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1.  Molecular Evidence of Infections with Babesia gibsoni Parasites in Japan and Evaluation of the Diagnostic Potential of a Loop-Mediated Isothermal Amplification Method 
Journal of Clinical Microbiology  2004;42(6):2465-2469.
Detection and analysis of Babesia gibsoni infection were performed with whole-blood samples collected between July 2002 and July 2003 from 945 and 137 dogs from the Aomori and Okinawa Prefectures of Japan, respectively, by PCR and loop-mediated isothermal amplification (LAMP). On the basis of the criterion for positivity by PCR, 3.9% (37 of 945) and 10.9% (15 of 137) of the dogs had B. gibsoni DNA. All 37 positive animals from Aomori Prefecture were male Tosa dogs (Japanese mastiff). The 15 dogs from Okinawa Prefecture with positive PCR assay results were of various breeds, ages, and sexes. The 18S ribosomal DNA (18S rDNA) sequences from all samples showed 100% homology to each other and to published B. gibsoni sequences. The limits of detection of B. gibsoni parasitemia by the PCR and LAMP methods with an 18S rDNA-based primer set were 0.0005% each. A comparison of the PCR and LAMP methods with microscopic examination for the detection of B. gibsoni infections in blood samples from 945 field dogs in Aomori Prefecture and 137 field dogs in Okinawa Prefecture showed that 37 and 15 dogs, respectively, were positive by the PCR and LAMP methods and that 16 and 12 dogs, respectively, were positive by light microscopic examination. All samples found to be positive by microscopic examination were also positive by the PCR and LAMP methods. The results of the PCR and LAMP methods agreed for samples with positive results by either method. Moreover, nonspecific reactions were not observed by the LAMP method. These results suggest that the LAMP method provides a useful tool for the detection of B. gibsoni infections in dogs.
PMCID: PMC427837  PMID: 15184421
2.  Babesias of red deer (Cervus elaphus) in Ireland 
Veterinary Research  2011;42(1):7.
Blood samples were obtained from 38 wild red deer (Cervus elaphus) at two sites in Ireland and subjected to PCR analysis of the 18S rRNA gene followed by sequencing. Two fragments of the 18S rRNA gene were generated by two different PCR protocols and subsequent sequencing suggested that at least six of the deer were infected by a babesia that, in those loci, is indistinguishable from Babesia divergens, an important tick-borne pathogen of cattle and of zoonotic significance. Additionally, a B. odocoilei-like parasite was detected in three samples and a babesia that did not match any sequences in the GenBank database was found in five samples. Neither B. capreoli nor B. venatorum (EU1) were found. There have been several reports of B. divergens occurring in deer species, including red deer, roe deer (Capreolus capreolus) and reindeer (Rangifer tarandus). However, in view of recent re-sequencing of bovine-origin samples deposited previously in GenBank, it is unlikely that any of these sequences from deer are B. divergens. The present study describes the only deer piroplasm detected so far that shows complete identity with B. divergens, in just over half of the 18S rRNA gene. The entire gene of this deer parasite should be analysed and transmission experiments undertaken before the infectivity of B. divergens for red deer can be confirmed.
PMCID: PMC3037898  PMID: 21314977
3.  Diversity of Babesia Infecting European Sheep Ticks (Ixodes ricinus) 
Journal of Clinical Microbiology  2001;39(9):3395-3397.
Questing Ixodes ricinus (Acari: Ixodidae) adult and nymphal ticks collected in various parts of Slovenia were tested for the presence of babesial parasites with a PCR assay based on the nuclear small subunit rRNA gene (nss-ribosomal DNA [rDNA]). Thirteen of 135 ticks were found to contain babesial DNA. Sequence determination and analysis of amplified portions of nss-rDNA revealed their identity with Babesia microti and a high degree of homology with Babesia odocoilei and Babesia divergens. The results of this study represent the first genetic evidence of B. microti and B. divergens-like parasites in I. ricinus ticks in Europe.
PMCID: PMC88357  PMID: 11526189
4.  Canine babesiosis in northern Portugal and molecular characterization of vector-borne co-infections 
Parasites & Vectors  2010;3:27.
Protozoa and bacteria transmitted by arthropods, including ticks and phlebotomine sand flies, may cause a wide range of canine vector-borne diseases. Dogs can be simultaneously or sequentially infected with multiple pathogens. Canine babesiosis caused by Babesia canis canis and Babesia canis vogeli is known to occur in Portugal. This study assessed, by means of blood smear examination, PCR and DNA nucleotide sequencing, the presence of Babesia spp. and co-infecting agents Leishmania, Anaplasma/Ehrlichia and Hepatozoon in 45 dogs from northern Portugal clinically suspected of babesiosis.
Forty-four dogs (98%) had infection with B. canis canis and one with B. canis vogeli. Co-infections were detected in nine animals (20%). Eight dogs were found infected with two vector-borne agents: six with B. canis canis and Leishmania infantum; one with B. canis canis and Ehrlichia canis; and one with B. canis canis and Hepatozoon canis. Another dog was infected with three vector-borne pathogens: B. canis vogeli, E. canis and L. infantum. Overall, L. infantum was found in seven (16%), E. canis in two (4%), and H. canis in one (2%) out of the 45 dogs with babesiosis. Almost 90% of the 45 cases of canine babesiosis were diagnosed in the colder months of October (18%), November (27%), December (20%), February (13%) and March (9%). Co-infections were detected in February, March, April, May, October and November. Twenty-two (50%) out of 44 dogs infected with B. canis were found infested by ticks including Dermacentor spp., Ixodes spp. and Rhipicephalus sanguineus. Mortality (9%) included two co-infected dogs that died spontaneously and two with single infections that were euthanized.
Babesia canis canis is the main etiological agent of canine babesiosis in northern Portugal. A higher sensitivity of Babesia spp. detection was obtained with PCR assays, compared to the observation of blood smears. Twenty percent of the dogs were co-infected with L. infantum, E. canis or H. canis. Furthermore, this is the first molecular identification of H. canis in dogs from northern Portugal.
PMCID: PMC2865458  PMID: 20377861
5.  Babesia Species Occurring in Austrian Ixodes ricinus Ticks▿  
Applied and Environmental Microbiology  2008;74(15):4841-4846.
Babesiosis is a tick-transmitted disease of veterinary and medical importance. The first Austrian case of human babesiosis was recently recorded. In the current study, ticks at all life cycle stages (instars), including 853 Ixodes ricinus and 11 Haemaphysalis concinna ticks, from sampling sites throughout Austria were examined for the presence of Babesia spp. by using 18S rRNA gene PCR and sequencing, and the overall mean infection rate was 51.04%. The infection rates for sampling sites were highly variable, ranging from 0% to almost 100%. Different instars and different sexes were infected almost equally. Babesia isolates occurring in Austrian ticks were identified as Babesia divergens, Babesia divergens-like, and Babesia sp. strain DD by sequencing a fragment of the heat shock protein 70 gene and internal transcribed spacer regions 1 and 2. To our knowledge, this is the first investigation of Babesia spp. in Austrian ticks.
PMCID: PMC2519353  PMID: 18539787
6.  Prevalence and diversity of Babesia spp. in questing Ixodes ricinus ticks from Norway 
Parasites & Vectors  2012;5:156.
Ixodes ricinus ticks transmit Babesia species to vertebrate hosts. Using molecular tools we were able to detect the presence of this piroplasmid in its vector. The aims of this study were to investigate the prevalence and identity of Babesia species in questing ticks collected in various areas of Norway.
DNA from questing l. ricinus ticks were examined with a realtime PCR for the presence of Babesia. Positive samples of tick DNA were identified to species using PCR, and sequence analysis.
From a total of 1908 questing l. ricinus ticks, 17 (0.9%) indicated the presence of Babesia spp. after realtime-PCR screening. Ixodes ricinus harbouring Babesia spp. was detected in 9 out of 22 localities. Further molecular analyses of DNA from these positive ticks indicate the presence of Babesia venatorum, B. divergens, B. capreoli and a currently undescribed Babesia in Norwegian ticks. The most prevalent was B. venatorum found in 71% of the positive ticks.
A total of 17 out of 1908 (0.9%) ticks were positive for Babesia. Our data confirm that there are several Babesia species in ticks in Norway. Babesia venatorum was the most prevalent. This species has a zoonotic potential and may cause human babesiosis following a tick bite.
PMCID: PMC3439691  PMID: 22862883
Babesia spp; Questing Ixodes ricinus; Zoonosis; Piroplasmosis; Realtime PCR; Prevalence; Sequencing
7.  Molecular Characterization of a Non–Babesia divergens Organism Causing Zoonotic Babesiosis in Europe 
Emerging Infectious Diseases  2003;9(8):943-948.
In Europe, most reported human cases of babesiosis have been attributed, without strong molecular evidence, to infection with the bovine parasite Babesia divergens. We investigated the first known human cases of babesiosis in Italy and Austria, which occurred in two asplenic men. The complete 18S ribosomal RNA (18S rRNA) gene was amplified from specimens of their whole blood by polymerase chain reaction (PCR). With phylogenetic analysis, we compared the DNA sequences of the PCR products with those for other Babesia spp. The DNA sequences were identical for the organism from the two patients. In phylogenetic analysis, the organism clusters with B. odocoilei, a parasite of white-tailed deer; these two organisms form a sister group with B. divergens. This evidence indicates the patients were not infected with B. divergens but with an organism with previously unreported molecular characteristics for the 18S rRNA gene.
PMCID: PMC3020600  PMID: 12967491
babesiosis; Babesia divergens; Babesia odocoilei; Babesia venatorum; EU1; Italy; Austria; taxonomy; Ixodes ricinus; 18S rRNA gene; research
8.  Molecular Detection and Characterization of Tick-borne Pathogens in Dogs and Ticks from Nigeria 
Only limited information is currently available on the prevalence of vector borne and zoonotic pathogens in dogs and ticks in Nigeria. The aim of this study was to use molecular techniques to detect and characterize vector borne pathogens in dogs and ticks from Nigeria.
Methodology/Principal Findings
Blood samples and ticks (Rhipicephalus sanguineus, Rhipicephalus turanicus and Heamaphysalis leachi) collected from 181 dogs from Nigeria were molecularly screened for human and animal vector-borne pathogens by PCR and sequencing. DNA of Hepatozoon canis (41.4%), Ehrlichia canis (12.7%), Rickettsia spp. (8.8%), Babesia rossi (6.6%), Anaplasma platys (6.6%), Babesia vogeli (0.6%) and Theileria sp. (0.6%) was detected in the blood samples. DNA of E. canis (23.7%), H. canis (21.1%), Rickettsia spp. (10.5%), Candidatus Neoehrlichia mikurensis (5.3%) and A. platys (1.9%) was detected in 258 ticks collected from 42 of the 181 dogs. Co- infections with two pathogens were present in 37% of the dogs examined and one dog was co-infected with 3 pathogens. DNA of Rickettsia conorii israelensis was detected in one dog and Rhipicephalus sanguineus tick. DNA of another human pathogen, Candidatus N. mikurensis was detected in Rhipicephalus sanguineus and Heamaphysalis leachi ticks, and is the first description of Candidatus N. mikurensis in Africa. The Theileria sp. DNA detected in a local dog in this study had 98% sequence identity to Theileria ovis from sheep.
The results of this study indicate that human and animal pathogens are abundant in dogs and their ticks in Nigeria and portray the potential high risk of human exposure to infection with these agents.
Author Summary
In Nigeria, dogs are not only kept as pets, but are also used for hunting as well as a source of animal protein among some ethnic groups. Ticks are known to infest dogs and serve as vectors for some pathogens of zoonotic and veterinary importance. There is limited information on the prevalence and distribution of vector borne pathogens in dogs and ticks in Nigeria. The aim of the study was to detect and characterize vector borne pathogens in dogs and ticks from Nigeria using molecular methods. The results of this study showed a high estimate of vector borne pathogens in Nigerian dogs (77.3%) and ticks (63.3%). DNA of Candidatus N. mikurensis, an emerging pathogen of humans was detected in Rhipicephalus sanguineus and Heamaphysalis leachi ticks. Another human pathogen, Rickettsia conorii israelensis the causative agent of Mediterranean spotted fever was detected in Rhipicephalus sanguineus ticks. This is the first description of Candidatus N. mikurensis in Africa and Rickettsia conorii israelensis in Nigeria. These results indicate that the use of molecular techniques for the diagnosis of emerging infections in developing countries is of utmost importance in assisting physicians and veterinarians in making accurate diagnoses and providing the appropriate treatment for their patients.
PMCID: PMC3591325  PMID: 23505591
9.  Molecular analysis of the rRNA genes of Babesia spp and Ehrlichia canis detected in dogs from RibeirÃo Preto, Brazil 
Brazilian Journal of Microbiology  2009;40(2):238-240.
The partial DNA sequences of the 18S rRNA gene of Babesia canis and the 16S rRNA gene of Ehrlichia canis detected in dogs from Ribeirão Preto, Brazil, were compared to sequences from other strains deposited in GenBank. The E. canis strain circulating in Ribeirão Preto is identical to other strains previously detected in the region, whereas the subspecies Babesia canis vogeli is the main Babesia strain circulating in dogs from Ribeirão Preto.
PMCID: PMC3769722  PMID: 24031351
Babesia canis vogeli; Ehrlichia canis; ticks; rRNA gene; PCR
10.  Identification of Parasitic Communities within European Ticks Using Next-Generation Sequencing 
Risk assessment of tick-borne and zoonotic disease emergence necessitates sound knowledge of the particular microorganisms circulating within the communities of these major vectors. Assessment of pathogens carried by wild ticks must be performed without a priori, to allow for the detection of new or unexpected agents.
Methodology/Principal Findings
We evaluated the potential of Next-Generation Sequencing techniques (NGS) to produce an inventory of parasites carried by questing ticks. Sequences corresponding to parasites from two distinct genera were recovered in Ixodes ricinus ticks collected in Eastern France: Babesia spp. and Theileria spp. Four Babesia species were identified, three of which were zoonotic: B. divergens, Babesia sp. EU1 and B. microti; and one which infects cattle, B. major. This is the first time that these last two species have been identified in France. This approach also identified new sequences corresponding to as-yet unknown organisms similar to tropical Theileria species.
Our findings demonstrate the capability of NGS to produce an inventory of live tick-borne parasites, which could potentially be transmitted by the ticks, and uncovers unexpected parasites in Western Europe.
Author Summary
Diseases transmitted by ticks have diverse etiology (viral, bacterial, parasitic) and are responsible for high morbidity and mortality rates around the world, both in humans and animals. The emergence or re-emergence of tick-borne diseases is increasingly becoming a problem as the geographical distribution of several tick species is expanding, as well as the numbers of potential or known tick-borne pathogens are constantly evolving. It is thus necessary to know which microorganisms circulate within communities of this major vector to ensure adequate epidemiological surveillance. In this study, we evaluated the potential of Next-Generation Sequencing techniques (NGS) to produce, without a priori, an inventory of both predicted and non-expected parasites carried by Ixodes ricinus, the most prevalent human biting tick in France. Our findings suggest that NGS strategies could be used to produce an inventory of live parasites residing in ticks from a selected area, thereby expanding our knowledge base of tick-associated parasites.
PMCID: PMC3967966  PMID: 24675738
11.  PCR-based Detection of Babesia spp. Infection in Collected Ticks from Cattle in West and North-West of Iran 
Babesiosis is a haemoparasitic disease of domestic and wild animals caused by species of the genus Babesia. Babesia bigemina, B. bovis and B. divergens are known to be pathogenic in cattle. The disease is transmitted during blood feeding by infected ticks and is the most economically important tick-borne disease in tropical and subtropical areas. Ixodid ticks are vectors in the transmission of babesiosis. The classic presentation is a febrile syndrome with apparent anemia and hemoglobinuria. This study was carried out to determine species of bovine Babesia spp. vector ticks collected from naturally occurring bovine babesiosis in West and North-West of Iran.
Two hundred and eleven ticks were collected from 113 cattle and ticks’ species were identified using the standard taxonomic keys. After DNA extraction from salivary glands of each tick, the presence of Babesia spp. infection in ticks was examined by PCR method using primers derived from the gene encoding rhoptry protein.
Rhipicephalus sanguineus and R. bursa ticks were infected with bovine Babesia spp.
Rhipicephalus spp. may play a major role in the transmission of infection of bovine Babesia spp. in West and North-West of Iran.
PMCID: PMC3875879  PMID: 24409438
Babesia; Rhipicephalus; bovine; PCR; Iran
12.  Incidence of Cercopithifilaria bainae in Dogs and Probability of Co-Infection with Other Tick-Borne Pathogens 
PLoS ONE  2014;9(2):e88198.
Cercopithifilaria bainae is a filarioid parasite that infects dogs, being transmitted by Rhipicephalus sanguineus group ticks in many countries of the Mediterranean basin. This study assessed the incidence density rate (IDR) of infection by C. bainae in dogs and the probability of co-infection with other tick-borne pathogens (i.e., Anaplasma platys, Babesia vogeli and Hepatozoon canis), in an area of high endemicity in southern Italy.
Methodology/Principal Findings
From March 2011 to October 2012, a field study involving 58 young dogs naturally exposed to tick infestation was conducted. Skin and blood samples obtained from each dog six times during an 18-month period were tested for C. bainae by parasite detection within skin snip sediments, with subsequent confirmation through PCR and DNA sequencing. Dogs examined monthly for ticks and A. platys, B. vogeli and H. canis were microscopically and/or molecularly diagnosed and after the first and the second summer seasons, the IDR for positive animal-month at risk was 3.8% and 1.7% in November 2011 and October 2012, respectively. All 58 C. bainae-infected dogs were simultaneously infected with at least one other tick-borne pathogen. After the first summer season (assessment in November 2011), a C. bainae-infected dog had a 33% probability of being infected with H. canis or A. platys, whereas after the second tick season (assessment in October 2012) the probability of co-infection was 78%, 22% and 11% for H. canis, A. platys and B. vogeli, respectively.
Our data indicate that tick-infested dogs are at risk of acquiring infection by C. bainae. In addition, the detection of C. bainae microfilariae indicates a prior tick exposure and, should stimulate testing for other tick-borne disease causing pathogens.
PMCID: PMC3912174  PMID: 24498437
13.  Wild Cervids Are Host for Tick Vectors of Babesia Species with Zoonotic Capability in Belgium 
Babesiosis is a tick-borne disease caused by different species of intraerythrocytic protozoan parasites within the genus Babesia. Different species of Babesia are described as potentially zoonotic and cause a malaria-like disease mainly in immunocompromised humans. Interest in the zoonotic potential of Babesia is growing and babesiosis has been described by some authors as an emergent zoonotic disease. The role of cervids to maintain tick populations and act as a reservoir host for some Babesia spp. with zoonotic capability is suspected. To investigate the range and infection rate of Babesia species, ticks were collected from wild cervids in southern Belgium during 2008. DNA extraction was performed for individual ticks, and each sample was evaluated for the absence of PCR inhibition using a PCR test. A Babesia spp. genus-specific PCR based on the 18S rRNA gene was applied to validated tick DNA extracts. A total of 1044 Ixodes ricinus ticks were collected and 1023 validated samples were subsequently screened for the presence of Babesia spp. DNA. Twenty-eight tick samples were found to be positive and identified after sequencing as containing DNA representing: Babesia divergens (3), B. divergens-like (5), Babesia sp. EU1 (11), Babesia sp. EU1-like (3), B. capreoli (2), or unknown Babesia sp. (4). This study confirms the presence of potentially zoonotic species and Babesia capreoli in Belgium, with a tick infection rate of 2.7% (95% CI 1.8,3.9%). Knowledge of the most common reservoir source for transmission of zoonotic Babesia spp. will be useful for models assessing the risk potential of this infection to humans.
PMCID: PMC3319931  PMID: 22214270
Babesia species; Belgium; Cervids; Tick; Zoonotic
14.  Ehrlichiosis, Babesiosis, Anaplasmosis and Hepatozoonosis in Dogs from St. Kitts, West Indies 
PLoS ONE  2013;8(1):e53450.
Although tick-borne diseases are important causes of morbidity and mortality in dogs in tropical areas, there is little information on the agents causing these infections in the Caribbean.
We used PCRs to test blood from a cross-section of dogs on St Kitts for Ehrlichia (E.) canis, Babesia (B.) spp., Anaplasma (A.) spp. and Hepatozoon (H.) spp. Antibodies against E. canis and A. phagocytophilum/platys were detected using commercial immunochromatography tests. Records of the dogs were examined retrospectively to obtain clinical and laboratory data.
Principal findings
There was serological and/or PCR evidence of infections of dogs with E. canis (27%; 46/170), Babesia spp. (24%; 90/372) including B. canis vogeli (12%; 43/372) and B. gibsoni (10%; 36/372), A. platys (11%; 17/157) and H. canis (6%; 15/266). We could not identify the Babesia sp. detected in nine dogs. There was evidence of multiple infections with dual infections with E. canis and B. canis vogeli (8%; 14/179) or B. gibsoni (7%; 11/170) being the most common. There was agreement between immunochromatography and PCR test results for E. canis for 87% of dogs. Only 13% of exposed dogs had signs of a tick-borne disease and 38% had laboratory abnormalities. All 10 dogs presenting for a recheck after treatment of E. canis with doxycycline were apparently healthy although all remained seropositive and six still had laboratory abnormalities despite an average of two treatments with the most recent being around 12 months previously. Infections with Babesia spp. were also mainly subclinical with only 6% (4/67) showing clinical signs and 13% (9/67) having laboratory abnormalities. Similarly, animals with evidence of infections with A. platys and H. canis were largely apparently healthy with only occasional laboratory abnormalities.
Dogs are commonly infected with tick-borne pathogens in the Caribbean with most having no clinical signs or laboratory abnormalities.
PMCID: PMC3546050  PMID: 23335965
15.  Evidence of new pathogenic Theileria species in dogs 
Babesiosis, caused by tick-borne haematozoan organisms of the genus Babesia, is a parasitic disease of domestic and wild mammals. Canine Babesidae have historically been classified as “large Babesia” (Babesia canis) and “small Babesia” (Babesia gibsoni) based on the size of their intraerythrocytic forms. Recent publications, however, suggest that the diversity of piroplasm species infecting dogs might be greater than previously appreciated. Sequencing and phylogenetic analysis of the ssrRNA gene has revealed that canine piroplasms are found in three clades, ‘true’ Babesia sp. (B. canis and B. gibsoni), Theileria annae and Theileria-like group (B. conradae). This newly recognised piroplasm T. annae appears to be hyperendemic in northwest Spain. The vector for this emergent canine infection remains undescribed, although the Ixodeshexagonus is suspected based on their presence upon the dogs in NW Spain and the relative absence of others. These observations have introduced a major change in the approach to the epidemiology of babesiosis in dogs. More clinical samples and data will need to be collected and analyzed to understand the importance of Theileria species in dogs.
PMCID: PMC3081695  PMID: 21526030
Babesia gibsoni; Canine babesiosis; Ixodeshexagonus; Molecular detection; Sequence analysis; Thrombocytopenia; Theileria annae
16.  Simultaneous Detection of Bovine Theileria and Babesia Species by Reverse Line Blot Hybridization 
Journal of Clinical Microbiology  1999;37(6):1782-1789.
A reverse line blot (RLB) assay was developed for the identification of cattle carrying different species of Theileria and Babesia simultaneously. We included Theileria annulata, T. parva, T. mutans, T. taurotragi, and T. velifera in the assay, as well as parasites belonging to the T. sergenti-T. buffeli-T. orientalis group. The Babesia species included were Babesia bovis, B. bigemina, and B. divergens. The assay employs one set of primers for specific amplification of the rRNA gene V4 hypervariable regions of all Theileria and Babesia species. PCR products obtained from blood samples were hybridized to a membrane onto which nine species-specific oligonucleotides were covalently linked. Cross-reactions were not observed between any of the tested species. No DNA sequences from Bos taurus or other hemoparasites (Trypanosoma species, Cowdria ruminantium, Anaplasma marginale, and Ehrlichia species) were amplified. The sensitivity of the assay was determined at 0.000001% parasitemia, enabling detection of the carrier state of most parasites. Mixed DNAs from five different parasites were correctly identified. Moreover, blood samples from cattle experimentally infected with two different parasites reacted only with the corresponding species-specific oligonucleotides. Finally, RLB was used to screen blood samples collected from carrier cattle in two regions of Spain. T. annulata, T. orientalis, and B. bigemina were identified in these samples. In conclusion, the RLB is a versatile technique for simultaneous detection of all bovine tick-borne protozoan parasites. We recommend its use for integrated epidemiological monitoring of tick-borne disease, since RLB can also be used for screening ticks and can easily be expanded to include additional hemoparasite species.
PMCID: PMC84950  PMID: 10325324
17.  Molecular diagnosis of Theileria and Babesia species infecting cattle in Northern Spain using reverse line blot macroarrays 
Piroplasmosis in cattle is caused by tick-borne haemoprotozoan parasites of the genera Theileria and Babesia. Molecular detection techniques offer higher sensitivity and specificity than microscopy examination methods and serological tests. A reverse line blot (RLB) macroarray that included generic and species-specific probes for Theileria annulata, Theileria buffeli, Babesia bovis, Babesia bigemina, Babesia divergens and Babesia major was used to study the presence and identity of the piroplasm species infecting 263 bovine blood samples from 79 farms, most of them in Northern Spain. Microscopy examination of blood smears and haematology were also performed whenever possible to identify animals with parasitaemia.
RLB hybridisation identified infection in 54.0% of the samples, whereas only 28.8% were positive by microscopy examination. The most frequently found species was T. buffeli, present in 42.6% of the samples. T. annulata was found in 22 samples (8.4%) from 12 farms, including 9 farms (14 samples) located in Northern Spain where presence of the vector is not very common. Babesia infections were less frequently detected: B. major was found in 3.0% of the samples, B. bigemina in 2.7%, B. bovis in 2.3% and B. divergens in 1.1%. Mixed infections were detected in 14 samples, accounting for six different combinations of species.
This is the first report in which B. major and B. divergens have been detected in Spain using molecular identification techniques and the first time that B. bovis has been detected in Northern Spain. The detection of T. annulata in Northern Spain suggests that the distribution of Mediterranean theileriosis might be changing. Samples with positive RLB hybridisation but negative microscopy had haematology values within the normal ranges suggesting that they corresponded to chronic carriers that may serve as reservoirs of the infection. In this sense, sensitive and specific laboratorial tests like RLB that clearly identify the parasite and can detect subclinical infections are essential to establish good control measures.
PMCID: PMC1482696  PMID: 16684356
18.  Development and Evaluation of a Seminested PCR for Detection and Differentiation of Babesia gibsoni (Asian Genotype) and B. canis DNA in Canine Blood Samples 
Journal of Clinical Microbiology  2003;41(9):4172-4177.
Canine babesiosis has recently been recognized as an emerging infectious disease of dogs in North America. We sought to develop a seminested PCR to detect and differentiate Babesia gibsoni (Asian genotype), B. canis subsp. vogeli, B. canis subsp. canis, and B. canis subsp. rossi DNA in canine blood samples. An outer primer pair was designed to amplify an ∼340-bp fragment of the 18S rRNA genes from B. gibsoni (Asian genotype), B. canis subsp. vogeli, B. canis subsp. rossi, and B. canis subsp. canis but not mammalian DNA. Forward primers were designed that would specifically amplify a smaller fragment from each organism in a seminested PCR. The practical limit of detection was 50 organisms/ml of mock-infected EDTA anticoagulated whole blood. The primer pair also amplified an ∼370-bp fragment of the B. gibsoni (USA/California genotype) 18S rRNA gene from the blood of an experimentally infected dog with a high percentage of parasitemia. Amplicons were not detected when DNA extracted from the blood of a dog that was naturally infected with Theileria annae at a low percentage of parasitemia was amplified. Due to limited sensitivity, this test is not recommended for the routine diagnosis of B. gibsoni (USA/California genotype) or T. annae. The PCR test did not amplify Toxoplasma gondii, Neospora caninum, Leishmania infantum, Cryptosporidium parvum, or canine DNA under any of the conditions tested. The seminested PCR test was able to detect and discriminate B. gibsoni (Asian genotype), B. canis subsp. vogeli, B. canis subsp. canis, and B. canis subsp. rossi DNA in blood samples from infected dogs.
PMCID: PMC193857  PMID: 12958243
19.  Ribosomal DNA probe for differentiation of Babesia microti and B. gibsoni isolates. 
Journal of Clinical Microbiology  1992;30(5):1210-1215.
The objective of this study was to determine whether different isolates of Babesia microti could be distinguished from morphologically similar isolates of B. gibsoni by using a ribosomal DNA (rDNA) probe. A Babesia-specific rDNA probe was obtained by polymerase chain reaction amplification of sequences from B. microti DNA using universal primers directed against highly conserved portions of the eukaryotic 16S-like rRNA gene. The chemiluminescent rDNA probe hybridized to Southern blots of restriction endonuclease-digested DNA preparations of different isolates of B. gibsoni from infected dogs and B. microti from infected humans and white-footed mice. Restriction fragment length polymorphisms served to differentiate these species. Although the hybridization patterns seen with DNAs from six B. microti isolates did not vary, those of the five B. gibsoni isolates did indicate genotypic variation. We concluded that isolates of B. microti and B. gibsoni can be differentiated on the basis of restriction fragment length polymorphism detected with a chemiluminescent rDNA probe.
PMCID: PMC265252  PMID: 1349901
20.  Human babesiosis in ireland: Further observations and the medical significance of this infection 
British Medical Journal  1969;4(5686):768-770.
Three splenectomized persons in Yugoslavia, California, and Ireland have been reported to be infected by three different Babesia species; two cases were fatal. In a study of the site where the fatal infection was contracted in Ireland, blood samples from 36 persons who had recently been bitten by ticks were inoculated into two splenectomized calves; no response to Babesia divergens was detected. Field-collected Ixodes ricinus ticks inoculated into another splenectomized calf resulted in fever and recovery of the agent of tick-borne fever (Cytoecetes phagocytophilia). This attempt to determine the presence of latent infection in human beings with intact spleens should be repeated on a larger scale in areas with a demonstrably high incidence of Babesia in ticks and animals. Few places in the world are free of piroplasms; their presence may present a hazard to splenectomized persons or to those whose splenic function is deficient.
PMCID: PMC1630245  PMID: 4902496
21.  Detection of Two Zoonotic Babesia microti Lineages, the Hobetsu and U.S. Lineages, in Two Sympatric Tick Species, Ixodes ovatus and Ixodes persulcatus, Respectively, in Japan 
The species Babesia microti, commonly found in rodents, demonstrates a high degree of genetic diversity. Three lineages, U.S., Kobe, and Hobetsu, are known to have zoonotic potential, but their tick vector(s) in Japan remains to be elucidated. We conducted a field investigation at Nemuro on Hokkaido Island and at Sumoto on Awaji Island, where up to two of the three lineages occur with similar frequencies in reservoirs. By flagging vegetation at these spots and surrounding areas, 4,010 ticks, comprising six species, were collected. A nested PCR that detects the 18S rRNA gene of Babesia species revealed that Ixodes ovatus and I. persulcatus alone were positive. Lineage-specific PCR for rRNA-positive samples demonstrated that I. ovatus and I. persulcatus carried, respectively, the Hobetsu and U.S. parasites. No Kobe-specific DNA was detected. Infected I. ovatus ticks were found at multiple sites, including Nemuro and Sumoto, with minimum infection rates (MIR) of ∼12.3%. However, all I. persulcatus ticks collected within the same regions, a total of 535, were negative for the Hobetsu lineage, indicating that I. ovatus, but not I. persulcatus, was the vector for the lineage. At Nemuro, U.S. lineage was detected in 2 of 139 adult I. persulcatus ticks (MIR, 1.4%), for the first time, while 48 of I. ovatus ticks were negative for that lineage. Laboratory experiments confirmed the transmission of Hobetsu and U.S. parasites to hamsters via I. ovatus and I. persulcatus, respectively. Differences in vector capacity shown by MIRs at Nemuro, where the two species were equally likely to acquire either lineage of parasite, may explain the difference in distribution of Hobetsu throughout Japan and U.S. taxa in Nemuro. These findings are of importance in the assessment of the regional risk for babesiosis in humans.
PMCID: PMC3346458  PMID: 22389378
22.  Transport of Babesia venatorum-infected Ixodes ricinus to Norway by northward migrating passerine birds 
Bovine babesiosis is regarded as a limited health problem for Norwegian cows, and the incidence has decreased markedly since the 1930s. Rare cases of babesiosis in splenectomised humans from infection with Babesia divergens and B.venatorum have been described. The objective of this study was to determine whether birds can introduce Babesia-infected ticks. There are between 30 and 85 million passerine birds that migrate to Norway every spring.
Passerine birds were examined for ticks at four bird observatories along the southern Norwegian coast during the spring migrations of 2003, 2004 and 2005. The presence of Babesia was detected in the nymphs of Ixodes ricinus by real-time PCR. Positive samples were confirmed using PCR, cloning and phylogenetic analyses.
Of 512 ticks examined, real-time PCR revealed five to be positive (1.0%). Of these, four generated products that indicated the presence of Babesia spp.; each of these were confirmed to be from Babesia venatorum (EU1). Two of the four B. venatorum-positive ticks were caught from birds having an eastern migratory route (P< 0.001).
Birds transport millions of ticks across the North Sea, the Skagerrak and the Kattegat every year. Thus, even with the low prevalence of Babesia-infected ticks, a substantial number of infected ticks will be transported into Norway each year. Therefore, there is a continuous risk for introduction of new Babesia spp. into areas where I. ricinus can survive.
PMCID: PMC3132728  PMID: 21699719
23.  Human Babesiosis in Japan: Epizootiologic Survey of Rodent Reservoir and Isolation of New Type of Babesia microti-Like Parasite 
Journal of Clinical Microbiology  2001;39(12):4316-4322.
We have carried out epizootiologic surveys at various sites in Japan to investigate wild animals that serve as reservoirs for the agents of human babesiosis in the country. Small mammals comprising six species, Apodemus speciosus, Apodemus argenteus, Clethrionomys rufocanus, Eothenomys smithii, Crocidura dsinezumi, and Sorex unguiculatus, were trapped at various places, including Hokkaido, Chiba, Shiga, Hyogo, Shimane, and Tokushima Prefectures. Animals harboring Babesia microti-like parasites were detected in all six prefectures. Inoculation of their blood samples into hamsters gave rise to a total of 20 parasite isolates; 19 were from A. speciosus, and the other 1 was from C. rufocanus. Sequencing of the parasite small-subunit rRNA gene (rDNA) sequence revealed that 2 of the 20 isolates were classified as Kobe type because their rDNAs were identical to that of the Kobe strain (the strain from the Japanese index case). The other 18 isolates were classified as a new type, designated the Hobetsu type, because they all shared an identical rDNA sequence which differed significantly from both that of Kobe-type isolates and that of northeastern United States B. microti (U.S. type). The parasites with Kobe-, Hobetsu- and U.S.-type rDNAs were phylogenetically closely related to each other but clearly different from each other antigenically. The isolates from rodents were demonstrated to be infective for human erythrocytes by inoculation into SCID mice whose erythrocytes had been replaced with human erythrocytes. The results suggest that a new type of B. microti-like parasite, namely, the Hobetsu type, is the major one which is prevalent among Japanese wild rodents, that A. speciosus serves as a major reservoir for both Kobe- and Hobetsu-type B. microti-like parasites, and that C. rufocanus may also be an additional reservoir on Hokkaido Island.
PMCID: PMC88542  PMID: 11724838
24.  A study on the determination of risk factors associated with babesiosis and prevalence of Babesia sp., by PCR amplification, in small ruminants from Southern Punjab (Pakistan) 
Babesiosis is a parasitic infection due to the multiplication of tick borne parasite, Babesia sp., in erythrocytes of host, which includes a wide variety of vertebrates including small ruminants causing decreased livestock output and hence economic losses. The objective of the present study was to establish a PCR based method for the detection of Babesia sp. in small ruminant population in Southern Punjab and to determine the risk factors involve in the spread of babesiosis. A total of 107 blood samples were collected from 40 sheep and 67 goats in seven districts of Southern Punjab from randomly selected herds. Data on the characteristics of the animals and the herd were collected through questionnaires. 36 blood samples (34% of total) produced the DNA fragment specific for 18S rRNA gene of Babesia sp., by PCR amplification, of which 20 were sheep and 16 were goats. Samples from all seven district contained Babesia positive samples and prevalence varied between 18 to 68%. It was observed that male animals (P = 0.009) and young animals under one year of age (P = 0.01) were more prone to the parasite. It was observed that herds consist of more than 15 animals (P = 0.007), composed of mixed species of small ruminants (P = 0.022), associated with dogs (P = 0.003) and dogs having ticks on their bodies (P = 0.011) were among the major risk factors for the spread of babesiosis in small ruminants.
PMCID: PMC3671477  PMID: 21894263
sheep; goats; PCR amplification; Babesia sp; ovin; caprin; PCR; amplification; Babesia sp
25.  Babesia spp. Identified by PCR in Ticks Collected from Domestic and Wild Ruminants in Southern Switzerland 
Applied and Environmental Microbiology  2006;72(10):6503-6507.
Concurrent infections with vector-borne pathogens affected a cattle herd in Switzerland, and one of the pathogens was identified as Babesia bigemina, which had never been observed in this country before. Therefore, a survey of the occurrence of ruminant Babesia spp. and their tick vectors in Switzerland was conducted. A total of 2,017 ticks were collected from sheep, goats, cattle, and wild ruminants (deer, roe deer, and chamois) in southern parts of Switzerland and identified morphologically. The vast majority of the ticks (99.2%) were Ixodes ricinus, but 14 ticks from sheep and goats were identified as Dermacentor marginatus and two ticks from wild ruminants were identified as Hemaphysalis punctata. PCR analyses of 700 ticks revealed the presence of Babesia divergens (n = 6), Babesia sp. genotype EU1 (n = 14), and B. major (n = 2), whose suggested occurrence was confirmed in this study by molecular analysis, and the presence of novel Babesia sp. genotype CH1 (n = 4), which is closely related to B. odocoilei and to Babesia sp. genotype RD61 reported from North America. The identification of B. divergens and B. major in ticks collected from wild ruminants cast doubt on the postulated strict host specificity of these bovine Babesia species. Furthermore, the zoonotic Babesia sp. genotype EU1 was detected in ticks collected from domestic animals but was obtained predominantly from ticks collected from wild ruminants. More than one tick containing DNA of different Babesia spp. were collected from two red deer. Hence, the role of these game animals as reservoir hosts of Babesia spp. seems to be important but requires further investigation.
PMCID: PMC1610307  PMID: 17021198

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