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We tested the hypothesis that age, breed, and sex are related to hematology, biochemistry, acute phase proteins (APPs), seroreactivity and level of parasitemia in dogs with an acute phase response (APR) due to Babesia canis infection. The study enrolled 61 privately owned dogs that naturally acquired B. canis infection. Groups were formed according to the age: young dogs less than one year, and adult dogs more than one year old. Moreover, the group of males was compared to females and purebred to mixed breed dogs. Seroreactivity was tested with immunofluorescence antibody test, level of parasitemia with real-time polymerase chain reaction (real-time PCR), hematology, and biochemistry with automatic analyzers, serum amyloid A with enzyme-linked immunosorbent assay, fibrinogen with heat precipitation and ceruloplasmin and paraoxonase-1 with manual spectrophotometric methods. For protein separation agarose gel electrophoresis was used. The main changes in the whole population of B. canis-infected dogs were fever, pancytopenia, and change in APPs level. One-third of young, and 96% of adult dogs were seropositive (P < 0.001). The level of parasitemia was higher in the young dogs (P < 0.001). Erythroid lineage parameters (P < 0.01), and leukocytes (P < 0.05) were lower in the young, when compared to the adult dogs. Young dogs had lower total globulins (P < 0.001), β- and γ-globulins (P < 0.001), and higher α-globulins (P = 0.022) than adult dogs. Young dogs had higher concentrations of phosphate (P = 0.003) and cholesterol (P < 0.001) and lower amylase (P = 0.014) and lipase activity (P = 0.020) than adult ones. Male dogs had lower neutrophil count than females (P = 0.035), and purebred dogs had more band neutrophils than mixed breed dogs (P = 0.004). In conclusion, dogs with natural Babesia canis infection at a young age have more severe anemia and APR including leukopenia than adults. Male and purebred dogs might also have more severe APR than females and mix-breeds, as they have more pronounced changes related to the myeloid lineage., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Canine babesiosis is a rapidly spreading tick-borne disease in Europe, which entails protozoan parasites invading red blood cells. Small extracellular vesicles (EVs) (< 200 nm) were isolated from the serum of 15 healthy and 15 by Babesia canis naturally infected dogs aimed to distinguish EV characteristics and protein profiles. There were no significant differences (P = 0.05) observed in the mean sizes and concentrations of serum EVs between the healthy and canine babesiosis groups. Despite a higher number of Canis lupus proteins detected in EVs from serum of diseased dogs, there were no statistically significant differences (P < 0.05) in the number of protein IDs between the experimental groups. We successfully identified 211 Canis lupus proteins across both experimental groups, of which 147 Canis lupus proteins were validated as being EV-associated. This data set is accessible via the ProteomeXchange PXD047647. EVs isolated from serum of B. canis infected dogs were Cd9 + , Cd63 + , Cd81 + , and Cd82 + . Furthermore, 73 Canis lupus proteins were validated as EV-associated and specific for EVs isolated from serum of B. canis-infected dogs. These were predominantly membrane and cytosolic proteins, and innate and adaptive immune system-related proteins, especially those involved in adhesion and proteoglycan mechanisms like integrins. Enrichment was also observed for proteins involved in vascular and cellular responses, including signalling pathways such as VEGF, VEGFR, and the LKB1 network. When only blood-related sites of EV expression were evaluated, the origins of EV proteins were mostly cells of immune system. These were dendritic cells, neutrophils, B cells, monocytes and platelets. In general, proteins were enriched in pathways that collectively regulate various cellular processes, including immune responses, communication, signal transduction, membrane trafficking, and apoptosis. Serum EVs and their protein cargo may have an important role in both the invasion of B. canis and the host's response to the parasitic infection, nevertheless, additional experimental research is warranted. The overall count of identified EV proteins of parasitic origin, meeting cut off criteria of two peptides and 1 % FDR, was relatively low., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Vladimir Mrljak reports financial support was provided by European Commission. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Equine piroplasmosis (EP) is caused by Theileria equi and Babesia caballi, transmitted by tick vectors. Horses can suffer an acute, subacute, and chronic forms of the disease, with clinical signs such as poor performance, fever, pale mucosal membranes, and jaundice. The diagnosis of EP subclinical cases is complex due to the sensitivity of real-time PCR and the limited parasite load in some carriers, making it challenging to differentiate them from seropositive, PCR negative (S+PCR-) individuals. This study aimed to describe haematological and biochemical changes in asymptomatic EP carriers, EP S+PCR- horses and control horses (EP seronegative and PCR negative). It also investigated potential haemato-biochemical markers to aid in distinguishing true EP carriers alongside molecular and serological tests. A comprehensive haematology and biochemistry profile was conducted on 410 sera and EDTA blood samples, comprising 130 EP positives by real-time PCR and competitive ELISA (cELISA) (carriers), 130 EP negatives by real-time PCR but positive to cELISA (S+PCR-) and 150 EP negative horses to real-time PCR and c-ELISA (controls). Our study confirmed that a haematological and biochemistry profile could help to differentiate between EP carriers/S+PCR- from healthy horses. Carriers and S+PCR- horses showed significant increases in the white blood cell count (WBC), high total proteins (TP) and total globulins (GLOB) concentration, and liver function markers compared to controls. Additionally, the evaluation of uric acid (UA) suggested oxidative stress in carrier horses. However, no useful haemato-biochemical diagnostic markers were identified to aid the challenging differentiation of EP carriers and S+PCR- horses, highlighting the need for improvement in molecular/serological diagnosis for these horses., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Eliazar Camino reports financial support was provided by Complutense University of Madrid. Eliazar Camino reports a relationship with Complutense University of Madrid that includes: funding grants., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Inflammation is a hallmark of the acute Babesia canis infection. Promatrix metalloproteinase (proMMP)-2 and -9 are involved in inflammation, but their levels have not been analyzed in canine babesiosis. We hypothesized that in dogs infected with B. canis, serum proMMP-2 and -9 levels change between presentation and recovery. Degree of the change differs if dogs develop systemic inflammatory response syndrome (SIRS). This study included 24 dogs with an acute B. canis infection, at presentation and after two weeks. We used routine hematology and biochemistry methods, spectrophotometry for the acute-phase proteins, microscopy for parasitemia and zymography for (pro)MMPs. In vitro endothelial cells and leukocyte short-term cultures, and platelet lysates were used to detect specific MMP activity. Statistical analyses included Wilcoxon test for paired samples, Mann-Whitney U test and Spearman's rank correlation. Our results showed that endothelial cells, leukocytes and platelets are the source of proMMP-2 and proMMP-9. Furthermore, both proMMPs were lower at presentation than after recovery (p < 0.001). At presentation, proMMP-9 levels correlated with parasitemia (rho = -0.616, p = 0.009), total leukocyte (rho = 0.704, p < 0.001) and neutrophil counts (rho = 0.741, p < 0.001). Extent of alterations in proMMP-2 levels between presentation and recovery was lower (p = 0.038) in dogs with SIRS than in non-SIRS dogs, while levels of proMMP-9 were comparable between these groups. Our conclusion is that during the acute B. canis infection, low serum levels of proMMP-2 and proMMP-9 at presentation reflect thrombocytopenia and leukopenia. Decreased proMMP-2 level could be associated with SIRS., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Canine babesiosis, a tick-borne haemoprotozoan disease of dogs, is of significance globally due to its rapid spread. A precise confirmatory diagnosis is required to curtail the rapid spread of infection. Our study described the evaluation of recombinant BgSA3 protein based indirect ELISA for sero-diagnosis and sero-surveillance of Babesia gibsoni infection in dogs. A partial BgSA3 gene segment (1921 bp) of B. gibsoni, encoding for recombinant truncated BgSA3 (75 kDa) protein devoid of predicted signal peptide (23 aa) at N-terminus and transmembrane region (20 aa) at C-terminus, was expressed in E. coli using a pET28a(+) vector. The rBgSA3 protein purified under native conditions using Ni-NTA superflow cartridge was confirmed by SDS-PAGE and Western blotting using sera from dogs infected/uninfected with B. gibsoni, and erythrocyte lysate/ plasma from infected/uninfected dogs. The rBgSA3 protein was specific only to B. gibsoni antibodies but did not react with uninfected sera. Further, rBgSA3 protein was evaluated for sero-diagnosis/sero-surveillance using Indirect-ELISA format. There was no cross reactivity to B. vogeli, E. canis, H. canis and D. repens infected dogs serum samples. The diagnostic sensitivity and specificity of rBgSA3 based I-ELISA was found to be 86.4 and 93.1 % respectively, in comparison with cytb based PCR assay. Additionally, rBgSA3-ELISA evaluated using survey serum samples (n = 287), detected 11.85 % samples as positive. In conclusion, B. gibsoni infection, an emerging disease is prevalent in the present study area and the standardized rBgSA3 protein based indirect-ELISA was found to be a specific and sensitive test for large scale sero-diagnosis and sero-surveillance of B. gibsoni infection in dogs., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Equine piroplasmosis (EP) is a tick-borne protozoan disease caused by Theileria equi and/or Babesia caballi. Clinical signs (fever, pale mucosal membranes, jaundice), anemia and hyperbilirubinemia have been associated with the disease. EP is widespread, has a significant economic impact on the equine industry and remains endemic in Spain. This study was carried out with samples belonging to 140 horses residing in Spain and showing common clinical signs of EP. A blood smear microscopic examination and a comparison between the different results obtained by competitive Enzyme-Linked Immunosorbent Assay (cELISA), real-time Polymerase Chain Reaction (PCR) and hematological and biochemical (direct and total bilirubin) screening were conducted. EP positivity rates by cELISA and PCR were 50.7% and 42.9%, respectively, whereas only 9% of the horses were positive in the microscopic analysis. A significantly higher number of B. caballi-positive horses were detected by cELISA than PCR, and Kappa value was higher for T. equi (k = 0.575) than for B. caballi (k = 0.401). For the first time, an association between a high ELISA inhibition percentage (IP) and a positive PCR result for B. caballi was determined. Although most authors have described T. equi as more pathogenic than B. caballi, we found that horses parasitized by B. caballi showed a more severe hemolytic anemia, whereas T. equi infections were mostly associated with leukocytosis. The hemogram and clinical chemistry could guide the veterinary surgeon towards the diagnosis of T. equi or B. caballi since horses showed a significant leukocytosis or anemia and hyperbilirubinemia, respectively; however PCR would be the test of choice in order to confirm the diagnosis. Information about the importance of a correct diagnosis of EP using a combination of techniques is essential in order to allow the early detection of cases and prevent the spread of the disease, as well as to avoid the common practice of treating horses without a laboratory diagnosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Canine babesiosis caused by Babesia canis sensu stricto became an emerging disease of dogs across Europe calling for attention also in countries where it was an only rare imported disease. An easy accessibility of molecular methods and the growing amount of sequencing data led to the description of intraspecific variability in 18S rDNA sequences designated as "genotypes". Using material from a homogenous cohort of dogs with microscopically confirmed canine babesiosis caused by B. canis, we evaluated Babesia intraspecific variability and amplification sensitivity of three different genes (18S rDNA, COI, Cytb) to assess their potential as diagnostic or phylogenetic markers. In raw sequencing data obtained, we observed at least 3 ambiguous positions in up to 86% of chromatograms within the ∼560 bp fragment of 18S rDNA suggesting the existence of several, not identical copies of this gene. Our COI haplotype analysis resulted in a star-like pattern indicating a recent origin of most haplotypes, but not supporting the existence of two dominant haplotypes. Similarly, the Cytb sequences obtained from samples with all variants of 18S rDNA were identical. We corroborate previous observations from three other European countries and bring the evidence of the existence of 18S rDNA paralogs in B. canis genome replacing currently used "genotype" theory., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Babesiosis, theileriosis, and anaplasmosis are the most common tick-borne diseases in sheep. The majority of anaplasmosis and theileriosis are subclinical; however, babesiosis causes severe infections in small ruminants. Although there are many reports of co-infections with the agents of these diseases, their clinical severity compared with either of the infections alone is unknown. Within the host, interactions between co-infecting species may cause variations in clinical presentation and response to therapy. The aim of this study was to determine the tick-borne agents in sheep located at sites where fatal disease outbreaks caused by babesiosis have commonly been reported. Two hundred and nine sheep with clinical signs suggestive of ovine babesiosis were included in the study. The initial diagnosis of haemoparasites was based on clinical symptoms and microscopy and was confirmed using PCR assays. The blood samples were examined for the presence of Babesia ovis (B. ovis), Anaplasma ovis (A. ovis), A. phagocytophilum, and Theileria ovis (T. ovis). The results showed 86.12% of the animals were infected with one or more pathogens. B. ovis was the dominant pathogen. Overall, the infection rate of B. ovis, A. ovis, T. ovis, and A. phagocytophilum was 70.81%, 56.94%, 21.05%, and 2.39%, respectively. The infection rate of B. ovis alone (31.11%) was higher than A. ovis (9.44%) or T. ovis (1.67%) alone. Co-infections were found at a higher percentage (57.78%) than single infections (42.22%). A. ovis was detected in the blood of a high percentage (98.07%) of co-infected animals. Coexistence of B. ovis and A. ovis (34.45%) was more common than other combinations of species. There was a noticeably low level of co-occurrence between B. ovis and T. ovis (1.11%). During the study, 11 sick animals did not survive despite treatment. Seven were infected with B. ovis alone, three had a dual infection with B. ovis and A. ovis, and one had B. ovis, A. ovis, and T. ovis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Piroplasmosis is caused by several species of protozoa such as the Babesia microti-like piroplasm (Bml), an emerging blood protozoan also known as Theileria annae or Babesia vulpes. Infection by Bml was first reported in dogs in Spain where it is endemic today. Recently, a high prevalence of Bml has been increasingly detected in red foxes (Vulpes vulpes) in European countries. The objective of this study was to determine infection levels of this parasite in foxes from Galicia, NW Spain, and ticks species infestation in these carnivores, where they are so far unknown. Samples of blood, spleen and ticks (if present) were taken from 237 hunted red foxes in the Galicia region. Blood smears were prepared for direct parasite observation, and spleen and tick samples were examined by nested PCR. Prevalences of Bml infection in Galician red foxes were estimated at 72% (171/237) by PCR and 38.23% (26/68) by direct observation. Among 837 ticks collected, the main tick identified was Ixodes hexagonus (present in 82.4% of the foxes) followed by Ixodes ricinus (12.3%), Dermacentor reticulatus (12.3%) and Rhipicephalus sanguineus sensu lato (3.5%). From 34 foxes testing positive for Bml, 616 ticks were collected: positive Bml PCR results were obtained in 55.6% (227/408) of ticks collected from 9 foxes, while the 208 ticks from the remaining 25 infected foxes returned negative PCR results. Given that canine piroplasmosis is endemic in this area, our observations point to the red fox as the main reservoir for Bml infection and the high proportion of I. hexagonus among ticks collected from red foxes suggests its likely role as vectors of B. microti-like piroplasm in this region. Further studies are needed for a better understanding of the link between the wild and domestic life cycles of this piroplasm., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Rhipicephalus microplus is a vector of cattle tick fever, a disease caused by the protozoans Babesia bovisand B. bigemina, and also anaplasmosis, produced by the Rickettsiales Anaplasma marginale. These tick-borne pathogens cause considerable losses to Brazilian livestock breeders and represent an obstacle to the expanded use of taurine breeds due to their higher sensitivity to ticks and hemoparasites compared to zebu breeds. Differences in the susceptibility to hemoparasites were also verified within breeds, suggesting that may be possible to select a most resistant phenotype. Therefore, repeatability of R. microplus counts and copy number of hemoparasites DNA were estimated, along with correlations between themselves, aiming to verify if those measures can be used as parameters to classify animals according to their parasite resistance degrees. Forty-two Canchim females kept on pastures naturally infested by ticks were evaluated for the level of infestation by R. microplus and infection by B. bovis, B. bigemina, and A. marginale. Twenty-four evaluations were performed once a month, for adult female ticks counts and blood samplings. The experimental period was divided into four phases, according to the animals age range: Phase 1: 8 to 13 months (collections 1 to 6); phase 2: 14 to 19 months (collections 7 to 12); phase 3: 20 to 25 months (collections 13 to 18), and phase 4: 26 to 31 months (collections 19 to 24). Blood samples were submitted to absolute quantification of hemoparasites DNA sequences using qPCR. The hemoparasite and tick counts data were transformed for normalization and were analyzed using mixed models. Among three species of hemoparasites studied, A. marginale presented the highest level of infection. During phase 3, B. bigemina presented higher infection levels (p < 0.05) compared to B. bovis, whereas no differences were observed in other phases. Estimated repeatabilities for parasite infection levels varied from low to moderate during our experiment. There were low correlations between tick counts and parasite infection levels, and between parasite infection levels from different species by themselves. Based on these results, under conditions of the present study, we suggest that it is possible to identify animals presenting a most resistant phenotype against infection by both hemoparasites and ticks. Moreover, the animal age may be an important factor related to resistance against these pathogens. The data obtained shed more light on the resistance to hemoparasites studied., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Babesia sp. BQ1 (Lintan) is one of the parasites isolated from infected sheep in China that belongs to the B. motasi-like phylogenetic group. The rhoptry-associated-protein 1 (rap-1) locus in this group consists of a complex organization of 12 genes of three main types: 6 rap-1a variants intercalated with 5 identical copies of rap-1b and a single 3' ending rap-1c gene. In the present study, transcription analysis performed by standard RT-PCR demonstrated that the three different rap-1 gene types and the four rap-1a variants were transcribed by the parasite cultivated in vitro. Peptides, specific for each rap-1 type gene, were selected in putative linear B-epitopes and used to raise polyclonal rabbit antisera. Using these sera, the same expression pattern of RAP-1 proteins was found in parasites cultivated in vitro or collected from acute infection whereas only RAP-1a67 was detectable in merozoite extracts. However, ELISA performed with recombinant RAP-1a67, RAP-1b or RAP-1c and sera from infected sheep demonstrated that RAP-1a67 is the main RAP-1 recognized during infection, even if some infected sheep also recognized RAP-1b and/or RAP-1c., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The gene encoding Babesia ovis surface protein D (BoSPD) was cloned from B. ovis cDNA library. This gene encodes a polypeptide chain of 155 amino acids, including a predicted 22 amino acid signal peptide. Sequence analysis of the BoSPD suggested that it is a surface protein with no known domains. BLAST analysis followed by multiple alignments showed four orthologs from other Apicomplexan species and suggested that BoSPD is specific for B. ovis. BoSPD-based PCR was then developed to specifically detect B. ovis in experimentally-infected sheep and Rhipicephalus bursa ticks, as well as in field samples. The PCR enabled detection of B. ovis at a calculated parasitemia of 0.0016% and was shown to be specific for B. ovis. Moreover, the BoSPD PCR allowed detection of prolonged subclinical infection in experimentally-infected lambs and in dissected organs of experimentally-infected ticks. Finally, the PCR was used to detect parasitemia in blood samples from naturally-infected sheep and in R. bursa ticks collected from sheep in an infected flock. These results suggest that the BoSPD gene sequence can be used as a specific and sensitive marker, allowing detection of subclinical parasitemia in sheep and in ticks. Based on its predicted properties, BoSPD may be considered as a candidate for anti-B. ovis vaccine development or a target for anti-B.ovis treatment., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The development of sensitive surveillance technologies using PCR-based detection of microbial DNA, such as the reverse line blot assay, can facilitate the gathering of epidemiological information on tick-borne diseases, which continue to hamper the productivity of livestock in many parts of Africa and elsewhere. We have employed a reverse line blot assay to detect the prevalence of tick-borne parasites in an intensively studied cohort of indigenous calves in western Kenya. The calves were recruited close to birth and monitored for the presence of infectious disease for up to 51 weeks. The final visit samples from 453 calves which survived for the study period were analyzed by RLB. The results indicated high prevalences of Theileria mutans (71.6%), T. velifera (62.8%), Anaplasma sp. Omatjenne (42.7%), A. bovis (39.9%), Theileria sp. (sable) (32.7%), T. parva (12.9%) and T. taurotragi (8.5%), with minor occurrences of eight other haemoparasites. The unexpectedly low prevalence of the pathogenic species Ehrlichia ruminantium was confirmed by a species-specific PCR targeting the pCS20 gene region. Coinfection analyses of the seven most prevalent haemoparasites indicated that they were present as coinfections in over 90% of the cases. The analyses revealed significant associations between several of the Theileria parasites, in particular T. velifera with Theileria sp. sable and T. mutans, and T. parva with T. taurotragi. There was very little coinfection of the two most common Anaplasma species, although they were commonly detected as coinfections with the Theileria parasites. The comparison of reverse line blot and serological results for four haemoparasites (T. parva, T. mutans, A. marginale and B. bigemina) indicated that, except for the mostly benign T. mutans, indigenous cattle seem capable of clearing infections of the three other, pathogenic parasites to below detectable levels. Although the study site was located across four agroecological zones, there was little restriction of the parasites to particular zones., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Ovine babesiosis is a fatal disease characterized by severe progressive hemolytic anemia. In order to clarify the causal mechanisms implicated in anemia, this study was aimed to assess the antioxidant status and erythrocyte oxidative stress in sheep suffering from ovine babesiosis. Babesia infection was confirmed both with Giemsa's staining blood smears and semi-nested PCR amplified region of 18S rRNA gene. Thirty-eight Iranian sheep, naturally infected with Babesia spp., were considered as the infected group and divided into four subgroups according to parasitemia rates (<1%, 1-2%, 2-3% and >3%), and the same number non-infected animals were selected as the control group. Blood samples were taken and hematological parameters, activities of antioxidant enzymes including erythrocyte glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT), glucose-6-phosphate dehydrogenase (G6PD), total antioxidant capacity (TAC), median corpuscular fragility (MCF), and serum concentrations of some trace minerals (copper, iron, zinc, manganese, and selenium) were measured. In addition, as an index of lipid peroxidation, the level of malondialdehyde (MDA) was measured. The results revealed a significant decrease (P<0.01) in RBC count, packed cell volume (PCV) and Hb concentration as well as the activities of erythrocyte GSH-Px, SOD, CAT, G6PD, TAC, MCF and serum concentrations of Cu, Zn, Mn and Se in the infected sheep. In contrast, significantly increased (P<0.01) levels of MDA and erythrocyte osmotic fragility as well as serum concentration of iron were recorded in the infected animals. Overall, the observed remarkable decrease in the antioxidant enzyme activities, median corpuscular fragility and substantial elevated levels of lipid peroxidation associated with the notable increase in parasitemia indicate high exposure of RBCs to oxidative damage in Babesia infected sheep. These results indicate that the disturbed antioxidant defense mechanisms in ovine babesiosis can promote the development of anemia., (Copyright © 2014. Published by Elsevier B.V.)

This study demonstrated the genetic diversity among MSA-2c, MSA-2a1 and MSA-2b proteins of Babesia bovis isolates obtained from bovine blood and Rhipicephalus annulatus tick samples. The least identities that were observed among the deduced amino acid sequences of MSA-2c, MSA-2a1 and MSA-2b were 55, 63, and 71%, respectively. During the study four B. bovis calves, aged about 1 month, were found to be infected with virulent field strains and developed babesiosis. Probably, the calves had received insufficient antibodies, or the antibodies raised against the vaccine strain did not cross-protect against virulent field isolates. The complete msa-2 locus from the Israeli B. bovis vaccine strain and two field isolates were characterized. Similarly to the Australian strains and isolates, the msa-2 loci of the examined Israeli strain and isolates had only two msa-2 genes - msa-2c and msa-2a/b - located between msa-2c and orfB. Several of the examined samples, contained different MSA-2 genotypes concurrently. No obvious geographical relationships among isolates from various regions of Israel were established. Moreover, in the phylogenetic analyses, the Israeli deduced MSA-2 amino acid sequences of the three examined genes were clustered together with sequences derived from other countries, proving that the msa-2 gene sequences of B. bovis shared the same genetic characters worldwide. The present study clearly showed that the MSA-2 proteins of B. bovis isolates from Israel were genetically distinct from the vaccine strains. Thus, further research will be needed in order to understand the genetic diversity mechanisms of B. bovis, and the immunological responses of the infected animals., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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 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.)

Although cardiac pathology and consequently elevated serum cardiac troponin I (cTnI) have been reported, clinically it remains difficult to diagnose cardiac involvement in canine babesiosis. Thus the use of cardiac biomarkers would be useful in determining if a dog with babesiosis also has concurrent cardiac dysfunction. The objectives of this study were to determine plasma N terminal brain natriuretic peptide (NT-proBNP) in canine babesiosis and if it is correlated with cTnI. Three groups of dogs with babesiosis were used: mild uncomplicated (Group 1), severe uncomplicated (Group 2), and complicated (Group 3), and a control group (Group 4) with 15 dogs per group. Each animal had the following determined: serum urea and creatinine, urea: creatinine ratio, cystatin-C, cTnI, blood lactate, plasma NT-proBNP, fractional shortening (FS), and blood pressure. The median NT-proBNP value in Groups 1-4 was 246, 650, 638, and 106 pmol/l. All 3 babesiosis groups had a statistically elevated NT-proBNP level compared to the control group and Groups 2 and 3 showed significantly higher values compared to Group 1. Median cTnI in Group 1-3 was 0.39, 0.4, and 1.45 ng/ml, respectively with the control group having concentrations below the detection limit (0.2 ng/ml). There was a significant difference in cTnI concentration between the control group and group 3 but no statistical difference between the other babesiosis groups. The study concluded that dogs with babesiosis showed elevated levels NT-proBNP and the more severe the disease process the greater the elevation. This elevation is earlier or independent of the increased cTnI., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Ehrlichiosis and babesiosis are tick-borne diseases, caused mainly by Ehrlichia canis and Babesia canis, respectively, with a worldwide occurrence in dogs, whose main vector is the brown-dog tick, Rhipicephalus sanguineus. The present work aimed to detect the presence of E. canis and Babesia sp. in 91 dog blood samples in Colombia, by molecular and serological techniques. We also performed sequence alignment to indicate the identity of the parasite species infecting these animals. The present work shows the first molecular detection of E. canis and B. vogeli in dogs from Colombia. Immunoglobulin-G (IgG) antibodies to E. canis and Babesia vogeli were found in 75 (82.4%) and 47 (51.6%) sampled dogs, respectively. Thirty-seven (40.6%) and 5 (5.5%) dogs were positive in PCR for E. canis and Babesia sp., respectively. After sequencing, amplicons showed 99% of identity with isolates of E. canis and B. vogeli. The phylogenetic trees based on 16S rRNA-Anaplasmataceae sequences and 18S rRNA-piroplasmid sequences supported the identity of the found E. canis and B. vogeli DNAs, respectively. The present work shows the first molecular detection of E. canis and B. vogeli in dogs in Colombia., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The present study aimed to assess antioxidant status and oxidative stress in sheep naturally infected with Babesia ovis. Red blood cell (RBC) count, hemoglobin (Hb) concentration, packed cell volume (PCV), piroplasm parasitemia percentage, malondialdehyde (MDA) concentration, erythrocyte superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) activities and total antioxidant capacity (TAC) were determined in 52 sheep naturally infected with B. ovis as well as same number of healthy sheep in West-Azerbaijan province, Iran. Microscopic examination of Giemsa-stained peripheral blood smears revealed B. ovis infection. The parasitological diagnosis was confirmed using polymerase chain reaction (PCR) analysis by amplifying a partial small subunit ribosomal RNA (ssu rRNA) gene sequence of B. ovis of 52 diseased sheep, 18 (34.61%), 11 (21.15%), 16 (30.76%) and 7 (13.48%) had <1%, 1-2%, 2-3% and >3% parasitemia, respectively. Compared to controls, the activities of erythrocyte GSH-Px, SOD, TAC and CAT showed a significant decrease, whereas the concentration of MDA in erythrocytes of infected sheep increased significantly. Parasitemia rate was positively correlated with MDA and negatively correlated with PCV, SOD, CAT, GSH-Px and TAC. Also, MDA was negatively correlated with PCV, SOD, catalase, GSH-Px and TAC. The study demonstrated that B. ovis plays an important role in the occurrence of oxidative damage to RBCs and anemia in ovine babesiosis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The aim of this study was to evaluate the platelet count, coagulation time and platelet activity in dogs experimentally infected with Rangelia vitalii during the acute phase of the disease. For this study, 12 young dogs (females) were used, separated in two groups. Group A (uninfected control) was composed by healthy dogs (n=5), and group B consisted of R. vitalii-infected animals (n=7). After being inoculated with R. vitalii-infected blood, animals were monitored by blood smear examinations, which showed intra-erythrocytic forms of the parasite five days post-inoculation (PI). Blood samples were collected on days 0, 10, 20 and 30 PI. The material collected was placed in tubes containing EDTA for quantification of platelets, citrate anticoagulant platelet aggregation, and measuring the clotting time. Right after blood collection on days 10 and 20 PI, dogs were anesthetized for collecting bone marrow samples. A significant reduction (P<0.01) of the number of platelets was observed in R. vitalii-infected blood, when compared with uninfected dogs on days 10 and 20 PI. Additionally, macro-platelets were observed only in infected dogs. Prothrombin time and activated partial thromboplastin time did not differ between infected and uninfected dogs. The megakaryocyte count increased (P<0.01) significantly in infected dogs when compared with uninfected ones on days 10 and 20 PI. Platelet aggregation decreased (P<0.01) significantly in infected dogs in comparison to the control on days 10 and 20 PI. Therefore, rangeliosis in dogs causes a severe thrombocytopenia during the acute phase of infection. This platelets reduction probably occurred due to splenic sequestration and/or immune-mediated thrombocytopenia., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Bovine babesiosis is a tick-transmitted hemoprotozoan disease that is mainly caused by Babesia bovis and Babesia bigemina and is characterized by significant morbidity and mortality worldwide. The disease is widespread in the northeastern region of Thailand, where an increasingly large part of the livestock is composed of water buffaloes. The present study was therefore conducted to investigate the epidemiological distribution of B. bovis and B. bigemina in water buffaloes in the northeastern region of Thailand. A total of 305 buffalo blood samples were randomly collected from five provinces and simultaneously analyzed by the nested PCR (nPCR) assay, ELISA, and IFAT techniques. The overall prevalence of B. bovis and B. bigemina was 11.2% and 3.6% by nPCR, 14.7% and 5.9% by ELISA, and 16.8% and 5.6% by IFAT, respectively. The high concordance between the molecular and the serological detection tests revealed the specificity and sensitivity of the diagnostic assays used for the detection of infection as well as the endemic stability status of the parasites in the surveyed areas. Statistically significant differences in the prevalence of the two infections were observed on the basis of age and location but not gender. Our data provide valuable information regarding the epidemiology of B. bovis and B. bigemina infection in water buffaloes in the northeastern region of Thailand which will likely be very beneficial for management and control programs of this disease., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The main objective of this study was to demonstrate the occurrence of equine piroplasmosis (EP) in horses of Lara State, Venezuela, and to correlate it with the factors host's sex and age in order to know the epidemiology of this disease at the Venezuelan Centroccidental Region. Antibody levels to Babesia caballi and Theileria equi were assessed in 360 equine serum samples, collected from 9 municipalities of Lara State, using an ELISA technique with recombinant antigens and monoclonal antibodies (Mabs). Antibodies to B. caballi were found in 254 horses (70.6%), whereas 181 animals (50.3%) were detected as seropositives to T. equi. In addition, 128 samples (35.56%) were seropositives to both hemoparasites. There were no significant differences between the seropositivity to B. caballi and T. equi with the factors sex and age of the horses. These results show that Lara State is an enzootic area for equine piroplasmosis, and are a contribution to a partial knowledge of the dynamic of this disease in Venezuela., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Ehrlichia canis and Babesia canis vogeli are two tick-borne canine pathogens with a worldwide importance. Both pathogens are transmitted by Rhipicephalus sanguineus, the brown dog tick, which has an increasing global distribution. A multiplex quantitative real-time PCR (qPCR) assay for the simultaneous detection of the tick-borne pathogens E. canis and B. canis vogeli was developed using dual-labeled probes. The target genes were the 16S rRNA of E. canis and the heat shock protein 70 (hsp70) of B. canis vogeli. The canine beta actin (ACTB) gene was used as an internal control gene. The assay was conducted without using any pre-amplification steps such as nested reactions. The sensitivity of each reaction in the multiplex qPCR assay was tested in the presence of high template concentrations of the other amplified genes in the same tube and in the presence of canine DNA. The detection threshold of the multiplex assay was 1-10 copies/μl in all channels and the amplifications of the B. canis hsp70 and ACTB were not affected by the other simultaneous reactions, while minor interference was observed in the amplification of the E. canis 16S rRNA gene. This assay would be useful for diagnostic laboratories and may save time, labor and costs., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Serum samples were collected from a total of 544 equids that included 524 horses, 13 mules, and 7 ponies from various regions of mainland Greece and were examined by competitive-inhibition ELISA (cELISA) to evaluate the level of exposure of Greek equids to Theileria (Babesia) equi and/or Babesia caballi, the causative agents of piroplasmosis. Association between seropositivity and host-related factors of species, gender, age, origin, activity and location were investigated. The overall seroprevalence was 11.6% (9.1-14.6%) with 95% confidence limit. The seroprevalence for T. equi and B. caballi was found to be 11% (8.6-14%) and 2.2% (1.2-3.9%), respectively. The animal-related factors significantly linked with seropositivity were the species, activities of farming, racing, recreation, and geographic location in Attica, Macedonia, Peloponnese and Thessaly region (p<0.05). The relative risks for the presence of T. equi, B. caballi and mixed infection in mules compared to horses was 8.39, 33.58 and 40.31, respectively. The infection level for T. equi, B. caballi and mixed infection were significantly higher in farm equids than in racing equids (p<0.05). Also, the rate of infection of T. equi was higher in farm equids than recreational equids (p<0.05). The relative risk of T. equi infection between farming equids and equids used only for recreation activity was 3.25-1, while the relative risk of B. caballi infection was 0.14-1 for racing animals relative to recreation animals. The region with the highest level of infection to both parasites was Thessaly (38.8% T. equi and 6.1% B. caballi), followed by Peloponnese (10.4% T. equi and 3.9% B. caballi), Attica region (8.3% T. equi and 0.6% B. caballi) and finally Macedonia the region with the lowest prevalence (6.6% T. equi and 4.4% B. caballi). A higher seroprevalence rate was found among local animals compared to imported equids, indicating that equine piroplasm infection is enzootic in Greece. T. equi seroprevalence was significantly different and higher among increasing age groups of equids, suggesting persistent infections or lower transmission levels whereby animals may need to be exposed longer before acquiring the infection. Competent tick vectors Rhipicephalus bursa and Rhipicephalus sanguineus for the transmission of equine piroplasmosis were recovered from horses and dogs, respectively., ((c) 2010 Elsevier B.V. All rights reserved.)

A detailed haematological study of dogs that were infected with low, moderate or high numbers of Babesia canis-infected red blood cells was performed in an attempt to elucidate the pathogenesis early after B. canis infection. Results showed that upon infection the C-reactive protein (CRP) level in plasma increased prior to the detection of parasites in the blood indicative of an acute phase reaction. The response was further characterised by fever, fibrinogenaemia, thrombocytopenia and leucopoenia. Thrombocytopenia was associated with increased coagulation time. Infected dogs also developed life threatening hypotension, and dogs that were infected with the highest dose of B. canis-infected red blood cells had to be treated chemotherapeutically. Hypotension was associated with a reduced packed cell volume (PCV). This reduction of PCV correlated with reduced plasma creatinin concentration, suggesting that the plasma volume was increased, affecting both the erythrocyte and creatinin concentration in the plasma. Importantly, the onset of the response but not the dynamics of the response was dependent on the infectious dose i.e. curves obtained with different doses of infected erythrocytes appeared to be shifted in time but had a similar shape. This indicates that infection triggered a preset inflammatory response.

A gene encoding a 29-kDa protein of Babesia orientalis (BoP29) was isolated by immunoscreening the B. orientalis cDNA expression library using monoclonal antibodies and its nucleotide sequence of the cDNA was 971bp with an open reading frame of 750bp. The results of BLASTn and BLASTx in NCBI showed that this cDNA sequence had similarity with Babesia bovis hypothetical protein (BBOV&#95;II005480). Western blotting showed that the recombinant protein (rBoP29) had positive seroreactivity to B. orientalis infected buffalo sera. An indirect enzyme-linked immunosorbent assay (ELISA) was developed using the rBoP29 as antigen. This method could detect specific antibody variation in the sera from the buffaloes experimentally infected with B. orientalis. Analysis of 132 sera samples collected from buffaloes showed that there was no significant difference in relative effectiveness of rBoP29-ELISA and PCR (chi(2)=0.910; P=0.897) in identifying positive samples. These results indicate that the BoP29 is a novel antigen of B. orientalis, and rBoP29 might be a good antigen candidate for diagnosis of B. orientalis infection in buffalo.

Bovine babesiosis is caused by Babesia bovis and B. bigemina in Argentina. These protozoans are prevalent north of parallel 30 degrees S, where their natural vector Rhipicephalus (Boophilus) microplus is widespread. To prevent babesiosis outbreaks in endemic areas, an increasing population of 4-10-month-old calves are vaccinated with low virulence B. bovis R1A (BboR1A) and B. bigemina S1A (BbiS1A) strains. In non-endemic areas, an additional calf population is also vaccinated and boostered as adults, before they are relocated to R. microplus-endemic areas of the country. Serological tests are currently utilized not only to determine the status of natural Babesia spp. infections, but also to confirm the infection caused by vaccine strains. For this purpose, an indirect enzyme immunoassay (ELISA) based on the recombinant major surface antigen-2c (rMSA-2c) of B. bovis expressed in Escherichia coli, was standardized using sera from Babesia spp. experimentally infected cattle. ELISA(rMSA-2c) was validated using sera obtained weekly during 336 days from steers primed and boostered with BboR1A and/or BbiS1A on days 0 and 154, then compared with the immunofluorescent-antibody test (IFAT). Western blot (WB) protein analysis was used to confirm the specificity of the immune response to rMSA-2c. The sensitivity and specificity for ELISA(rMSA-2c) were 92 and 96% after the Babesia spp. priming and 88 and 73% after the boostering immunization, respectively. The sensitivity and specificity for IFAT were 99 and 90% after priming and 92 and 98% after boostering, respectively. Unlike IFAT, ELISA(rMSA-2c) detected a remarkable delayed booster response and a significant drop in specificity between 35 and 84 days after the booster immunization. Simultaneously, 87.5% of cattle boostered with B. bigemina showed cross-reactions in the ELISA(rMSA-2c), particularly between 63 and 77 days after the inoculation. A reaction against E. coli was observed, since bands of approximately 40 and/or 42kDa were detected using sera from cattle before and after Babesia spp. inoculations. ELISA(rMSA-2c) showed to be useful between 42 and 98 days after priming with Babesia spp. live vaccine to evaluate the success of infecting cattle. However, after boostering the test showed low specificity.

The aims of this study were to determine the presence of Babesia spp. in blood samples from Italian dogs with clinical signs compatible with tick-borne diseases by means of PCR-restriction fragment length polymorphism (RFLP) and describe the clinicopathological findings of dogs with Babesia infection. We evaluated the majority of canine babesiosis cases by means of clinical history, physical examination, hematological, biochemical, serum electrophoresis, urinalysis and hemostatic tests. Forty-five out of 164 canine blood samples studied were positive to Babesia PCR-RFLP with the following results: Babesia canis canis (n=34) and Babesia canis vogeli (n=11). The majority of B. c. canis infections were detected in Northern Italy (29.1%; 30/103). B. c. vogeli cases were detected mainly in Central and Southern Italy (16.3%; 10/61). Only one B. c. vogeli was detected in Northern Italy (0.9%; 1/103). Three positive samples to B. c. canis and four positive samples to B. c. vogeli were selected for sequencing of a fragment of the 18S rRNA gene (410bp) for further molecular characterization. The sequence obtained from all seven dogs was 99/100% homologous to sequences from B. c. canis and B. c. vogeli, respectively, present in GenBank. Sixty-two percent of dogs infected with B. c. canis had recently travelled on a hunting trip to East European countries. The main acute clinical signs were dehydration, apathy, anorexia and fever. The majority of dogs infected with B. c. canis presented at initial clinical examination mild to severe thrombocytopenia, hyperfibrinogenemia, mild to moderate normocytic-normochromic non-regenerative anemia, hemolysis and neutropenia. The urinalysis showed hemoglobinuria in 13/19 dogs suggesting intravascular hemolysis. Dogs with B. c. canis infection had high levels of C-reactive protein. Hypoalbuminemia was present in 17/26 dogs. The 11 cases of B. c. vogeli infection did not present a homogenous clinicopathological pattern. B. c. vogeli infections were observed in young dogs causing hemolytic anemia and in adult/old does that frequently presented predisposing factors such as splenectomy or immunocompromised conditions. In conclusion, this study demonstrates the presence of B. c. canis and B. c. vogeli in Italian sick dogs and differences in clinicopathological pattern in these two species of B. canis.

Canine babesiosis represents an important veterinary medical problem. This study describes the molecular characterization of babesial parasites detected in eight clinically suspected dogs from northern Portugal, affected by lethargy, muscle tremors, weight loss, pale mucous membranes, hyperthermia or red-coloured urine. Microscopic examination of peripheral blood smears showed large intraerythrocytic piroplasms morphologically compatible with Babesia canis in all eight animals. DNA was extracted from blood on filter paper, and a Babesia spp. infection confirmed by polymerase chain reaction (PCR) amplification of a 408bp fragment of the 18S rRNA gene. Analysis of PCR-derived sequences revealed that seven dogs were infected with B. canis canis and one with B. canis vogeli. This is the first molecular identification report of both the species B. canis and the subspecies B. canis canis and B. canis vogeli in dogs from Portugal.

A longitudinal study was conducted in Southeast Uganda for 14 months on 640 Zebu cattle kept under natural tick challenge, with a view to identifying clinical features for prediction of seroconversion to Anaplasma marginale, Babesia bigemina and Theileria parva infections. Physical examination, condition scoring and tick counts were undertaken on all cattle every 4 weeks. In addition, 5300 sera were collected and analysed for antibodies against A. marginale, B. bigemina and T. parva infections using the enzyme-linked immunosorbent assay (ELISA). The major clinical features compiled included weight loss, fever (rectal temperature), anaemia (packed cell volume), pallor of mucous membranes, lymph node enlargement, staring coat, diarrhoea and lacrymation. The risk factors included tick challenge at village level, sex, age, Rhipicephalus spp. density and Boophilus spp. density on individual animals. Using a binary logistic regression model, the clinical features and risk factors were analysed. The results suggest that increasing rectal temperature was associated with increased probability for seroconversion to A. marginale, while high level of Rhipicephalus spp. density and increasing packed cell volume (PCV) were significantly associated with reduced probability of seroconversion. Although statistically significant, none of the factors had large effects, with odds ratios (OR) of 0.87, 1.15 and 0.98 for Rhipicephalus spp. density, rectal temperature and PCV, respectively. For B. bigemina infection, a high level of Boophilus spp. density, anaemia and staring coat were significantly associated with increased probability of seroconversion (OR 1.50, 1.78, 1.37, respectively). Presence of lacrymation and old age were associated with reduced probability of seroconversion (OR 0.52, 0.86 respectively). For T. parva infection, lymph node enlargement (OR 1.30) was associated with increased probability of seroconversion, while high Rhipicephalus spp. density and increasing packed cell volume (PCV) were associated with reduced probability of seroconversion (OR 0.68 and 0.98, respectively). In conclusion, presence and intensity of the respective tick vectors for tick-borne diseases, age and clinical features such as anaemia, fever, staring coat, lymph node enlargement and lacrymation are indicators for seroconversion to A. marginale, B. bigemina and T. parva infections in cattle. These indicators for seroconversion could be exploited in the development of decision support tools for clinical diagnosis of tick-borne diseases.

This prospective, interventional, case-controlled study sought to determine the association between adrenocortical function and mortality in dogs with naturally occurring Babesia rossi babesiosis. Sixty-eight dogs with canine babesiosis were studied and fifteen normal dogs were used as controls. Blood samples were obtained from the jugular vein in each dog prior to treatment, at admission to hospital, for the measurement of basal plasma ACTH (adrenocorticotrophic hormone) and serum cortisol concentrations. Immediately thereafter, each dog was injected intravenously with 5 microg/kg of ACTH (tetracosactrin). A second blood sample was taken 1h later for serum ACTH-stimulated cortisol measurement and the resultant calculation of delta cortisol by subtracting basal from ACTH-stimulated cortisol. Diagnosis of babesiosis was confirmed by polymerase chain reaction (PCR) and reverse line blot (RLB). Three outcomes were defined: hospitalization with subsequent death (n=4); hospitalization followed by recovery (n=48); and treatment as an outpatient (n=16). Basal cortisol, but not ACTH-stimulated cortisol, was significantly higher in patients compared to control dogs. Basal- and ACTH-stimulated serum cortisol concentrations were significantly higher in the dogs that died, compared to hospitalized dogs that survived and compared to dogs treated as outpatients. There was no significant difference in delta cortisol concentrations or cortisol to ACTH ratios across outcome groups in dogs suffering from B. rossi babesiosis However, dogs with delta cortisol concentrations below 83 nmol/l had significantly higher cortisol to ACTH ratios compared to dogs with delta cortisol concentrations above 83 nmol/l. These findings of increased basal- and ACTH-stimulated cortisol and increased cortisol to ACTH ratios confirm the absence of adrenal insufficiency and concur with those in human malaria.

Hypoglycaemia has been identified as a life-threatening metabolic complication in almost 20% of severely ill dogs suffering from babesiosis due to Babesia canis rossi infection, and has been correlated with mortality. Hyperinsulinaemia as a result of inappropriate insulin secretion may precipitate hypoglycaemia, and has been suggested as a possible cause of hypoglycaemia in human and murine malaria. This prospective, cross-sectional, observational study, including 94 dogs with naturally occurring virulent babesiosis, sought to identify the presence of inappropriate insulin secretion in hypoglycaemic canine babesiosis. Pre-treatment jugular blood samples were collected for simultaneous determination of plasma glucose and insulin concentrations. Animals were retrospectively divided into three groups: hypoglycaemic (BG<3.3 mmol/L; n=16), normoglycaemic (BG 3.3-5.5 mmol/L; n=62), and hyperglycaemic (BG>5.5 mmol/L; n=16). The median insulin concentrations for the hypoglycaemic, normoglycaemic, and hyperglycaemic groups were 10.7 pmol/L, 10.7 pmol/L, and 21.7 pmol/L, respectively. Statistical analysis revealed no significant difference in insulin concentration between the three groups. Additionally, the median insulin concentration in the hypoglycaemic and normoglycaemic groups was below the detection limit of the assay, suggesting that insulin secretion was appropriately low (i.e. undetectable) in these cases. Only two dogs had inappropriately elevated insulin concentrations. One of these dogs was hypoglycaemic. We conclude that hyperinsulinaemia is an infrequent cause of hypoglycaemia in virulent canine babesiosis. Other causes of hypoglycaemia, such as increased glucose consumption, depletion of hepatic glycogen stores, and hepatic dysfunction with impaired gluconeogenesis, are speculated to play more important roles in the pathophysiology of hypoglycaemia in canine babesiosis.

In this study, we developed two immunochromatographic tests (ICTs), which are nitrocellulose membrane-based immunoassays for the convenient and rapid serodiagnosis of bovine babesiosis caused by Babesia bovis (BoICT) and Babesia bigemina (BiICT). The efficacy of two ICTs was evaluated using 13 positive sera from experimentally infected cattle with B. bovis or B. bigemina. Clear results showed that the BoICT and ELISA detected antibodies in sera collected from 14 to 93 days post-infection, while BiICT and ELISA detected from 13 to 274 days post-infection. In additon, non-infected cattle, Neospora caninum, and Cryptosporidium parvum were negative in two ICTs. To evaluate the field utility of the ICTs, we tested 186 field bovine sera collected from cattle living in Yanbian (China) and Mato Grosso do Sul (Brazil). The results of ICTs were compared to those of classical serodiagnostic methods, enzyme-linked immunosorbent assay (ELISA) and the indirect immunofluorescence assay (IFAT). The overall concordances of BoICT were determined as 92.5 and 90.3% when the results of ELISA and IFAT were set as the reference standards, respectively. In contrast, those of BiICT showed 96.8 and 92.5% relative to the results of standard ELISA and IFAT, respectively. Conventional and rapid diagnosic devices for bovine babesiosis may provide a valuable tool in clinical and field applications.

This prospective, cross-sectional, observational study was designed to determine the association between the hormones of the pituitary-adrenal and pituitary-thyroid axes and outcome in dogs with naturally occurring Babesia canis rossi babesiosis. Ninety-five dogs with canine babesiosis were studied and blood samples were obtained from the jugular vein in each dog prior to treatment at admission to hospital. Serum cortisol, adrenocorticotrophic hormone (ACTH), thyroxine, free thyroxine and thyrotropin (TSH) concentrations were measured. Diagnosis was confirmed by polymerase chain reaction and reverse line blot and dogs infected with Babesia canis vogeli or Ehrlichia canis were excluded. Three outcomes were defined: hospitalization with subsequent death (n=7); hospitalization followed by recovery (n=56); and treatment as an outpatient (n=32). Serum cortisol and ACTH concentrations were significantly higher in the dogs that died, compared to hospitalized dogs that survived and compared to dogs treated as outpatients. Serum T4 and free T4 concentrations were significantly lower in the dogs that died, compared to the hospitalized dogs that survived and compared to dogs treated as outpatients. Serum TSH concentrations were not significantly different between any of the groups. Mortality was significantly associated with high cortisol and high ACTH concentrations and with low T4 and fT4 concentrations in dogs suffering from B. canis rossi babesiosis.

In the presented study we evaluated the hematological changes in samples of blood obtained from 248 dogs naturally infected with large Babesia. The evaluation included red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), leucocyte counts, thrombocyte counts, mean platelet volume (MPV), morphology of erythrocytes and leucogram. The most common disorders in affected dogs were thrombocytopenia and anisocytosis. The count of erythrocytes below reference values was detected in 26.2% of dogs and 31.4% of affected animals presented hematocrit below the reference values. Hemoglobin concentration below the reference values was noted in 29% of dogs, an increase of MCHC above normal values was detected in 21% of examinated dogs and MCV below normal values was recognized in 2% of dogs. 60.5% of dogs presented anisocytosis, 25% poikilocytosis, 23.8% polychromasia, 19.7% hypochromia and 4.4% erythroblastosis. Thrombocytopenia was detected in 99.5% of dogs, but only 15.3% of examined animals showed increase of MPV, which suggests a response of the bone marrow. 36.3% of dogs had neutropenia, and 21.8% presented a left shift, 14.9% had the lymphocytosis and 7.2% lymphopenia.

The erythrocyte sedimentation rate (ESR), white blood cell count (WBC), haematocrit (HCT) and platelet number (PLT) were quantified and compared with the acute phase proteins (APPs) in dogs naturally infected with Babesia canis and healthy dogs. Both groups were treated with imidocarb dipropionate on the day of admission and both groups were monitored for all parameters on the admission day and on the first, second, third, fourth and seventh days in order to determine the presence of an acute phase reaction, to assess the diagnostic value of these markers in uncomplicated canine babesiosis and to evaluate the use of APPs in treatment monitoring. It was demonstrated that an acute phase response occurs in dogs naturally infected with Babesia canis, with significant increases in the concentration of major acute phase proteins. The serum concentration of C-reactive protein (CRP), serum amyloid A (SAA) and the erythrocyte sedimentation rate (ESR) decreased daily after treatment and approached reference range values by the eighth day. PLT and haematocrit (HCT) increased daily after treatment and approached reference range values by the fourth day. WBC and haptoglobin increased after treatment and then decreased from the third and fourth days, respectively, to the eighth day. The diagnostic sensitivity of CRP, SAA and PLT was significantly higher compared to haptoglobin, ESR, HCT and the WBC count. CRP and SAA were of clinical use in monitoring the response to antibabesial treatment.

As a continuation of our studies on molecular epizootiology of piroplasmosis in Spain and other countries, we present in this contribution the finding of new hosts for some piroplasms, as well as information on their 18S rRNA gene sequences. Genetic data were complemented with sequences of apocytochrome b gene (whenever possible). The following conclusions were drawn from these molecular studies: Theileria annulata is capable of infecting dogs, since it was diagnosed in a symptomatic animal. According to cytochrome b sequences, isolates from cows and dog present slight differences. The same isolates showed, however, identical sequence in the 18S rRNA gene. This exemplifies well the usefulness of the mitochondrial gene for examining infra-specific variation. Babesia bovis is an occasional parasite of equines, since it was detected in two symptomatic horses. We found evidence of genetic polymorphism occurring in the 18S rRNA gene of Spanish T. equi-like and B. ovis isolates. B. bennetti from Spanish seagull is loosely related to B. ovis, and might represent a genetically distinct branch of babesids. A partial sequence of a cytochrome b pseudogene was obtained for the first time in Babesia canis rossi from South Africa. The pseudogene is distantly related to B. bigemina cytochrome b gene. These new findings confirm the ability of some piroplasms to infect multiple hosts, as well as the existence of a relatively wide genetic polymorphisms with respect to the cytochrome b gene. On the other hand, the existence of mtDNA-like pseudogenes of possible nuclear location in piroplasms is interesting due to their possible impact on molecular phylogeny studies.

This observational study of 100 dogs naturally infected with Babesia canis rossi determined whether severity of parasitaemia was associated with outcome of infection and documented the relative distribution of parasitised red blood cells (pRBC) in capillary and venous circulation. The association between increased parasitaemias and outcome with a clinically compromised circulation was also investigated. Outcome was defined as either hospitalisation with death, or hospitalisation with eventual recovery or treatment as an outpatient. Dogs were enrolled if large babesias were found on stained thin capillary blood smears made from an ear prick. Thin venous smears were prepared from jugular or cephalic blood. Parasitaemias were manually counted and expressed as the percent pRBC. Ten dogs died, 50 recovered after hospitalisation and 40 were treated as outpatients. Venous sampling site did not affect venous parasitaemia (P=0.6). Both capillary and venous parasitaemias of dogs that died were significantly higher than those of dogs that recovered after hospitalisation (P=0.002) and dogs that were treated as outpatients (P<0.0001). When assessing the whole group, capillary parasitaemia (median 0.61%, range <0.05-71.6%, interquartile range (IQR) 0.22-3.75%) was significantly higher than venous parasitaemia (median 0.14%, range 0-30.6%, IQR 0.046-0.52%) with P<0.0001. The 21 dogs with a clinically compromised circulation were more likely to die (P<0.0001) and had significantly higher capillary (median 5.98%, range 0.09-71.6%, IQR 2.44-19.41%) and venous (median 2.81%, range <0.05-30.6%, IQR 0.17-9.03%) parasitaemias than the 79 dogs with a clinically normal circulation (capillary median parasitaemia 0.38%, range <0.05-12.87%, IQR 0.16-1.42%; venous median parasitaemia 0.096%, range 0-6.13%, IQR <0.05-0.33%; P<0.0001). This study shows that high parasitaemia is significantly associated with death in B c rossi infected dogs. The previous clinical suspicion that capillary parasitaemias are usually higher than venous parasitaemias is confirmed. Thus capillary samples are the most appropriate diagnostic samples. Prior observations that a clinically compromised circulation is associated with death are confirmed. Despite the highly significant association between compromised circulation and higher parasitaemia, it is thought unlikely that parasite burden is the sole trigger for circulatory collapse.

Blood smear examination, flow cytometry, duplex Polymerase Chain Reaction (PCR), and duplex nested PCR (nPCR) were evaluated for detection of Babesia bigemina and Babesia bovis infections in cattle vaccinated with live attenuated strains. Two groups of four cattle were immunized with either B. bigemina (Bi) or B. bovis (Bo). On day 23 post inoculation (PI), Bi cattle were vaccinated with B. bovis (BiBo) and Bo cattle were vaccinated with B. bigemina (BoBi). Babesia bigemina was first detected by blood smear examination 7.5+/-3.5 days PI in the Bi group and 32.2+/-1.7 days PI in the BoBi group. The first occurrence of B. bovis in blood smears was 8.0 days PI in the Bo group and 36.0+/-2.6 days PI in the BiBo group. Flow cytometry detected parasitized erythrocytes on day 1.7+/-1.5 and 2.2+/-1.5 PI in the Bi and Bo groups, respectively, but did not discriminate between the two Babesia spp. Duplex PCR detected B. bigemina on day 4.0+/-0.8 and 26.0+/-0.8 PI in the Bi and BoBi groups, respectively, and B. bovis on day 4.0 and 25.3+/-0.5 PI in the Bo and BiBo groups, respectively. The duplex nPCR detected B. bigemina on 3.0+/-0.8 and 25.0+/-0.0 days PI in the Bi and BoBi groups, respectively, and 4.7+/-1.7 and 27.7+/-6.2 days PI in the Bo and BiBo groups, respectively. Duplex nPCR outperformed the other tests in terms of specificity and sensitivity, indicating that it is the most useful method for identifying Babesia spp. in cattle following vaccination.

Babesiosis is a world-wide zoonosis caused by tick-borne hematozoan parasites of the genus Babesia. Canine Babesidae have historically been classified as "large Babesia" (Babesia canis) and "small Babesia" (Babesia gibsoni) based on the size of their intraerythrocytic forms. Genetic sequencing technology using the polymerase chain reaction (PCR) has allowed further subdivision. B. gibsoni has three strains: "Asia", "California" and a recently identified small babesial-like parasite, Theileria annae. This newly recognised piroplasm appears to be hyperendemic in northwest Spain. In order to provide some insight into the situation, all the cases diagnosed in our laboratory (NW of Spain) during 2003 were evaluated. Our study (62 samples) shows the existence of a piroplasm morphologically different from B. canis and similar to B. gibsoni, which is genetically related to T. annae. Severe regenerative anemia and thrombocytopenia are almost constant characteristics of infection with T. annae in dogs. In many cases azotemia is found. Abnormally high serum concentrations of urea and creatinin, together with elevated concentrations of inorganic phosphorus, hypoalbuminemia, hypercholesterolemia, proteinuria, high protein/creatinin and presence of hyaline and granular casts in the microscopic examination of urine sediment suggest a glomerular component to the disease. We conclude that observational research and clinical trials should be conducted in order to improve our understanding of this emerging disease in order to provide some insight into the best therapeutic practices.

There are few extensive studies about clinicopathological findings of spontaneous canine babesiosis caused by a large form of the parasite found in Europe. To further characterize and describe clinicopathological findings in dogs affected with this large form of Babesia in northeastern Italy, we evaluated 23 Italian dogs with canine babesiosis by means of clinical history, physical examination, hematological, biochemical, hemostatic tests, serum electrophoresis and urinalysis. Seventeen dogs (74%) had recently traveled on a hunting trip (within 5-15 days of being presented to the clinic) to Bosnia and Herzegovina (n=7), to Croatia (n=8) and to Hungary (n=2). The duration of clinical signs ranged from 1 to 5 days prior to the arrival at the clinic. The main clinical signs were dehydration (100%), apathy (74%), anorexia or decrease appetite (70%) and fever (68%). The anemia was present in 74% of the dogs and classified as mild (35%), moderate (59%) and severe (6%). In all cases, the anemia was normocytic and normochromic. Only three dogs presented erythrocyte regeneration. Seventy percent of dogs had hemolytic anemia and 30% had non-hemolytic anemia. Sixty-nine percent of dogs showed leucopenia and 74% neutropenia. Leucocitosis, due to mature neutrophilia and lymphocytosis, was present in one dog. Activated lymphocytes were noted in 61% of dogs. In all dogs, thrombocytopenia and an elevated hyperfibrinogenemia were present. Significant prolonged activated partial thromboplastin time (aPTT) was only found in one case. In four dogs, both plasma fibrinogen/fibrin degradation products (FDPs) and D-Dimer were increased. Antithrombin (AT) was slightly decreased in 11 of the 23 dogs. In the majority of cases, mild elevation of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatinekinase (CK), total bilirubin and lactic acid and decrease of total iron and total iron binding capacity (TIBC) were present. In conclusion, the main clinicopathological findings were a mild to severe thrombocytopenia, a mild to moderate hemolytic anemia, neutropenia and hyperfibrinogenemia.

In this study, a pair of oligonucleotide primers were designed according to the nucleotide sequence of the small subunit ribosomal RNA (ssu rRNA) gene of Babesia ovis isolated from sheep in eastern Turkey. The primers were used to detect parasite DNA from blood samples of B. ovis-infected sheep and goats by polymerase chain reaction (PCR). A 549-bp DNA fragment was specifically amplified from blood samples from sheep and goats, naturally infected with B. ovis. No PCR products resulted from Babesia motasi, T. ovis, Theileria sp. OT1, Theileria sp. OT3, T. lestoquardi, B. canis, B. microti,T. annulata or normal sheep leucocytes DNA using these specific primers. B. ovis-infected erythrocytes with 1% parasitemia were subjected to 10-fold serial dilutions (from 10(-1) to 10(-9)) using an uninfected sheep erythrocytes, and DNA was extracted from each diluted sample for testing the sensitivity of the PCR. The PCR was sensitive enough to detect parasite DNA from the dilution of 10(-5) with 0.00001% parasitemia. This is more sensitive than examining 200 fields under light microscopy. In addition, 98 field samples collected from small ruminanats in eastern Turkey were tested for B. ovis infection. Four samples were positive Babesia spp. in blood smears, 21 samples were positive for B. ovis DNA by PCR. These results indicate that the PCR provides a useful diagnostic tool for the detection of B. ovis infection in sheep and goats.

Blood samples and ticks were collected from 48 cattle and 74 horses from seven sites in the Peten region of Guatemala. Data on body condition, mucous membrane capillary refill time and tick infestation levels were recorded for each animal in the study. Horses had significantly higher levels of tick infestation than cattle, as well as poorer body condition scores. Seroprevalence of Babesia spp. was 95.8% for B. bovis in cattle, 89.6% for B. bigemina in cattle, and 92.7% for B. equi in horses. Seroprevalence of Anaplasma marginale in cattle was 87.5%, similar to reports in animals from other regions of Central America. This is the first time that A. phagocytophilum has been reported in animals from this region, with overall PCR-prevalence of 27.6% in cattle and horses, and seroprevalence of 28.4% (52% in cattle and 13% in horses). An agent was identified with serological cross-reactivity and close genetic relatedness to Ehrlichia ruminantium, but further testing confirmed that the agent in Guatemalan cows was not the agent of heartwater. Ticks were identified to species with the predominant species identified on cattle as Boophilus microplus and Amblyomma cajennense, while Anocentor nitens and A. cajennense were most commonly found on horses. Prevalence of infection, tick infestation levels, host factors and environmental data were analyzed for association; A. nitens was significantly associated with A. phagocytophilum prevalence by village.

Babesia spp. infections were investigated in Bos taurus x Bos indicus dairy cows and calves and in Boophilus microplus engorged female ticks and eggs. Blood samples and engorged female ticks were collected from 25 cows and 27 calves. Babesia spp. was detected in ticks by microscopic examination of hemolymph of engorged female and by squashes of egg samples. Cattle infection was investigated in blood thin smears and by DNA amplification methods (PCR and nested PCR), using specific primers for Babesia bovis and Babesia bigemina. Merozoites of B. bovis (3 animals) and B. bigemina (12 animals) were detected exclusively in blood smears of calves. DNA amplification methods revealed that the frequency of B. bigemina infection in calves (92.6%) and in cows (84%) and of B. bovis in calves (85.2%) and in cows (100%) did not differ significantly (P > 0.05). Babesia spp. infection was more frequent in female ticks and eggs collected from calves (P < 0.01) than from cows, especially in those which had patent parasitemia. Hatching rates of B. microplus larvae were assessed according to the origin of engorged females, parasitemia of the vertebrate host, frequency and intensity of infection in engorged female tick, and frequency of egg infection. Hatching rate was lower in samples collected from calves (P < 0.01) than from cows, and in those in which Babesia spp. was detected in egg samples (P < 0.01).

With the aim of developing more simple diagnostic alternatives, a differential single-round and multiplex polymerase chain reaction (PCR) method was designed for the simultaneous detection of Babesia caballi and Babesia equi, by targeting 18S ribosomal RNA genes. The multiplex PCR amplified DNA fragments of 540 and 392 bp from B. caballi and B. equi, respectively, in one reaction. The PCR method evaluated on 39 blood samples collected from domestic horses in Mongolia yielded similar results to those obtained from confirmative PCR methods that had been established earlier. Thus, the single-round and multiplex PCR method offers a simple tool for the differential diagnosis of B. caballi and B. equi infections in routine diagnostic laboratory settings as well as in epidemiological studies.

The purpose of this study was to demonstrate the occurrence of equine piroplasmosis in Mongolia, a country in which the disease occurs epidemically in different climatic conditions. Antibodies to Babesia equi and B. caballi were determined in serum samples of 254 pastured horses in different locations of Mongolia using an enzyme-linked immunosorbent assay with recombinant antigens. One hundred and eighty-five (72.8%) and 102 (40.1%) of all serum samples were positive for B. equi and B. caballi infections, respectively. In addition, 78 (30.7%) samples were positive for both B. equi and B. caballi infections. These results indicate that equine piroplasmosis is widespread in Mongolia. To our knowledge, this is the first report describing an epidemiological study on equine piroplasmosis in different geographic regions in Mongolia.

The present work describes the detection and first molecular characterization of Babesia vogeli in dogs, naturally infected in Brazil and even in South America. Microscopic examination of Giemsa-stained peripheral blood smears collected from dogs originating from four different locations in Brazil revealed the presence of large Babesia merozoites and trophozoites (>2.5 microm). DNA was extracted from infected blood samples and PCR amplifications of the 18S rDNA were carried out. As a reference, DNA from an isolate of B. vogeli originated from Egypt was used. PCR products were purified and sequenced. The DNA sequences demonstrated 100% identity among the Brazilian isolates. Comparisons with the 18S rDNA sequence of the B. vogeli isolate from Egypt and with other B. vogeli sequences from Spain, France, Japan, Australia and South Africa confirmed the affiliation of all Brazilian isolates to the species B. vogeli.

Giemsa-stained thin blood smears prepared monthly from cattle, sheep and goats in the Greater Accra region of Ghana between May 1994 and December 1996 were examined for presence of tick-borne haemoparasites. The majority of animals were less than 2 months old at the start of the survey. Monthly and cumulative incidences are presented of Anaplasma sp., Babesia bigemina, Borrelia sp., Eperythrozoon sp., Theileria mutans and Theileria velifera in cattle, Anaplasma sp., Borrelia sp., and Theileria sp. in sheep, and Anaplasma sp. in goats. T. mutans was the commonest parasite in cattle, with 100% incidence in calves by 10 months of age, and Anaplasma was commonest in small ruminants. The relative prevalence of these haemoparasites in blood smears from cattle, sheep and goats sampled on a single occasion at sites in all 10 regions of Ghana was found to be similar, though actual infection rates were lower. Packed cell volume (PCV) measurements from the sampled animals are also presented; no seasonal trends were evident in the PCV of the cattle, sheep and goats sampled monthly. In animals sampled on a single occasion, mean PCV was significantly higher in cattle and sheep without detectable haemoparasite infection, and in cattle was lowest in animals positive for both Babesia and Anaplasma, while there was no difference in mean PCV levels between parasitised and non-parasitised goats.