Two Species of Canine Babesia in Australia: Detection and Characterization by PCR

The haemoprotozoan Babesia canis has been recognized in Australia for many years, and a second, smaller species has recently been discovered. Amplification and sequencing of a partial region of the 18S small subunit ribosomal RNA (rRNA) gene enabled detection and characterization of the large and small canine babesiae of Australia for the first time. Isolates from northern Australia were genetically characterized to be 99% homologous to Babesia canis vogeli, confirming previous speculation about the subspecies of B. canis endemic to Australia. The partial 18S rRNA gene sequence amplified from isolates obtained in southeastern Australia was genetically identical to Babesia gibsoni, a species not previously known in Australia. The polymerase chain reaction (PCR) used was shown to be specific to Babesia and had a high sensitivity, detecting DNA at a parasitemia of approximately 0.0000027%. This study also reports the first known detection and characterization of B. canis DNA in Rhipicephalus sanguineus ticks using PCR

Cysteine proteinase C1A paralog profiles correspond with phylogenetic lineages of pathogenic piroplasmids

Piroplasmid parasites comprising of Babesia, Theileria, and Cytauxzoon are transmitted by ticks to farm and pet animals and have a significant impact on livestock industries and animal health in tropical and subtropical regions worldwide. In addition, diverse Babesia spp. infect humans as opportunistic hosts. Molecular phylogeny has demonstrated at least six piroplasmid lineages exemplified by B. microti, B. duncani, C. felis, T. equi, Theileria sensu stricto (T. annulata, T. parva, and T. orientalis) and Babesia sensu stricto (B. bovis, B. bigemina, and B. ovis). C1A cysteine-proteinases (C1A-Cp) are papain-like enzymes implicated in pathogenic and vital steps of the parasite life cycle such as nutrition and host cell egress. An expansion of C1A-Cp of T. annulata and T. parva with respect to B. bovis and B. ovis was previously described. In the present work, C1A-Cp paralogs were identified in available genomes of species pertaining to each piroplasmid lineage. Phylogenetic analysis revealed eight C1A-Cp groups. The profile of C1A-Cp paralogs across these groups corroborates and defines the existence of six piroplasmid lineages. C. felis, T. equi and Theileria s.s. each showed characteristic expansions into extensive families of C1A-Cp paralogs in two of the eight groups. Underlying gene duplications have occurred as independent unique evolutionary events that allow distinguishing these three piroplasmid lineages. We hypothesize that C1A-Cp paralog families may be associated with the advent of the schizont stage. Differences in the invertebrate tick host specificity and/or mode of transmission in piroplasmid lineages might also be associated with the observed C1A-Cp paralog profiles

Survey of vector-borne agents in feral cats and first report of Babesia gibsoni in cats on St Kitts, West Indies

Background: As there is little data on vector-borne diseases of cats in the Caribbean region and even around the world, we tested feral cats from St Kitts by PCR to detect infections with Babesia, Ehrlichia and spotted fever group Rickettsia (SFGR) and surveyed them for antibodies to Rickettsia rickettsii and Ehrlichia canis. Results: Whole blood was collected from apparently healthy feral cats during spay/ neuter campaigns on St Kitts in 2011 (N = 68) and 2014 (N = 52). Sera from the 52 cats from 2014 were used to detect antibodies to Ehrlichia canis and Rickettsia rickettsii using indirect fluorescent antibody tests and DNA extracted from whole blood of a total of 119 cats (68 from 2011, and 51 from 2014) was used for PCRs for Babesia, Ehrlichia and Rickettsia. We could not amplify DNA of SFG Rickettsia in any of the samples but found DNA of E. canis in 5% (6/119), Babesia vogeli in 13% (15/119), Babesia gibsoni in 4% (5/119), mixed infections with B. gibsoni and B. vogeli in 3% (3/119), and a poorly characterized Babesia sp. in 1% (1/119). Overall, 10% of the 52 cats we tested by IFA for E. canis were positive while 42% we tested by indirect fluorescent antibody (IFA) for R. rickettsii antigens were positive. Conclusions: Our study provides the first evidence that cats can be infected with B. gibsoni and also indicates that cats in the Caribbean may be commonly exposed to other vector-borne agents including SFGR, E. canis and B. vogeli. Animal health workers should be alerted to the possibility of clinical infections in their patients while public health workers should be alerted to the possibility that zoonotic SFGR are likely circulating in the region

Clinical investigation on Theileria equi and Babesia caballi infections in Italian donkeys

Background: Interest in the welfare and diseases of donkeys is constantly increasing in several countries. Despite this, clinical research into donkeys needs to be in continual development since they show different reactions compared to horses in many conditions, including infectious diseases, and need specific clinical and therapeutic approaches. No reports are currently available on clinical and clinical pathology data regarding donkeys with natural piroplasms infection. Results: Venous blood samples were taken from one hundred and thirty eight donkeys and underwent indirect fluorescent antibody test (IFAT) to detect IgG antibodies against Theileria equi and Babesia caballi and real-time polimerase chain reaction (PCR) to detect Babesia spp. and Theileria spp.. Clinical examinations, haematological analyses and serum bilirubin evaluation were also performed and compared with positive or negative status. A seroprevalence of 40.6% and 47.8% was found for T. equi and B. caballi, respectively; double positivity was detected in 19.6% of the animals. PCR results showed that 17.4% of the animals tested positive for T.equi and 3.6% for B. caballi with no double positivity. Twelve donkeys (8.7%) had clinical signs consistent with chronic forms of the disease and no acute forms were detected. Fifty-eight donkeys had haematological and serum bilirubin alterations and 56 (96.6%) of them were IFAT and/or PCR positive. Changes in erythrocyte number, packed cell volume, hemoglobin concentration, mean corpuscular hemoglobin, platelets number and total bilirubin were significantly associated with positive and symptomatic animals. Conclusion: Nonspecific clinical presentation seems to be very common in donkeys and several clinical pathology alterations persist after natural infection. Therefore, apparently healthy donkeys can have masked but severe clinical pathology alterations. Acute forms are very seldom observed in donkeys. Clinical monitoring of chronically infected donkeys is recommended since such animals represent a risk both for transmission to other animals and for their own health; furthermore, their production performances could be reduced. The study should also be intended as a contribution for veterinary practitioners because it describes the most usual clinical presentations and laboratory findings of equine piroplasmosis in naturally infected donkeys in endemic areas

Diverse tick-borne microorganisms identified in free-living ungulates in Slovakia

Background: Free-living ungulates are hosts of ixodid ticks and reservoirs of tick-borne microorganisms in central Europe and many regions around the world. Tissue samples and engorged ticks were obtained from roe deer, red deer, fallow deer, mouflon, and wild boar hunted in deciduous forests of south-western Slovakia. DNA isolated from these samples was screened for the presence of tick-borne microorganisms by PCR-based methods. Results: Ticks were found to infest all examined ungulate species. The principal infesting tick was Ixodes ricinus, identified on 90.4% of wildlife, and included all developmental stages. Larvae and nymphs of Haemaphysalis concinna were feeding on 9.6% of wildlife. Two specimens of Dermacentor reticulatus were also identified. Ungulates were positive for A. phagocytophilum and Theileria spp. Anaplasma phagocytophilum was found to infect 96.1% of cervids, 88.9% of mouflon, and 28.2% of wild boar, whereas Theileria spp. was detected only in cervids (94.6%). Importantly, a high rate of cervids (89%) showed mixed infections with both these microorganisms. In addition to A. phagocytophilum and Theileria spp., Rickettsia helvetica, R. monacensis, unidentified Rickettsia sp., Coxiella burnetii, "Candidatus Neoehrlichia mikurensis", Borrelia burgdorferi (s.l.) and Babesia venatorum were identified in engorged I. ricinus. Furthermore, A. phagocytophilum, Babesia spp. and Theileria spp. were detected in engorged H. concinna. Analysis of 16S rRNA and groEL gene sequences revealed the presence of five and two A. phagocytophilum variants, respectively, among which sequences identified in wild boar showed identity to the sequence of the causative agent of human granulocytic anaplasmosis (HGA). Phylogenetic analysis of Theileria 18S rRNA gene sequences amplified from cervids and engorged I. ricinus ticks segregated jointly with sequences of T. capreoli isolates into a moderately supported monophyletic clade. Conclusions: The findings indicate that free-living ungulates are reservoirs for A. phagocytophilum and Theileria spp. and engorged ixodid ticks attached to ungulates are good sentinels for the presence of agents of public and veterinary concern. Further analyses of the A. phagocytophilum genetic variants and Theileria species and their associations with vector ticks and free-living ungulates are required.Fil: Kazimírová, Mária. Slovak Academy of Sciences. Institute of Zoology; EslovaquiaFil: Hamšíková, Zuzana. Slovak Academy of Sciences. Institute of Zoology; EslovaquiaFil: Spitalská, Eva. Slovak Academy of Sciences. Institute of Virology. Biomedical Research Center,; EslovaquiaFil: Minichová, Lenka. Slovak Academy of Sciences. Institute of Virology. Biomedical Research Center,; EslovaquiaFil: Mahríková, Lenka. Slovak Academy of Sciences. Institute of Zoology; EslovaquiaFil: Caban, Radoslav. Široká ; EslovaquiaFil: Sprong, Hein. National Institute for Public Health and Environment.Laboratory for Zoonoses and Environmental Microbiology; Países BajosFil: Fonville, Manoj. National Institute for Public Health and Environment.Laboratory for Zoonoses and Environmental Microbiology; Países BajosFil: Schnittger, Leonhard. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Kocianová, Elena. Slovak Academy of Sciences. Institute of Virology. Biomedical Research Center,; Eslovaqui

Genome-wide diversity and gene expression profiling of Babesia microti isolates identify polymorphic genes that mediate host-pathogen interactions

Babesia microti, a tick-transmitted, intraerythrocytic protozoan parasite circulating mainly among small mammals, is the primary cause of human babesiosis. While most cases are transmitted by Ixodes ticks, the disease may also be transmitted through blood transfusion and perinatally. A comprehensive analysis of genome composition, genetic diversity, and gene expression profiling of seven B. microti isolates revealed that genetic variation in isolates from the Northeast United States is almost exclusively associated with genes encoding the surface proteome and secretome of the parasite. Furthermore, we found that polymorphism is restricted to a small number of genes, which are highly expressed during infection. In order to identify pathogen-encoded factors involved in host-parasite interactions, we screened a proteome array comprised of 174 B. microti proteins, including several predicted members of the parasite secretome. Using this immuno-proteomic approach we identified several novel antigens that trigger strong host immune responses during the onset of infection. The genomic and immunological data presented herein provide the first insights into the determinants of B. microti interaction with its mammalian hosts and their relevance for understanding the selective pressures acting on parasite evolution

Canine Babesioses in noninvestigated areas of Serbia

During the years 2012-2014, a total of 158 outdoor dogs from Pančevo and Đurđevo (northern Serbia) and Niš and Prokuplje (southern Serbia) were submitted to molecular analyses (PCR and sequencing) for canine babesioses. An overall prevalence of 21.5% was found, due to the species Babesia sp. 'spanish dog' (10.1%), B. gibsoni (5.7%), B. canis vogeli (1.9%), B. caballi (1.9%), and B. microti (1.9%). In addition, sequence analysis showed the presence of Hepatozoon canis in a dog from Niš. No significant difference between infected and noninfected dogs was found by age, sex, and place of residence, whereas there was difference regarding the presence of ticks (p<0.005) and application of preventive measures such as applying of antitick drugs/devices. Moreover, a significant difference was established by area: Dogs from Prokuplje showed infection rates (59.1%) higher than dogs from Pančevo (11.9%), Niš (4.5), and Đurđevo (where infected dogs were not found), and a different geographical distribution of the species was found. The presence of so many Babesia species and the first identification of H. canis will allow investigations on the pathogenic role played by each one and suggests entomological studies on the tick species that are more suitable vectors for each of them. Finally, the presence of so many infected dogs offers the opportunity of evaluating the hypothesis of a possible zoonotic role of babesial species affecting dogs

Prevalence of tick-borne haemoparasites in small ruminants in Turkey and diagnostic sensitivity of single-PCR and RLB

Background: Tick-borne haemoparasitic diseases (TBHDs), caused by Theileria, Babesia, Anaplasma and Ehrlichia, are common in regions of the world where the distributions of host, pathogen and vector overlap. Many of these diseases threaten livestock production and some also represent a concern to human public health. The primary aim of this study was to determine the prevalence of the above-mentioned pathogens in a large number of blood samples (n = 1979) collected from sheep (n = 1727) and goats (n = 252) in Turkey. A secondary aim was to assess the diagnostic sensitivity of a number of species-specific polymerase chain reaction (PCR) tests and the reverse line blotting (RLB) assay. DNA samples were screened using species-specific PCR for the presence of Theileria ovis, Theileria sp. MK, T. lestoquardi, T. uilenbergi, T. luwenshuni, Babesia ovis, Anaplasma ovis and A. phagocytophilum while RLB was undertaken to test for the presence of all known Theileria, Babesia, Anaplasma and Ehrlichia species. The diagnostic sensitivity of these two approaches was then compared in terms of their ability to detect single species and mixed infections. Results: Overall, 84 and 74.43% of the small ruminants sampled were identified as hosting one or more pathogen(s) by species-specific PCR and RLB respectively. The presence of Theileria sp. OT1, T. luwenshuni and T. uilenbergi in Turkey was revealed for the first time while the presence of Babesia motasi, B. crassa and T. separata in Turkish small ruminants was confirmed using molecular methods. A high prevalence of mixed infection was evident, with PCR and RLB approaches indicating that 52.24 and 35.42% of animals were co-infected with multiple species, respectively. More than 80% of the mixed infections contained T. ovis and/or A. ovis. The RLB approach was found to be capable of detecting mixed infections with species such as Theileria sp. OT1, Theileria sp. OT3, T. separata, B. crassa and Babesia spp. Conclusion: The results indicated that pathogens causing TBHDs are highly prevalent in sheep and goats in Turkey. The diagnostic sensitivity of species-specific single PCR was generally higher than that of RLB. However, the latter approach was still capable of identifying a high proportion of individuals containing mixed-species infections. The use of species-specific single PCR is recommended to accurately estimate pathogen prevalence and to identify co-infected hosts

The evolutionary dynamics of variant antigen genes in Babesia reveal a history of genomic innovation underlying host-parasite interaction

Babesia spp. are tick-borne, intraerythrocytic hemoparasites that use antigenic variation to resist host immunity, through sequential modification of the parasite-derived variant erythrocyte surface antigen (VESA) expressed on the infected red blood cell surface. We identified the genomic processes driving antigenic diversity in genes encoding VESA (ves1) through comparative analysis within and between three Babesia species, (B. bigemina, B. divergens and B. bovis). Ves1 structure diverges rapidly after speciation, notably through the evolution of shortened forms (ves2) from 5′ ends of canonical ves1 genes. Phylogenetic analyses show that ves1 genes are transposed between loci routinely, whereas ves2 genes are not. Similarly, analysis of sequence mosaicism shows that recombination drives variation in ves1 sequences, but less so for ves2, indicating the adoption of different mechanisms for variation of the two families. Proteomic analysis of the B. bigemina PR isolate shows that two dominant VESA1 proteins are expressed in the population, whereas numerous VESA2 proteins are co-expressed, consistent with differential transcriptional regulation of each family. Hence, VESA2 proteins are abundant and previously unrecognized elements of Babesia biology, with evolutionary dynamics consistently different to those of VESA1, suggesting that their functions are distinct