-infected cat,

Conclusion

New sequences of Spanish isolates of C. felis, B. caballi and (to a lesser extent) B. equi show a relatively high degree of genetic divergence within the group of piroplasms. Correct taxonomic interpretation of such findings needs a wide-range search and molecular analyses of piroplasm isolates in different geographic locations. The discovery of these new piroplasm variants is relevant in veterinary medicine since it may help to explain both immunodiagnostic and therapeutic failures.

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Cited by (99)

Cytauxzoonosis
2022, Greene's Infectious Diseases of the Dog and Cat, Fifth Edition
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  First Described: Cytauxzoonosis was first recognized in domestic cats from Missouri during the mid-1970s.
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  Cause: Cytauxzoon felis is a hematoprotozoal pathogen of domestic and wild felids.
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  Affected Hosts: Only felids are infected with C. felis. Domestic cats often develop a severe disease but occasionally only a mild, brief or unapparent illness. The bobcat reservoir host is believed to typically demonstrate mild illness but can die as a result of infection. Other felids (e.g., cougars, tigers, lions) can also become infected.
- •
  Arthropod Vectors: Amblyomma americanum (lone star tick) is the primary vector for pathogen transmission, although Dermacentor variabilis (American dog ticks) may also be competent vectors.
- •
  Geographic Distribution: In the United States, cytauxzoonosis is identified in the midwest, south-central, south-eastern, and the mid-Atlantic states; the geographic range has expanded with the expanded range of A. americanum ticks. Cytauxzoonosis is also reported in South America (especially Brazil). Other species of Cytauxzoon cause infection in felidae in Eurasia.
- •
  Route of Transmission: Cytauxzoon felis is transmitted by the feeding of vector ticks.
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  Major Clinical Signs: Cytauxzoonosis is a rapidly progressive febrile illness characterized by anorexia, lethargy or depressed mentation, elevation of the third eyelid, vocalization, increased respiratory effort, icterus, pallor, lymphadenomegaly, splenomegaly or hepatomegaly, and sometimes seizures.
- •
  Differential Diagnosis: Tularemia is an important differential for outdoor cats. Depending on clinical signs present, other differentials include hemotropic mycoplasmosis, cholangiohepatitis, histoplasmosis, sepsis, primary IMHA, oxidative toxicities, toxoplasmosis, and FIP.
- •
  Human Health Significance: None.
Phylogenetic analysis, genetic diversity and geographical distribution of Babesia caballi based on 18S rRNA gene
2021, Ticks and Tick-borne Diseases
The present investigation was aimed to study the presence of Babesia caballi clades upon phylogenetic analysis of all available V4 hypervariable 18S rRNA gene sequences in GenBank in addition to the intra- and interclade genetic diversity in B. caballi and the distribution of parasite clades in different countries. Out of altogether 155 small-subunit ribosomal RNA gene sequences of B. caballi available in the database, only 92 sequences with a complete V4 hypervariable region (>293 bp) were used in multiple sequence alignment. The phylogenetic tree placed all the sequences into two distinct clades with high bootstrap values which are designated as B. caballi clades A and B. Clade A was further divided into two subclades A1 and A2 with 98% bootstrap support. On the contrary, clade B contained multiple small subclades which either lacked bootstrap support or did not have enough bootstrap support to further group them into subclades. All the sequences of B. caballi were 91.5-100% identical with each other. Clade B manifested a comparatively higher genetic diversity (95.2-100% identity) amongst sequences as opposed to clade A (97.3-100% identity). Moreover, it indicated 91.5-93.5%, 92.9-94.6% and 91.5-94.6% nucleotide identity with B. caballi subclades A1, A2, and clade A, respectively. Significant nucleotide variations were observed in one region, between nucleotide positions 126-178, in some of the sequences. A total of 21 molecular signature residues were identified in the V4 hypervariable region. The alignment report of the V4 hypervariable region of 18S rRNA gene of clades A and B exhibited nucleotide variation at nine and 24 places, respectively. The distribution map of all the clades of B. caballi is also reported. The number of 18S rRNA gene sequences employed in the study is relatively high compared to previous studies. Therefore, a fair comparison of definite genetic variations between isolates/sequences from different countries was carried out.
Three new species of Cytauxzoon in European wild felids
2021, Veterinary Parasitology
Protists of the genus Cytauxzoon infect a wide variety of wild and domestic felids worldwide. While the American Cytauxzoon felis has been well described, data on the European isolates of Cytauxzoon are still scant. The aim of the current study was to determine the genetic diversity of European Cytauxzoon spp. in wild felids across Europe by analyzing one nuclear and two mitochondrial genes, along with representative complete mitochondrial genomes. Overall, 106 biological samples from wild felids (92 from Felis silvestris and 14 from Lynx lynx) from Germany, Romania, Czech Republic, and Luxembourg were collected and screened for the presence of Cytauxzoon spp. using nested PCR protocols, targeting the highly conserved 18S rDNA, mitochondrial cytochrome b (CytB) and cytochrome c oxidase subunit I (COI) genes. Furthermore, 18 previously confirmed wild felid biological samples from Europe, and comparative material from USA positive for C. felis, were included in the study. In 18S rDNA sequences analyses, Cytauxzoon spp. from felids formed two separate clades of New World and Old World isolates, with a low inner diversity of the European clade. In contrast to 18S rDNA, the phylogenetic analyses of CytB and COI genes affirmatively revealed three highly supported clades, resulting in three defined genotypes. Similar intra- and interspecific variability of CytB and COI genes was observed in the case of different Babesia spp. Considering geography, host species and analyses of three genes, we conclude that the three detected genotypes of Cytauxzoon in European wild felids represent three new species, which we herein describe.
Phylogenetic analysis and geographical distribution of Theileria equi and Babesia caballi sequences from horses residing in Spain
2020, Ticks and Tick-borne Diseases
The intraerythrocytic protozoans Theileria equi and Babesia caballi are the causative agents of equine piroplasmosis (EP), one of the most important equine tick-borne diseases due to its significant impact on global international horse trade. Although EP is known to be endemic in Spain, previous phylogenetic studies have only been conducted for limited geographical regions. Therefore, the objective of this study was to evaluate the genetic diversity and distribution of these parasite species nationwide. This was performed by amplification of the 18S small subunit (SSU) rRNA gene from 100 EP positive equine blood samples using a nested PCR protocol, and sequencing the obtained amplicons. Seventy-seven T. equi and six B. caballi isolates were successfully sequenced and phylogenetic analysis revealed that the T. equi isolates grouped into the previously described clades A (n = 21/77), D (n = 1/77) and E (n = 55/77), while B. caballi isolates were placed into clades A (n = 5/6) and B (n = 1/6). Isolates from T. equi clade D and B. caballi clade B have not previously been reported in Spain. A greater intra-clade diversity (97.3–98.3 % identity) was observed between T. equi clade E isolates compared to those within clade A (99.7–100 % identity). Additionally, a multivariable logistic regression model was used to analyse associations between the clade of T. equi infection and available epidemiological data. Horses residing in Spanish northern regions were statistically more likely to be infected with T. equi clade E (p = 0.01). We conclude that while extensive sequence variation of equine piroplasms exists in Spanish infected horses, a requirement for increased equine movement controls between Spain and EP-endemic countries should be considered.
Translocation a potential corridor for equine piroplasms in Cape mountain zebra (Equus zebra zebra)
2019, International Journal for Parasitology: Parasites and Wildlife
Citation Excerpt :
A molecular epidemiology study of equine piroplasmosis in South Africa has identified as many as 25 distinct T. equi sequences belonging to three distinct genotypes (Bhoora et al., 2010a). In addition, eleven distinct T. equi sequences has been observed in horses from Sudan (Salim et al., 2010) and genetic variants within T. equi has been identified in Spanish horses based on 18 S sequence data (Criado-Fornelio et al., 2004; Nagore et al., 2004). In addition, Githaka et al. (2014) reported an organism potentially related to T. equi was transmissible to waterbuck in Kenya.
Translocation of animals in fragmented habitats is an important means of dispersal and gene flow, however, the movement of animals has led to the spread of various diseases globally and wildlife are often the reservoirs of these diseases. Currently, Cape mountain zebra are translocated within South Africa as a management method for augmentation of isolated and fragmented populations. The movement of pathogens due to translocations in local regions have gone largely unchecked, particularly where there may still be isolated regions that can be negatively affected. Equine piroplasmosis is a tick-borne disease caused by Theilaria equi and/or Babesia caballi reported to occur in equids (Bhoora et al., 2010; Zweygarth et al., 2002). Here, the presence of T. equi and B. caballi was detected in 137 clinically healthy Cape mountain zebra from three South African reserves, Mountain Zebra National Park (MZNP), De Hoop Nature Reserve (DHNR) and Karoo National Park (KNP) using the multiplex EP real-time PCR (qPCR) assay. We observed 100% prevalence for T. equi and identified only one animal from MZNP with B. caballi. These results affirm that precautions should be taken prior to founding new populations of Cape mountain zebra and that potential farms and properties adjacent to prospective reserves should be screened for the presence of the organisms in order to mitigate risks of infection to domestic animals.
Revisiting the genotypes of Theileria equi based on the V4 hypervariable region of the 18S rRNA gene
2024, Frontiers in Veterinary Science

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^☆: Nucleotide sequence data reported in this paper are available in the GenBank™ database under the accession numbers AY150062, AY150063, AY150064, AY309955, AY309956, AY346369 and AY346370.

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Short communicationThe “expanding universe” of piroplasms☆

Abstract

Introduction

Section snippets

Clinical/biological samples

Molecular epizootiology

Molecular epizootiology

Conclusion

Vet. Parasitol.

Vet. Microbiol.

Vet. Parasitol.

Vet. Parasitol.

Vet. Parasitol.

Vet. Parasitol.

Int. J. Parasitol.

Mol. Biochem. Parasitol.

Vet. Parasitol.

Phylogeny and evolution of the piroplasms

Parasitology

Studies on failure of T strain live Babesia bovis vaccine

Aust. Vet. J.

PHYLIP: phylogeny inference package (Version 3.2)

Cladistics

Short communication
The “expanding universe” of piroplasms☆