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Serial blood passage of virulent Babesia bovis in splenectomized cattle results in attenuated derivatives that do not cause neurologic disease. Tick transmissibility can be lost with attenuation, but when retained, attenuated B. bovis can revert to virulence following tick passage. This study provides data showing that tick passage of the partially attenuated B. bovis T2Bo derivative strain further decreased virulence compared with intravenous inoculation of the same strain in infected animals. Ticks that acquired virulent or attenuated parasites by feeding on infected cattle were transmission fed on naive, splenectomized animals. While there was no significant difference between groups in the number of parasites in the midgut, hemolymph, or eggs of replete female ticks after acquisition feeding, animals infected with the attenuated parasites after tick transmission showed no clinical signs of babesiosis, unlike those receiving intravenous challenge with the same attenuated strain prior to tick passage. Additionally, there were significantly fewer parasites in blood and tissues of animals infected with tick-passaged attenuated parasites. Sequencing analysis of select B. bovis genes before and after tick passage showed significant differences in parasite genotypes in both peripheral blood and cerebral samples. These results provide evidence that not only is tick transmissibility retained by the attenuated T2Bo strain, but also it results in enhanced attenuation and is accompanied by expansion of parasite subpopulations during tick passage that may be associated with the change in disease phenotype., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Recently, several arthropod-borne infections have been introduced into previously non-endemic regions in Europe as the result of various global changes. At the same time, endemic regions are expanding and the risk of co-infections is rising, due to climate change that allows vectors to move and spread infectious diseases into new areas. The aim of the current study was to confirm simultaneous infections with Anaplasma phagocytophilum and/or Babesia canis canis in Dirofilaria-infected dogs from Slovakia, central Europe. Genomic DNA was isolated from 366 blood samples of microfilaraemic dogs without clinical signs of infection. Samples were further screened for the presence of canine tick-borne pathogens using PCR and sequencing. This survey revealed co-infection with four arthropod-borne pathogens, in particular, Dirofilaria repens, Dirofilaria immitis, A. phagocytophilum, and B. canis canis. While D. repens, responsible for canine subcutaneous dirofilariosis, is scattered through the whole territory of the country, D. immitis occurs only in endemic areas of southeastern and southwestern Slovakia in mixed infection with D. repens. Co-infection with A. phagocytophilum was reported in 3.27% of the dogs positive for D. repens; mixed infection with D. repens and B. canis canis was detected in 3.55% of the tested blood samples. Eastern Slovak Lowland represents a natural focus of B. canis canis and is a highly endemic area for canine dirofilariosis. The presence of triple infection with D. repens, A. phagocytophilum, and B. canis canis was detected in one dog originating from the eastern lowland region of Slovakia. This study highlights the importance of co-infected, clinically healthy dogs in the spreading of several different arthropod-borne pathogens and the necessity for detailed epidemiological surveys, especially in newly infested areas., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Babesia are tick-borne parasites that are increasingly considered as a threat to animal and public health. We aimed to assess the role of European free-ranging wild ruminants as maintenance mammalian hosts for Babesia species and to determine risk factors for infection. EDTA blood was collected from 222 roe deer (Capreolus c. capreolus), 231 red deer (Cervus e. elaphus), 267 Alpine chamois (Rupicapra r. rupicapra) and 264 Alpine ibex (Capra i. ibex) from all over Switzerland and analysed by PCR with pan-Babesia primers targeting the 18S rRNA gene, primers specific for B. capreoli and Babesia sp. EU1, and by sequencing. Babesia species, including B. divergens, B. capreoli, Babesia sp. EU1, Babesia sp. CH1 and B. motasi, were detected in 10.7% of all samples. Five individuals were co-infected with two Babesia species. Infection with specific Babesia varied widely between host species. Cervidae were significantly more infected with Babesia spp. than Caprinae. Babesia capreoli and Babesia sp. EU1 were mostly found in roe deer (prevalences 17.1% and 7.7%, respectively) and B. divergens and Babesia sp. CH1 only in red deer. Factors significantly associated with infection were low altitude and young age. Identification of Babesia sp. CH1 in red deer, co-infection with multiple Babesia species and infection of wild Caprinae with B. motasi and Babesia sp. EU1 are novel findings. We propose wild Caprinae as spillover or accidental hosts for Babesia species but wild Cervidae as mammalian reservoir hosts for B. capreoli, possibly Babesia sp. EU1 and Babesia sp. CH1, whereas their role regarding B. divergens is more elusive.

The population structure of original Babesia bigemina isolates and reference strains with a defined phenotypic profile was assessed using 18S rRNA and rap-1c genes. Two reference strains, BbiS2P-c (virulent) and BbiS1A-c (low virulence), were biologically cloned in vitro. The virulence profile of the strains and clones was assessed in vivo. One fully virulent and one low-virulence clone were mixed in identical proportions to evaluate their growth efficiency in vitro. Each clone was differentiated by two microsatellites and the gene gp45. The 18S rRNA and rap-1c genes sequences from B. bigemina biological clones and their parental strains, multiplied exclusively in vivo or in vitro, were compared with strain JG-29. The virulence of clones derived from the BbiS2P-c strain was variable. Virulent clone Bbi9P1 grew more efficiently in vitro than did the low-virulence clone Bbi2A1. The haplotypes generated by the nucleotide polymorphism, localized in the V4 region of the 18S rRNA, allowed the identification of three genotypes. The rap-1c haplotypes allowed defining four genotypes. Parental and original strains were defined by multiple haplotypes identified in both genes. The rap-1c gene, analyzed by high-resolution melting (HRM), allowed discrimination between two genotypes according to their phenotype, and both were different from JG-29. B. bigemina biological clones made it possible to define the population structure of isolates and strains. The polymorphic regions of the 18S rRNA and rap-1c genes allowed the identification of different subpopulations within original B. bigemina isolates by the definition of several haplotypes and the differentiation of fully virulent from low virulence clones., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Equine piroplasmosis (EP) caused by intraerythrocytic parasites (Theileria equi and Babesia caballi) is an emerging equine disease of world-wide distribution. In Pakistan, the prevalence and incidence of EP are unknown. In order to obtain the first insights into the prevalence of the disease, a total of 430 equids, including 33 mules, 65 horses and 332 donkeys, aging from ≤ 5 to ≥ 10 years of either sex, from five metropolises of Punjab, Pakistan, were serologically tested for the presence of antibodies directed against B. caballi and T. equi, using a competitive enzyme-linked immunosorbent assay (cELISA). Out of 430 equid serum samples tested, 226 (52.6%, 95% CI 47.7-57.4) were found cELISA positive for EP (T. equi and/or B. caballi infections). The overall seroprevalence of EP was 41.2% (95% CI 36.5-46.0) for T. equi and 21.6% (95% CI 17.8-25.8) for B. caballi. A small proportion of equids (10.2%, 95% CI 7.5-13.5) was seropositive for both T. equi and B. caballi. Seroprevalence of T. equi was significantly higher (P<0.01) in equines from the metropolis of Lahore (66.7%, 95% CI 54.3-77.6) and in horses (56.9%, 95% CI 44.0-69.2). Multivariable logistic regression model analysis indicated that factors associated with prevalence of EP were being an equine species kept in metropolis Lahore (OR=4.24, 95% CI 2.28-7.90), horse (OR=2.82, 95% CI 1.53-5.20) and male equids (OR=1.81, 95% CI 1.15-2.86)., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The achievements of sled dogs in competitions depend both on their training and on their health. Vector-borne infections may lead to anaemia, affect joints or heart muscle or even cause death. Between December 2009 and October 2010, one hundred and twenty six individual blood samples were collected from 26 sled dog kennels situated in different regions of Poland. The majority of samples were taken during the racing season (winter 2009/10). The prevalences of 3 vector-borne infections- including 2 'old pathogens' Anaplasma phagocytophilum and Babesia canis, and 'new pathogen' Hepatozoon canis-were estimated in sled dogs using PCR and nested PCR. Additionally, 25 serum samples originating from a subset of 3 kennels situated in a tick-borne encephalitis (TBE) endemic area (Mazowiecki region), were tested for antibodies against the tick-borne encephalitis virus (TBEV). Because of the recently reported occurrence of Dirofilaria repens in Central Poland and that of fatal cases of unknown aetiology in two of the kennels, blood samples collected from dogs at these kennels in 2010 and in February-May 2013 and from two unaffected kennels were checked for evidence of presence of this parasite. Babesia canis DNA was detected in 11 sled dogs (4 with clinical babesiosis, 7 asymptomatic; 8.7%) inhabiting mainly endemic regions of Poland (9/11 cases). Three serum samples originating from one location tested positive for TBEV antibodies (total seroprevalence: 3/25=12%, local seroprevalence: 3/12=25%). The risk of TBEV infection was associated with previous B. canis infections. Dirofilaria repens DNA was detected in 15 dogs (44%). Prevalence was especially high in two sled dog kennels situated near Grodzisk Mazowiecki (50-57%). No blood samples tested positive for A. phagocytophilum or H. canis DNA. The present study has established that the prevalence of vector-borne pathogens in working sled dogs is significant in the endemic regions and has justified the important role of surveillance of reservoir hosts in the epidemiology of TBE. Our results emphasize the need for regular monitoring for the presence of D. repens., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The achievements of sled dogs in competitions depend both on their training and on their health. Vector-borne infections may lead to anemia, affect joints or heart muscles or even cause death. Canine babesiosis is an emerging, quickly spreading tick-borne disease in Central Europe. Over a 13-year period (2000-2012) the occurrence of babesiosis cases was analyzed in one sled dog kennel situated in Kury, a village near Tłuszcz (N 52°24'56.78″, E 21°30'37.55″) in Central Poland. Twenty cases/episodes of babesiosis were noted among the 10-12 dogs living in the kennel. In 2000-2004, no cases of babesiosis were noted; the first two cases were noted in April 2005. Since that time, only one dog remained uninfected; 6 dogs were infected once, 3 dogs demonstrated symptoms of babesiosis twice, one dog was infected three times and one dog had it five times. Babesiosis appeared in Spring and Autumn, despite the application of anti-tick treatment. No fatal cases were recorded, but in one case a splenectomy was performed due to splenomegaly and spleen rupture. Additionally, the abundance of the main Babesia canis vector, the Dermacentor reticulatus tick, was estimated and monitored during a 4-year period (2008-2012) close to the dog kennel. The abundance of questing ticks was high in 2008 and 2009, but dropped by 10-fold between 2010 and 2012, when the abandoned meadow was cut and used as horse pasture by the local farmer. The regular occurrence, typical seasonal pattern and identification of B. canis DNA in questing tick from this locality confirmed the establishment of a new hyper enzootic region for canine babesiosis. The effectiveness and schedule of applied preventive measures were discussed., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The aim of this study is to determine the efficacy of exoantigens derived from Babesia gibsoni cultures to induce protective immunity against challenge exposure of virulent organisms. An attenuated B. gibsoni Oita strain was maintained in vitro by the microaerophilus stationary phase (MASP) method, and exoantigens-containing supernatant fluids were collected for preparation of the immunization. Two dogs received three subcutaneous immunizations with a 20-day interval of B. gibsoni exoantigens plus 0.5 mg saponin (Quil A). On day 68 after the prime immunization, the immunized dogs and control dogs were challenged intravenously with 2 × 10(8) virulent parasites of a homologous B. gibsoni strain. The results showed that exoantigens could induce a high degree of protection against virulent homologous challenge exposure. Two dogs immunized with exoantigens showed a lower parasitemia, accompanied by a slight decrease in the PCV that returned to normal values. Control dogs developed typical acute clinical signs, including severe anemia and hyperthermia. The immunization elicited humoral immune responses. In dogs immunized with exoantigens, the maximum antibody titer was 2,560 and 5,120 by indirect fluorescent antibody test (IFAT), respectively. Preliminary Western blot analysis of the immunogen revealed five dominant proteins of molecular weights of 18, 37, 43, 50, and 57 kDa. These results suggested that the culture-derived exoantigens were candidates for non-viable vaccine.

Canine babesiosis is a tick-borne disease caused by parasites of the genus Babesia. Tumour necrosis factor alpha (TNF-α) is a cytokine that plays a role in the pathogenesis of canine babesiosis. In this study, the authors determined the concentration of serum TNF-α in 11 dogs infected with Babesia canis and calculated Spearman's rank correlations between the concentration of TNF-α and blood pressure, and between TNF-α and indices of renal damage such as: fractional excretion of sodium (FE(Na(+))), urinary creatinine to serum creatinine ratio (UCr/SCr), renal failure index (RFI), urine specific gravity (USG) and urinary protein to urinary creatinine ratio (UPC). The results demonstrated statistically significant strong negative correlations between TNF-α and systolic arterial pressure (r = -0.7246), diastolic arterial pressure (r = -0.6642) and mean arterial pressure (r = -0.7151). Serum TNF-α concentration was also statistically significantly correlated with FE(Na(+)) (r = 0.7056), UCr/SCr (r = -0.8199), USG (r = -0.8075) and duration of the disease (r = 0.6767). The results of this study show there is an increase of serum TNF-α concentration during canine babesiosis, and the increased TNF-α concentration has an influence on the development of hypotension and renal failure in canine babesiosis. This probably results from the fact that TNF-α is involved in the production of nitric oxide and induction of vasodilation and hypotension, which may cause renal ischaemia and hypoxia, and finally acute tubular necrosis and renal failure.

Canine babesiosis is a tick-borne disease with world-wide significance caused by intraerythrocytic protozoa of the genus Babesia. The eicosanoids, as inflammatory mediators, are involved in the regulation of the immune response and inflammatory reaction. Metabolism of lipids is of great importance in babesiosis. In this study it was aimed to investigate the dynamics of serum concentration of prostaglandin E2 (PGE2), thromboxane B2 (TxB2), leukotriene B4 (LTB4), triglycerides, total cholesterol (Chol), HDL- and LDL-cholesterol in dogs naturally infected with Babesia canis and healthy dogs. Both groups were measured for all parameters on the admission day and on the first, second and seventh day of the disease. Dogs that were included in this study had systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS). It was demonstrated that the level of LTB4, PGE2, TxB2 in dogs naturally infected with B. canis significantly changed during the disease. The level of LTB4 was significantly higher during the study, while the concentration of PGE2 was significantly higher second, third and seventh day of disease in relation with healthy dogs. The level of TxB2 was significantly lower at the beginning of the disease, but after seven days concentration was significantly higher. Both group of patients with SIRS and MODS had significantly higher level of LTB4. Substained high concentrations of PGE2 were observed in dogs with MODS after therapy but not in dogs with SIRS, and LTB4 followed a similar tendency. On the other hand, increases in TxB2 were only significant in dogs with SIRS. The lipid profile in naturally infected dogs with B. canis infection was significantly changed. Further studies are needed to assess the prognostic values of lipid mediators in dogs with B. canis infection, and the ability of these markers to predict the progress of SIRS and MODS., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In this study, two pairs of oligonucleotide primers were designed according to the nucleotide sequence of the internal transcribed spacers (ITSs) of Babesia bigemina and B. bovis isolates from China. The primers were used in a multiplex PCR to detect parasite DNA in blood samples from cattle. There was no cross reactions with B. ovata, B. major, B. sp. Kashi, Theileria annulata, T. sergenti, T. sinensis or normal bovine DNA. The sensitivity of multiplex PCR assay was 1 pg and 10 pg DNA for B. bigemina and B. bovis, respectively. A total of 260 field blood samples collected from cattle in five provinces of China were analyzed by multiplex PCR and light microscopy. PCR testing revealed that 7.3% (19/260) and 5.8% (15/260) of cattle were positive for B. bigemina and B. bovis and 1.2% (3/260) of cattle were co-infected with B. bigemina and B. bovis. Using light microscopy, 2.3% (6/260) and 1.5% (4/260) of cattle were infected by B. bigemina and B. bovis, respectively, and no co-infection was found. The results showed that the multiplex PCR developed in the present study could be an alternative diagnostic tool for the detection of B. bovis and B. bigemina infection in cattle.

Water buffaloes are thought to be the reservoir hosts for several hemoprotozoan parasites that infect cattle. In the present study, we surveyed Sri Lankan bred water buffaloes for infections with Babesia bovis, Babesia bigemina, Theileria annulata, and Theileria orientalis using parasite-specific PCR assays. When 320 blood-derived DNA samples from water buffaloes reared in three different districts (Polonnaruwa, Mannar, and Mullaitivu) of Sri Lanka were PCR screened, B. bovis, B. bigemina, and T. orientalis were detected. While T. orientalis was the predominant parasite (82.5%), low PCR-positive rates were observed for B. bovis (1.9%) and B. bigemina (1.6%). Amplicons of the gene sequences of the Rhoptry Associated Protein-1 (RAP-1) of B. bovis, the Apical Membrane Antigen-1 (AMA-1) of B. bigemina, and the Major Piroplasm Surface Protein (MPSP) of T. orientalis were compared with those characterized previously in Sri Lankan cattle. While the B. bigemina AMA-1 sequences from water buffaloes shared high identity values with those from cattle, B. bovis RAP-1 sequences from water buffaloes diverged genetically from those of cattle. For T. orientalis, none of the MPSP sequence types reported previously in Sri Lankan cattle (types 1, 3, 5, and 7) were detected in the water buffaloes, and the MPSP sequences analyzed in the present study belonged to types N1 or N2. In summary, in addition to reporting the first PCR-based survey of Babesia and Theileria parasites in water buffaloes in Sri Lanka, the present study found that the predominant variants of water buffalo-derived B. bovis RAP-1 and T. orientalis MPSP sequences were different from those previously described from cattle in this country., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: 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., Conclusions: 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.

Babesia bovis is the causative agent of fatal babesiosis in cattle. In the present study, we investigated the genetic diversity of B. bovis among Philippine cattle, based on the genes that encode merozoite surface antigens (MSAs). Forty-one B. bovis-positive blood DNA samples from cattle were used to amplify the msa-1, msa-2b, and msa-2c genes. In phylogenetic analyses, the msa-1, msa-2b, and msa-2c gene sequences generated from Philippine B. bovis-positive DNA samples were found in six, three, and four different clades, respectively. All of the msa-1 and most of the msa-2b sequences were found in clades that were formed only by Philippine msa sequences in the respective phylograms. While all the msa-1 sequences from the Philippines showed similarity to those formed by Australian msa-1 sequences, the msa-2b sequences showed similarity to either Australian or Mexican msa-2b sequences. In contrast, msa-2c sequences from the Philippines were distributed across all the clades of the phylogram, although one clade was formed exclusively by Philippine msa-2c sequences. Similarities among the deduced amino acid sequences of MSA-1, MSA-2b, and MSA-2c from the Philippines were 62.2-100, 73.1-100, and 67.3-100%, respectively. The present findings demonstrate that B. bovis populations are genetically diverse in the Philippines. This information will provide a good foundation for the future design and implementation of improved immunological preventive methodologies against bovine babesiosis in the Philippines. The study has also generated a set of data that will be useful for futher understanding of the global genetic diversity of this important parasite., (© 2013.)

Infection with canine vector-borne pathogens was evaluated in dogs from four different regions of Costa Rica by PCR. Demographic data, clinical signs, packed cell volume values, and the presence of tick infestation were recorded for each dog. Forty seven percent (69/146) of the dogs were infected with at least one pathogen and 12% were co-infected with two pathogens. Ehrlichia canis was detected in 34%, Anaplasma platys in 10%, Babesia vogeli in 8%, and Hepatozoon canis in 7.5% of the blood samples. No infection was detected with Leishmania spp. in blood, skin scrapings or conjunctival swabs. Thirty percent of the dogs presented at least one clinical sign compatible with vector-borne disease, and of those, 66% were infected with a pathogen. Subclinical infections were determined in 58% of the infected dogs including 82% (9/11), 58% (29/50), 42% (5/12) and 36% (5/14) of the dogs with H. canis, E. canis, B. vogeli and A. platys infections, respectively. A distinct relationship was found between infection and anemia. The mean PCV values were 34.4% in dogs with no infection, 31.5% in those who had a single infection and 23% in those with co-infection. Co-infected dogs had significantly lower PCV values compared to non-infected and single-infected dogs (p<0.0001). Thirty five percent (51/146) of the dogs were infested with ticks, 82% of them were infested with Rhipicephalus sanguineus sensu lato and 18% with Amblyomma ovale. Dogs infected with A. platys, B. vogeli, or E. canis were significantly associated with R. sanguineus s.l. infestation (p<0.029). This is the first description of infections with B. vogeli and H. canis in Costa Rica as well as in Central America. The results of this study indicate that multiple vector-borne pathogens responsible for severe diseases infect dogs in Costa Rica and therefore, increased owner and veterinarian awareness are needed. Moreover, prevention of tick infestation is recommended to decrease the threat of these diseases to the canine population., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Genotypic diversity in Babesia bovis (cause of Asiatic redwater in cattle) vaccine strains and field isolates from South Africa were investigated using the Bv80 gene as well as microsatellites. The S11 vaccine strain possessed both A and B alleles of the Bv80 gene, as well as genotypic diversity within each allele type as defined by repeat variation resulting in different amplicon sizes. Rapid serial passage of vaccine strain from passage S10 to S24 resulted in loss of genotypic diversity that yielded a single allele A genotype with an amplicon size of 558 bp. This suggested that clonal selection occurred during rapid passaging. Extensive genotypic diversity exists in 44 field isolates characterized with both Bv80 A and B alleles, but can be readily distinguished from the S24 vaccine strain using either the Bv80 allele specific PCR assays or using multi-locus micro-satellite typing. This indicated that no recent documented clinical cases of Asiatic redwater were caused by the reversion to virulence of the current vaccine strain., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Peroxiredoxins (Prxs) are a family of antioxidant enzymes. Here, we cloned a 2-Cys Prx, BgTPx-1, from the canine Babesia parasite B. gibsoni. Sequence identity between BgTPx-1 and 2-Cys Prx of B. bovis was 81% at the amino acid level. Enzyme activity assay by using recombinant BgTPx-1 (rBgTPx-1) indicated that BgTPx-1 has antioxidant activity. Antiserum from a mouse immunized with rBgTPx-1 reacted with parasite lysates and detect a protein with a monomeric size of 22 kDa and also a 44 kDa protein, which might be an inefficiently reduced dimer. BgTPx-1 was expressed in the cytoplasm of B. gibsoni merozoites. These results suggest that the BgTPx-1 may play a role to control redox balance in the cytoplasm of B. gibsoni.

Chimeric primers, the sensitivity and specificity of which allow them to be used in both the clinical setting and the epizootological assessment of tick infection by a real-time polymerase chain reaction (RT-PCR) assay, have been designed against Babesia canis infection. The findings suggest that a large number of Babesia DNA copies are detectable in the blood in acute babesiosis. Some animals that had experienced babesiosis developed blood B. canis carriage--a small number oftrophozoites remained alive for a long time. When babesiosis was suspected, its diagnosis could be confirmed by RT-PCR in half of dogs with subclinical signs. The tick concentration of Babesia ranged from several hundred to a few thousand parasites. There were no significant differences in the number of Babesia parasites in the infected ticks in relation to their collection site. However, the occurrence of infected ticks was significantly higher in the places of constant contact with a canine population, which is indicative of the decisive role of dogs in the intensity of an epizootic process in the foci of B. canis infection.

Acute tubular necrosis (ATN) was described in canine babesiosis. Hypotension is considered as one of the factors which influence the development of hypoxic renal damage. In this study hypotension defined as mean arterial pressure (MAP) < 80 mmHg was detected in 7 out of 48 dogs (14.6%) infected with Babesia canis. Lower systolic arterial pressure (SAP), diastolic arterial pressure (DAP) and MAP were detected in azotaemic dogs infected with B. canis. Statistically significant negative correlations between blood pressures (SAP, DAP and MAP) and serum creatinine and urea concentrations showed the influence of decreased blood pressure on the development of azotaemia and is probably also associated with ATN in canine babesiosis.

Babesia bovis and Babesia bigemina are tick-borne hemoparasites causing babesiosis in cattle worldwide. This study was aimed at providing information about the occurrence and geographical distribution of B. bovis and B. bigemina species in cattle from Gauteng province, South Africa. A total of 268 blood samples collected from apparently healthy animals in 14 different peri-urban localities were tested using previously established nested PCR assays for the detection of B. bovis and B. bigemina species-specific genes encoding rhoptry-associated protein 1 (RAP-1) and SpeI-AvaI restriction fragment, respectively. Nested PCR assays revealed that the overall prevalence was 35.5% (95% confidence interval [CI]=± 5.73) and 76.1% (95% CI=± 5.11) for B. bovis and B. bigemina, respectively. PCR results were corroborated by sequencing amplicons of randomly selected samples. The neighbor-joining trees were constructed to study the phylogenetic relationship between B. bovis and B. bigemina sequences of randomly selected isolates. Analysis of phylogram inferred with B. bovis RAP-1 sequences indicated a close relationship between our isolates and GenBank strains. On the other hand, a tree constructed with B. bigemina gp45 sequences revealed a high degree of polymorphism among the B. bigemina isolates investigated in this study. Taken together, the results presented in this work indicate the high incidence of Babesia parasites in cattle from previously uncharacterised peri-urban areas of the Gauteng province. These findings suggest that effective preventative and control measures are essential to curtail the spread of Babesia infections among cattle populations in Gauteng.

In Ethiopia, ticks and tick-borne diseases are widely distributed and contribute to important economic losses. Several studies investigated the prevalence and species composition of ticks infesting ruminants; however, data on tick-borne pathogens are still scarce. During the study period from October 2010 to April 2011, a total of 1,246 adult ticks and 264 nymphs were collected from 267 cattle and 45 sheep in Bako District, western Oromia, Ethiopia. The study showed infestation of 228/267 (85.4 %) cattle and 35/45 (77.8 %) sheep with adult ticks. Overall, eight tick species, belonging to three genera (Amblyomma, Rhipicephalus, Hyalomma), were identified and Amblyomma cohaerens (n = 577), Rhipicephalus evertsi evertsi (n = 290), Rhipicephalus (Boophilus) decoloratus (n = 287), and Amblyomma variegatum (n = 85) were the more prevalent species. A statistically significant host preference in A. cohaerens for cattle and R. evertsi evertsi for sheep was noticed. Molecular detection of piroplasms, performed only for adult ticks of two species of the genus Rhipicephalus (R. evertsi evertsi and R. decoloratus), revealed an overall prevalence of 4 % (8/202) Theileria buffeli/sergenti/orientalis, 0.5 % (1/202) Theileria velifera, and 2 % (4/202) Theileria ovis. The study showed that tick infestation prevalence is considerably high in both cattle and sheep of the area, but with a low intensity of tick burden and a moderate circulation of mildly pathogenic piroplasm species.

Babesia ovis, an intraerythrocytic protozoan parasite transmitted by ticks, causes severe infections in sheep in tropical and subtropical regions of the world. Parasite-specific immunoreactive proteins have been used as antigen in the serological diagnosis of babesiosis. There is no study about determination of B. ovis-specific proteins in sheep. This study was planned to determine the immunoreactive proteins of B. ovis. In this study, two splenectomized lambs, and twelve seropositive sheep and five seronegative lambs for anti-B. ovis antibodies were used as materials. Infected blood samples at 5% of parasitemia from the two splenectomized lambs experimentally infected with a virulent B. ovis field strain were analyzed for B. ovis-specific proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (WB). B. ovis-specific five major proteins were recognized by anti-B. ovis serum but not by healthy sheep serum. They were of approximate molecular weights 154, 109, 77, 58, and 38 kDa. As the control samples, protein profiles of the blood extracts of two lambs before splenectomy operation were also blotted with the immune sera, but none of the five proteins was detected. These proteins were also immunoblotted with heterologous positive and negative sheep sera. All of twelve positive sera recognized the 109 kDa protein with 100 percent sensitivity. The 77 kDa protein reacted in 11 of 12 sera (91.6%). The sensitivities of the other 3 proteins ranged between 83.3% and 25%. The five protein bands immunoblotted with sera of the 5 negative lambs did not give any positive reaction. The results of this study revealed the presence of proteins recognized by the serum antibodies of experimentally and naturally infected sheep with B. ovis. Additional studies on the purification of these proteins and on subsequently their utilization in a serodiagnostic method are required to improve the serological diagnosis of ovine babesiosis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Artemisone was evaluated, in in vitro and in vivo, for control of bovine babesiosis caused by Babesia bigemina and Babesia bovis parasites. In vitro, artemisone reduced parasitemia in a dose-dependent manner: the inhibitory effects increased gradually, reaching a maximum inhibition of 99.6% and 86.4% for B. bigemina and B. bovis, respectively 72 h after initiation of treatment with initial parasitemia of 0.5%. In calves infected with either B. bigemina or B. bovis artemisone treatment was well tolerated and prevented development of acute babesiosis in all animals except for one B. bovis-infected calf. The treatment did not eliminate all blood parasites, and recovered animals carried a persistent low-level infection. Treatment with artemisone may be useful as an alternative drug for preventing the pathology that results from babesiosis, without interfering with acquired immune protection following recovery from an acute babesiosis infection or vaccination., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In order to determine the molecular and serological prevalence of Babesia bigemina and Babesia bovis, a total of 247 blood samples were collected from cattle and water buffalos in Beheira and Faiyum Provinces in Egypt and examined by nested polymerase chain reaction (nPCR) and enzyme-linked immunosorbent assay (ELISA). In cattle, the prevalence of B. bigemina and B. bovis was 5.30% and 3.97% by nPCR and 10.60% and 9.27% by ELISA, respectively, whereas those of water buffalos were 10.42% and 4.17% by nPCR and 15.63% and 11.46% by ELISA, respectively. Statistically significant differences in the prevalence of the two infections were observed on the basis of age and health status. Sequencing analysis revealed two genotypes for B. bovis spherical body protein-4. In conclusion, the current data provide valuable information regarding the epidemiology of B. bigemina and B. bovis infections in cattle and water buffalos from Egypt, which can be employed in developing future strategies for disease management and control., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The virulence of the Babesia gibsoni Oita isolate was attenuated by serial passages in vitro by using the microaerophilus stationary phase (MASP) technique. After 400 serial passages, the virulence of the isolate was found to be attenuated. This was evidenced by the response of two dogs inoculated intravenously with 10(9)B. gibsoni passaged isolate. Specific antibodies were produced at a titer of 1:20,480, as detected by the fluorescent antibody test (IFAT). These results suggested that the serial passages of B. gibsoni reduced its virulence while retaining its antigenicity. The dogs that were inoculated with the attenuated isolate (1 and 2) and two naïve dogs (3 and 4) were challenged by intravenous inoculation of 2×10(8) infected erythrocytes of the virulent Oita isolate. Protection afforded by exposure to the attenuated isolate was evidenced by a lower parasitemia in dogs 1 and 2 with a rapid decrease to nondetectable levels, accompanied by a slight decrease in the PCV that returned to normal values. Dogs 3 and 4 developed typical acute clinical signs, including severe anemia and hyperthermia. These results suggested that the attenuated isolate was a candidate for live vaccine., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Bovine babesiosis is a tick-borne disease caused mainly by Babesia bovis and Babesia bigemina, which are associated to considerable economic losses in cattle herds worldwide. Approximately 60% of buffalo herds in South America are located in Northern Brazil. Little is known about the impact of babesiosis on buffalo herds in Brazil. The present work aimed to verify the occurrence of B. bovis and B. bigemina in 542 water buffaloes in the state of Pará, Northern Brazil, using molecular and serological techniques. The percentage of seropositive animals for B. bovis and B. bigemina was 41.2% and 19.0%, respectively, by ELISA. B. bovis and B. bigemina DNA were detected in 15 and 16% of sampled buffaloes, respectively. A high correlation (Kappa index of 0.9) between serological and molecular tests suggests that the combination of the utilized techniques in the present study is suitable for babesiosis diagnosis in an endemic unstable area. Significantly difference of positivity for serological and molecular assays was verified to localities and reproductive status of sampled animals, but not between buffalo breeds. The immune status of sampled buffaloes associated to the circulation of babesiosis agents in sampled population suggests that the studied area is at risk to clinical babesiosis outbreaks. Furthermore, this study demonstrated that this region can be classified as endemically unstable., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In vitro interactions between atovaquone (ATV) and proguanil (PG) against Babesia gibsoni and the clinical efficacy of this combination therapy using Malarone(®) which is the antimalarial drug containing ATV and PG were evaluated. This combination showed synergism against uncloned wild-type and ATV-resistant B. gibsoni in vitro examinations using a modified fixed ratio method. Administration of Malarone(®) to experimentally B. gibsoni infected two dogs in chronic stage and three dogs in acute stage resulted in decrease in parasitemia, and clinical improvements were observed. However, all dogs showed relapse of parasitic infection with a single-nucleotide polymorphism in the cytchrome b gene (M121I). Some side effects were confirmed: self-limiting vomiting in two dogs and hyperphosphatasia in another dog. Mild increases in the levels of alanine aminotransferase were confirmed in two dogs. This is the first study to evaluate the interactions in vitro and the clinical efficacy of ATV and PG against canine B. gibsoni infection in dogs., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Bovine babesiosis is an economically significant disease that affects dairy farming operations in Thailand. In the present study, 1824 blood-DNA samples prepared from cattle bred in 4 different regions of the country (North, Northeast, Central, and South) were screened using a nested PCR for the specific detection of Babesia bovis. While the overall prevalence of B. bovis was 8.8%, the Central region of Thailand was found to be a high-risk area of the country, as the prevalence of the parasite was 15.0%. The positive rate was relatively higher among the animals of 1-5 years of age. The genetic diversity among the B. bovis parasites was also studied based on their MSA-2b gene, and the findings showed that the Thai sequences were dispersed across 8 of 13 total clades observed in the phylogram. Three of these clades were formed only of Thai sequences. Similarity among the deduced MSA-2b amino acid sequences determined in the present study was 68.3-100%. In conclusion, the present study found that all the locations surveyed were infected with B. bovis and that the parasite populations in Thailand were genetically diverse. Our findings highlight the need for further studies in Thailand to generate more information before a sound control strategy could be implemented against B. bovis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Objective: To describe a case of cerebral babesiosis and acute respiratory distress syndrome (ARDS) in a dog., Case Summary: A 5-year-old male neutered Scottish Terrier was referred to the emergency department of the Ecole Nationale Vétérinaire de Toulouse for evaluation of progressive dyspnea and clinical signs suggestive of central neurological disease. Thoracic radiographs showed a diffuse and heavy interstitial/alveolar lung pattern. Babesiosis was diagnosed based on blood smear evaluation. The dog died of cardiopulmonary arrest 6 hours after presentation. Cerebral babesiosis and ARDS were confirmed at necropsy. Major pathological findings included erythrocyte aggregation in the lungs, liver, and brain., New or Unique Information Provided: This case report describes an unusual clinical presentation of Babesia canis canis infection, the most common species associated with babesiosis in Europe. In addition, this is to our knowledge the first case of Babesia-associated ARDS confirmed by histopathology in a dog., (© Veterinary Emergency and Critical Care Society 2013.)

Hemoprotozoan infections often cause serious production losses in livestock. In the present study, we conducted a PCR-based survey of Babesia bovis, Babesia bigemina, Theileria annulata, Theileria orientalis, Trypanosoma evansi and Trypanosoma theileri, using 423 DNA samples extracted from blood samples of cattle (n=202), water buffaloes (n=43), sheep (n=51) and goats (n=127) bred in the Hue and Hanoi provinces of Vietnam. With the exception of T. annulata and T. evansi, all other parasite species (B. bovis, B. bigemina, T. orientalis and T. theileri) were detected in the cattle populations with B. bovis being the most common among them. Additionally, four water buffaloes and a single goat were infected with B. bovis and B. bigemina, respectively. The Hue province had more hemoprotozoan-positive animals than those from the Hanoi region. In the phylogenetic analyses, B. bovis-MSA-2b, B. bigemina-AMA-1 and T. theileri-CATL gene sequences were dispersed across four, one and three different clades in the respective phylograms. This is the first study in which the presence of Babesia, Theileria and Trypanosoma parasites was simultaneously investigated by PCR in Vietnam. The findings suggest that hemoprotozoan parasites, some of which are genetically diverse, continue to be a threat to the livestock industry in this country.

Tick-borne pathogens that cause persistent infection are of major concern to the livestock industry because of transmission risk from persistently infected animals and the potential economic losses they pose. The recent reemergence of Theileria equi in the United States prompted a widespread national survey resulting in identification of limited distribution of equine piroplasmosis (EP) in the U.S. horse population. This program identified Babesia caballi-seropositive horses using rhoptry-associated protein 1 (RAP-1)-competitive enzyme-linked immunosorbent assay (cELISA), despite B. caballi being considered nonendemic on the U.S. mainland. The purpose of the present study was to evaluate the suitability of RAP-1-cELISA as a single serological test to determine the infection status of B. caballi in U.S. horses. Immunoblotting indicated that sera from U.S. horses reacted with B. caballi lysate and purified B. caballi RAP-1 protein. Antibody reactivity to B. caballi lysate was exclusively directed against a single ∼50-kDa band corresponding to a native B. caballi RAP-1 protein. In contrast, sera from experimentally and naturally infected horses from regions where B. caballi is endemic bound multiple proteins ranging from 30 to 50 kDa. Dilutions of sera from U.S. horses positive by cELISA revealed low levels of antibodies, while sera from horses experimentally infected with B. caballi and from areas where B. caballi is endemic had comparatively high antibody levels. Finally, blood transfer from seropositive U.S. horses into naive horses demonstrated no evidence of B. caballi transmission, confirming that antibody reactivity in cELISA-positive U.S. horses was not consistent with infection. Therefore, we conclude that a combination of cELISA and immunoblotting is required for the accurate serodiagnosis of B. caballi.

Equine piroplasmosis is caused by one of 2 erythrocytic parasites Babesia caballi or Theileria equi. Although the genus of the latter remains controversial, the most recent designation, Theileria, is utilized in this review. Shared pathogenesis includes tick-borne transmission and erythrolysis leading to anemia as the primary clinical outcome. Although both parasites are able to persist indefinitely in their equid hosts, thus far, only B. caballi transmits across tick generations. Pathogenesis further diverges after transmission to equids in that B. caballi immediately infects erythrocytes, whereas T.equi infects peripheral blood mononuclear cells. The recent re-emergence of T.equi in the United States has increased awareness of these tick-borne pathogens, especially in terms of diagnosis and control. This review focuses in part on factors leading to the re-emergence of infection and disease of these globally important pathogens., (Published 2013. This article is a U.S. Government work and is in the public domain in the U.S.A.)

Fleas (Insecta: Siphonaptera) are obligate bloodsucking insects, which parasitize birds and mammals, and are distributed throughout the world. Several species have been implicated in pathogen transmission. This study aimed to monitor red foxes and the fleas isolated from them in the Palermo and Ragusa provinces of Sicily, Italy, as these organisms are potential reservoirs and vectors of pathogens. Thirteen foxes (Vulpes vulpes) and 110 fleas were analysed by polymerase chain reaction (PCR) to detect DNA of the pathogens Ehrlichia canis, Babesia microti, Rickettsia spp., Anaplasma phagocytophilum, Anaplasma platys, Anaplasma marginale and Anaplasma ovis. In the foxes, A. ovis was detected in only one animal, whereas the prevalence of the E. canis pathogen was 31%. B. microti and Rickettsia spp. were not detected. Of all of the collected fleas, 75 belonged to the species Xenopsylla cheopis, 32 belonged to Ctenocephalides canis, two belonged to Ctenocephalides felis and one belonged to Cediopsylla inaequalis. In the fleas, the following pathogens were found: A. ovis (prevalence 25%), A. marginale (1%), A. phagocytophilum (1%), Rickettsia felis (2%) and E. canis (3%). X. cheopis was the flea species most frequently infected with Anaplasma, in particular A. ovis (33%), A. marginale (1%) and A. phagocytophilum (1%). Both C. felis exemplars were positive for R. felis. E. canis was found in the lone C. inaequalis and also in 3% of the X. cheopis specimens. No fleas were positive for B. microti or A. platys. As foxes often live in proximity to domestic areas, they may constitute potential reservoirs for human and animal parasites. Further studies should be performed on fleas to determine their vectorial capacity., (© 2013 Blackwell Verlag GmbH.)

Objective: To evaluate the ability of atropine sulfate, butylscopolammonium bromide combined with metamizole sodium, and flunixin meglumine to ameliorate the clinical adverse effects of imidocarb dipropionate in horses., Animals: 28 horses with piroplasmosis., Procedures: 28 horses were randomly assigned to 4 equal groups according to the pretreatment administered. Fifteen minutes before administration of 2.4 mg of imidocarb dipropionate/kg IM, horses in the first group were pretreated with 0.02 mg of atropine sulfate/kg IV, the second group with a combination of 0.2 mg of butylscopolammonium bromide/kg IV and 25 mg of metamizole sodium/kg IV, the third group with 1.1 mg of flunixin meglumine/kg IV, and the fourth (control) group with 1 mL of saline (0.9% NaCl) solution/50 kg IV. Physical examination, including evaluation of rectal temperature, heart and respiratory rates, capillary refill time, mucous membrane color, hydration status, abdominal sounds, signs of abdominal pain, salivation, diarrhea, and number of defecations, was performed., Results: Imidocarb dipropionate use in the control group was associated with serious adverse effects including signs of abdominal pain (4/7 horses) and diarrhea (2/7). Horses pretreated with atropine had no diarrhea, but 6 had signs of abdominal pain. Only 1 horse that received butylscopolammonium-metamizole pretreatment had signs of abdominal pain and 3 had diarrhea, which was numerically but not significantly different than the control group. Of horses pretreated with flunixin, 3 had signs of abdominal pain and 3 had diarrhea., Conclusions and Clinical Relevance: A combination of butylscopolammonium bromide and metamizole sodium may be useful to ameliorate the adverse effects of imidocarb dipropionate in horses, although group size was small and significant differences from the control group were not found.

Ovine babesiosis and theileriosis are important hemoprotozoal diseases of sheep and goats in tropical and subtropical regions that lead to economic losses in these animals. PCR-restriction fragment length polymorphism (PCR-RFLP) is a reliable molecular diagnostic tool for discriminating Theileria or Babesia species in the same host. In this study, the DNA sequences of a ribosomal protein S8 (RPS8) gene from four species of piroplasms in China were used to develop a species-specific PCR-RFLP diagnostic tool. The sensitivity of the PCR assays was 0.1 pg DNA for B. motasi and 1 pg DNA for T. uilenbergi and 10 pg DNA for Babesia sp. Xinjiang-2005 and T. luwenshuni. The clear size difference of the PCR products allowed for a direct discrimination for B. motasi, Babesia sp. Xinjiang-2005 and ovine Theileria species (T. uilenbergi and T. luwenshuni), except that the mixed infection between T. uilenbergi and T. luwenshuni may be difficult to distinguish, simply after the electrophoretic separation of the amplification products. Further T. uilenbergi and T. luwenshuni diagnoses were made by digesting the PCR product with SacI. The established method could be applicable for the survey of parasite dynamics, and epidemiological studies as well as prevention and control of the disease., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Canine babesiosis (or piroplasmosis) is an emerging tick-borne disease caused by intraerythrocytic protozoa of the genera Babesia and Theileria. In dogs, infection by these parasites usually induces a haemolytic syndrome that can be fatal when complicated. Canine babesiosis prevalence is high in France, with Babesia canis thought to be the main etiological agent of the disease. This article presents the results of a multiregional prospective longitudinal survey on canine babesiosis conducted in France from October 2006 to December 2007. A total of 836 cases were reported by veterinarians using a multiple choice questionnaire and blood samples from 70 dogs were analyzed using PCR-RFLP to identify species responsible for canine babesiosis cases across the country. The main clinical signs reported were lethargy (98%), anorexia (98%) and hyperthermia ≥ 39 °C (80%) followed by pale mucous membranes (54%), modification of urine aspect (45%) and splenomegaly (33%). The dog population at risk was mainly represented by young dogs living in rural areas. Twenty-five out of the 70 blood samples (36%) tested by diagnostic PCR were found to contain Babesia/Theileria genus-specific DNA and all had profiles similar to that of Babesia canis genomic DNA after restriction fragment length polymorphism analyses. The survey results provide a reference for further molecular studies to assess the species and vectors involved in the transmission of the disease in France and across the Mediterranean basin., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Identification and molecular characterization of Babesia gibsoni proteins with potential antigenic properties are crucial for the development and validation of the serodiagnostic method. In this study, we isolated a cDNA clone encoding a novel B. gibsoni 76-kDa protein by immunoscreening of the parasite cDNA library. Computer analysis revealed that the protein presents a glutamic acid-rich region in the C-terminal. Therefore, the protein was designated as B. gibsoni glutamic acid-rich protein (BgGARP). A BLASTp analysis of a translated BgGARP polypeptide demonstrated that the peptide shared a significant homology with a 200-kDa protein of Babesia bigemina and Babesia bovis. A truncated BgGARP cDNA (BgGARPt) encoding a predicted 13-kDa peptide was expressed in Escherichia coli (E. coli), and mouse antisera against the recombinant protein were used to characterize a corresponding native protein. The antiserum against recombinant BgGARPt (rBgGARPt) recognized a 140-kDa protein in the lysate of infected erythrocytes, which was detectable in the cytoplasm of the parasites by confocal microscopic observation. In addition, the specificity and sensitivity of enzyme-linked immunosorbent assay (ELISA) with rBgGARPt were evaluated using B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. Moreover, 107 serum samples from dogs clinically diagnosed with babesiosis were examined using ELISA with rBgGARPt. The results showed that 86 (80.4%) samples were positive by rBgGARPt-ELISA, which was comparable to IFAT and PCR as reference test. Taken together, these results demonstrate that BgGARP is a suitable serodiagnostic antigen for detecting antibodies against B. gibsoni in dogs., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A locally bred, 12-year-old, intact female Satsuma dog presented with generalized alopecia. Erythema, crusts and desquamation were observed primarily on the truck. Papules and erosions were present in the pinnae, and there were multiple areas of skin necrosis on the right forelimb. The cutaneous lesions had not responded to treatment with systemic antibiotics and prednisolone. The dog also had progressive anemia. Babesia gibsoni was detected in the blood, and the dog was treated with antiprotozoal agents. The skin lesions and anemia improved, but relapsed after the treatment was discontinued. Histopathological examination of skin biopsies revealed findings suggestive of early leukocytoclastic vasculitis or ischemic vasculopathy.

Babesia bovis, the causative agent of severe bovine babesiosis, is endemic in Sri Lanka. The live attenuated vaccine (K-strain), which was introduced in the early 1990s, has been used to immunize cattle populations in endemic areas of the country. The present study was undertaken to determine the genetic diversity of merozoite surface antigens (MSAs) in B. bovis isolates from Sri Lankan cattle, and to compare the gene sequences obtained from such isolates against those of the K-strain. Forty-four bovine blood samples isolated from different geographical regions of Sri Lanka and judged to be B. bovis-positive by PCR screening were used to amplify MSAs (MSA-1, MSA-2c, MSA-2a1, MSA-2a2, and MSA-2b), AMA-1, and 12D3 genes from parasite DNA. Although the AMA-1 and 12D3 gene sequences were highly conserved among the Sri Lankan isolates, the MSA gene sequences from the same isolates were highly diverse. Sri Lankan MSA-1, MSA-2c, MSA-2a1, MSA-2a2, and MSA-2b sequences clustered within 5, 2, 4, 1, and 9 different clades in the gene phylograms, respectively, while the minimum similarity values among the deduced amino acid sequences of these genes were 36.8%, 68.7%, 80.3%, 100%, and 68.3%, respectively. In the phylograms, none of the Sri Lankan sequences fell within clades containing the respective K-strain sequences. Additionally, the similarity values for MSA-1 and MSA-2c were 40-61.8% and 90.9-93.2% between the Sri Lankan isolates and the K-strain, respectively, while the K-strain MSA-2a/b sequence shared 64.5-69.8%, 69.3%, and 70.5-80.3% similarities with the Sri Lankan MSA-2a1, MSA-2a2, and MSA-2b sequences, respectively. The present study has shown that genetic diversity among MSAs of Sri Lankan B. bovis isolates is very high, and that the sequences of field isolates diverged genetically from the K-strain., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Piroplasms are tick-borne hemoprotozoans with a major impact on extensive management systems. Detection of sub-clinical low-level carriers, which can act as source of infection for vector ticks, is key to protect livestock trade and facilitate preventive control programs. The purpose of this study was to develop a method for the detection of ovine piroplasms and to use it in a field study aimed at investigating piroplasms infection in semi-extensive production systems in the Basque Country (northern Spain)., Methods: A DNA bead-based suspension array using the Luminex xMAP technology that included a generic Theileria-Babesia control probe, 6 species-specific probes, and an internal control probe was developed to detect and identify piroplasms that infect sheep. To monitor piroplasm infection in clinically healthy sheep from 4 flocks that share communal mountain pastures, blood samples were collected during 2 grazing seasons., Results: Piroplasms were detected in 48% (214/446) of blood samples, nearly half of them (49.1%, 105/214) as mixed infections. Five different piroplasms were identified: Theileria sp. OT3 in 34.8% of the samples, Theileria ovis in 20.9%, and at lower prevalences Babesia motasi (12.3%), Theileria luwenshuni/OT1 (10.5%) and Babesia ovis (6.3%). Despite differences among flocks associated to differences in management, an increasing trend in the incidence of piroplasm infection with increasing age of animals after increased tick exposure was observed. This increment could be attributed to continued re-infection associated with re-exposure to ticks at grazing. Ticks were collected from animals (4 species) and vegetation (8 species), and associations between tick abundance seasonality and risk of infection with the different piroplasms were established., Conclusion: The multiplex Luminex xMAP procedure is a rapid and high throughput technique that provided highly specific and sensitive identification of single and mixed piroplasm infections in blood of sheep carriers. This study confirmed a situation of endemic stability for piroplasm infection in the region, where infection is present in the absence of clinical signs, and mountain grazing allows for sufficient inoculation rates to maintain such situation.

This is the first report of confirmed canine babesiosis in Latvia supporting the observed geographical expansion of this disease. Between 2009 and 2011 three dogs which have not traveled outside of Latvia were diagnosed with babesiosis. Hematological analysis and serological tests for granulocytic anaplasmosis, ehrlichiosis and borreliosis were negative (Idexx SNAP 4Dx test). Peripheral blood erythrocytes of the three dogs contained large Babesia that were identified as Babesia canis canis by PCR. Sequences of partial 18S rRNA gene were 98-100% similar to the sequences of B. canis canis isolated from dogs in other European countries. We conclude that these are the first autochthonous canine babesiosis cases reported from Latvia., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Cattle babesiosis is a tick-borne disease of cattle with the most severe form of the disease caused by the apicomplexan, Babesia bovis. Babesiosis is transmitted to cattle through the bite of infected cattle ticks of the genus Rhipicephalus. The most prevalent species is Rhipicephalus (Boophilus) microplus, which is distributed throughout the tropical and subtropical countries of the world. The transmission of B. bovis is transovarian and a previous study of the R. microplus ovarian proteome identified several R. microplus proteins that were differentially expressed in response to infection. Through various approaches, we studied the reaction of the R. microplus ovarian transcriptome in response to infection by B. bovis., Methods: A group of ticks were allowed to feed on a B. bovis-infected splenectomized calf while a second group fed on an uninfected splenectomized control calf. RNA was purified from dissected adult female ovaries of both infected and uninfected ticks and a subtracted B. bovis-infected cDNA library was synthesized, subtracting with the uninfected ovarian RNA. Four thousand ESTs were sequenced from the ovary subtracted library and annotated., Results: The subtracted library dataset assembled into 727 unique contigs and 2,161 singletons for a total of 2,888 unigenes, Microarray experiments designed to detect B. bovis-induced gene expression changes indicated at least 15 transcripts were expressed at a higher level in ovaries from ticks feeding upon the B. bovis-infected calf as compared with ovaries from ticks feeding on an uninfected calf. We did not detect any transcripts from these microarray experiments that were expressed at a lower level in the infected ovaries compared with the uninfected ovaries. Using the technique called serial analysis of gene expression, 41 ovarian transcripts from infected ticks were differentially expressed when compared with transcripts of controls., Conclusion: Collectively, our experimental approaches provide the first comprehensive profile of the R. microplus ovarian transcriptome responding to infection by B. bovis. This dataset should prove useful in molecular studies of host-pathogen interactions between this tick and its apicomplexan parasite.

Vector-borne diseases (VBDs) continue to threaten the worldwide livestock industry, but comprehensive epidemiological surveys on such diseases have not been conducted in the Philippines. In the present study, we screened 408 bovine blood samples from 9 areas in Cebu, Philippines, for various VBD pathogens using specific PCR assays. The results revealed prevalences of 54.7, 15.4, 10.0, and 12.0% for Anaplasma spp., Babesia bigemina, Babesia bovis, and Trypanosoma (Tr.) theileri, respectively. In contrast, none of the samples were positive for Trypanosoma (Tr.) evansi, Theileria (Th.) orientalis, and Theileria (Th.) annulata. Mixed infections were observed in 24.2% of the samples tested. Phylogenetic analysis based on the 16S rRNA gene revealed that the Anaplasma spp. sequences from the present study were genetically close either to Anaplasma marginale or Anaplasma phagocytophilum. In addition, B. bovis RAP-1 and Babesia bigemina AMA-1 gene sequences were identical and monophyletic to other known B. bovis and B. bigemina sequences. On the other hand, Tr. theileri cathepsin-L like protein gene sequences shared 97.1-100% identities with those from the USA and Brazil and clustered within a single genotype in the phylogenetic tree. The molecular identification of several VBD pathogens in Cebu cattle calls for the implementation of control measures to prevent the spread of these pathogens to nearby localities or islands, and ultimately, economic losses to the Philippine economy., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Urinary creatinine to serum creatinine (UCr/SCr) ratio and renal failure index (RFI) are useful indices of renal damage. Both UCr/SCr ratio and RFI are used in differentiation between prerenal azotaemia and acute tubular necrosis. In this work the authors calculated the UCr/SCr ratio and RFI in dogs infected with Babesia canis and the values of these indices in azotaemic dogs infected with the parasite. The results of this study showed significantly lower UCr/SCr ratio in dogs infected with B. canis than in healthy dogs. Moreover, in azotaemic dogs infected with B. canis the UCr/SCr ratio was significantly lower and the RFI was significantly higher than in non-azotaemic dogs infected with B. canis. The calculated correlation between RFI and duration of the disease before diagnosis and treatment was high, positive and statistically significant (r = 0.89, p < 0.001). The results of this study showed that during the course of canine babesiosis caused by B. canis in Poland acute tubular necrosis may develop.

As it feeds upon cattle, Rhipicephalus (Boophilus) microplus is capable of transmitting a number of pathogenic organisms, including the apicomplexan hemoparasite Babesia bovis, a causative agent of bovine babesiosis. The R. microplus female gut transcriptome was studied for two cohorts: adult females feeding on a bovine host infected with B. bovis and adult females feeding on an uninfected bovine. RNA was purified and used to generate a subtracted cDNA library from B. bovis-infected female gut, and 4,077 expressed sequence tags (ESTs) were sequenced. Gene expression was also measured by a microarray designed from the publicly available R. microplus gene index: BmiGI Version 2. We compared gene expression in the tick gut from females feeding upon an uninfected bovine to gene expression in tick gut from females feeding upon a splenectomized bovine infected with B. bovis. Thirty-three ESTs represented on the microarray were expressed at a higher level in female gut samples from the ticks feeding upon a B. bovis-infected calf compared to expression levels in female gut samples from ticks feeding on an uninfected calf. Forty-three transcripts were expressed at a lower level in the ticks feeding upon B. bovis-infected female guts compared with expression in female gut samples from ticks feeding on the uninfected calf. These array data were used as initial characterization of gene expression associated with the infection of R. microplus by B. bovis.

The focus of this study was the detection of equine piroplasmosis in Distrito Capital, Miranda, Aragua, Guárico and Apure States from Venezuela, using two methods: Competitive-Inhibition ELISA and multiplex PCR and the analysis of the possible differences in occurrence in relation to the primary purpose of the horses, which is related to varied degrees of exposure to tick. Antibody levels to Babesia caballi and Theileria equi were assessed in 694 equine serum samples using Competitive-Inhibition ELISA, while PCR assays were performed in 136 horses, using two sets of oligonucleotides to establish the presence of T. equi, B. caballi or both. The overall seroprevalence of equine piroplasmosis was 50.2%, antibodies to B. caballi were found in 161 horses (23.2%), whereas 97 (14.0%) were seropositive to T. equi and 90 (13.0%) were positives to both parasites (mixed infections). PCR determinations (n=136) showed a prevalence of 66.2%, distributed in 84 (61.8% positives) for T. equi and, 6 (4.4%) were positive to both parasites. The cELISA showed higher levels of prevalence of B. caballi and mixed infections, as compared to the PCR method. This discrepancy can be explained by the different parameters that are evaluated by each technique, PCR detect the parasite itself, while cELISA detects antibodies to the parasite. By PCR, the highest prevalence was found in Apure state, where 92.3% of the samples were positive to T. equi infections. In this locality, free grazing animals are used for livestock management. This high prevalence may be linked to the tick species present in that area. More epidemiological studies will be necessary to assess the epidemiological status of equine piroplasmosis in Venezuela., (Published by Elsevier B.V.)

The prevalence of piroplasm (order Piroplasmida) infection was assessed in blood and bone marrow samples from 91 red foxes (Vulpes vulpes) from northern, central and southern Portugal by means of molecular methods. PCR for the 18S rRNA gene of Babesia spp. followed by sequencing revealed 63 foxes positive for the Babesia microti-like piroplasm (syn. Theileria annae) (69.2%; 95% confidence interval [CI]: 58.7-78.5%) and one fox positive for Babesia canis (1.1%; 95% CI: 0.0-6.0%). Positivity to the B. microti-like piroplasm or B. canis in 43 blood samples (83.7%) was significantly higher (p<0.001) than in 43 paired bone marrow samples (20.9%). There were no statistically significant differences in the prevalence of infection between genders (p=0.219) or age groups (<2 years vs. ≥ 2 years) (p=1.0). This is the first report of the B. microti-like piroplasm in foxes from Portugal as well as the first report on detection by PCR and genotyping of B. canis in a red fox worldwide. A natural cycle of the B. microti-like piroplasm is suggested in red fox populations based on the high prevalence of the protozoan. Red foxes might be a reservoir of the B. microti-like piroplasm and a source of infection to dogs., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The passive transfer of antibodies from dams to offspring via colostrum is believed to play an important role in protecting neonatal mammals from infectious disease. The study presented here investigates the uptake of colostrum by 548 calves in western Kenya maintained under smallholder farming, an important agricultural system in eastern Africa. Serum samples collected from the calves and dams at recruitment (within the first week of life) were analysed for the presence of antibodies to four tick-borne haemoparasites: Anaplasma marginale, Babesia bigemina, Theileria mutans and Theileria parva. The analysis showed that at least 89.33% of dams were seropositive for at least one of the parasites, and that 93.08% of calves for which unequivocal results were available showed evidence of having received colostrum. The maternal antibody was detected up until 21 weeks of age in the calves. Surprisingly, there was no discernible difference in mortality or growth rate between calves that had taken colostrum and those that had not. The results are also important for interpretation of serosurveys of young calves following natural infection or vaccination., (Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Babesia parasites, mainly Babesia bovis and B. bigemina, are tick-borne hemoparasites inducing bovine babesiosis in cattle globally. The clinical signs of the disease include, among others, anemia, fever and hemoglobinuria. Babesiosis is known to occur in tropical and subtropical regions of the world. In this study, we aim to provide information about the occurrence and phylogenetic relationship of B. bigemina and B. bovis species in cattle from different locations in nine provinces of South Africa. A total of 430 blood samples were randomly collected from apparently healthy cattle. These samples were genetically tested for Babesia parasitic infections using nested PCR assays with species-specific primers., Results: Nested PCR assays with Group I primer sets revealed that the overall prevalence of B. bigemina and B. bovis in all bovine samples tested was 64.7% (95% CI = 60.0-69.0) and 35.1% (95% CI = 30.6-39.8), respectively. Only 117/430 (27.2%) animals had a mixed infection. The highest prevalence of 87.5% (95% CI = 77.2-93.5) for B. bigemina was recorded in the Free State province collection sites (Ficksburg, Philippolis and Botshabelo), while North West collection sites had the highest number of animals infected with B. bovis (65.5%; 95% CI = 52.7-76.4). Phylograms were inferred based on B. bigemina-specific gp45 and B. bovis-specific rap-1 nucleotide sequences obtained with Group II nested PCR primers. Phylogenetic analysis of gp45 sequences revealed significant differences in the genotypes of B. bigemina isolates investigated, including those of strains published in GenBank. On the other hand, a phylogeny based on B. bovis rap-1 sequences indicated a similar trend of clustering among the sequences of B. bovis isolates investigated in this study., Conclusion: This study demonstrates the occurrence of Babesia parasites in cattle from different provinces of South Africa. It was also noted that the situation of Babesia parasitic infection in cattle from certain areas within the surveyed provinces had either reached endemic stability or was progressing towards stability.

This study was carried out to investigate presence and distribution of Theileria and Babesia species via microscopic examination and reverse line blotting (RLB) techniques in sheep and goats in the Black Sea region of Turkey. For this purpose, 1,128 blood samples (869 sheep and 259 goats) were collected by active surveillance from sheep and goats in different provinces of various cities in the region in the years 2010 and 2011. Smears were prepared from the blood samples, stained with Giemsa, and examined under the light microscope for Theileria and Babesia piroplasms. The genomic DNAs were extracted from blood samples. The length of 360-430-bp fragment in the variable V4 region of 18S SSU rRNA gene of Theileria and Babesia species was amplified using the gDNAs. The polymerase chain reaction products were hybridized to the membrane-connected species-specific probes. A total of 38 animals (3.37%) including 34 sheep (3.91%) and 4 goats (1.54%) were found to be positive for Theileria spp. piroplasms in microscopic examination of smears while Babesia spp. piroplasm could not detected. Infection rates were 34.64% in sheep, 10.04% in goats, and totally 28.99% for Theileria ovis while 0.58% in sheep and totally 0.44% for Babesia ovis. However, Theileria sp. OT3 was detected in 2.65% of sheep and 2.04% of all animals; besides Theileria sp., MK had 0.58% prevalence in sheep and 0.77% in goats, with a total 0.62% with RLB. Although T. ovis and Theileria sp. MK were determined in both sheep and goats, B. ovis and Theileria sp. OT3 were observed only in the sheep. These results provide the first detailed molecular data for sheep and goat theileriosis and babesiosis in the region.