Molecular Detection of Animal Trypanosomes in Different Animal Species in the Visayas Region of the Philippines.

PURPOSE: Animal trypanosomosis is one of the most important parasitic diseases significantly affecting the Philippine economy. It is considered by the government to be the second most important disease of livestock after fasciolosis. A PCR-based molecular survey for trypanosomes in different animals in Bohol, Philippines, was performed to assess the prevalence of trypanosomosis in the area during the rainy and dry season. METHODS: A total of 269 blood samples were collected in two batches in rainy and dry season from different animal species in Ubay Stock Farm in Ubay, Bohol, the Philippines, including 151 samples from water buffaloes, 76 samples from cattle, 35 samples from goats, and 7 samples from horses. DNA was subsequently extracted from these blood samples, and two different PCR assays were employed to detect and identify trypanosomes DNA including ITS1 PCR and CatL PCR. RESULTS: Animal trypanosomes, Trypanosoma evansi and Trypanosoma theileri, were detected in water buffalo (37.7%) [95%CI: 30.4 - 45.7], cattle (44.7%) [95%CI: 34.1 - 55.9], and goats (34.3%) [95%CI: 20.8 - 50.8]. Only T. evansi was detected in horses (28.6%) [95% CI: 8.2 - 64.1]. No clinical signs were observed in all positive animals. CONCLUSION: This highlights the importance of domestic animals that can be infected with no signs but may act as reservoir animals and transmit trypanosomosis to susceptible animals. This study supports the importance of regular surveillance to estimate the prevalence of the disease, emphasizing its various dynamics in the affected areas and supporting efficient intervention measures.

Molecular Detection and Identification of Babesia spp., Theileria spp., and Anaplasma spp. in Sheep From Border Regions, Northwestern China.

Babesia, Theileria, and Anaplasma are important causative agents of tick-borne diseases that severely affect sheep. However, there is paucity in the occurrence genetic diversity of the infections of tick-borne diseases in sheep in border regions, northwestern China. In this study, nested polymerase chain reaction (nPCR) assays and gene sequencing were used to identify tick-borne Babesia spp., Theileria spp., and Anaplasma spp. infections in border regions, northwestern China. Out of 323 samples tested in this study, 225 (69.7%) sheep were infected with Babesia spp., Theileria spp., and Anaplasma spp. Two hundred six (63.8%), 60 (18.6%), 54 (16.7%), 51 (15.8%), 32 (9.9%), 19 (5.9%), and 16 (5.0%) were positive for A. ovis, B. motasi-like, A. bovis, T. uilenbergi, A. phagocytophilum, T. luwenshuni, and B. motasi-like Xinjiang, respectively. The most common dual infection was with A. ovis and B. motasi-like while the most frequent triple coinfection was A. ovis, B. motasi-like, and T. uilenbergi with coinfection rates of 17.0% (55/323) and 5.0% (16/323), respectively. Sequencing analysis indicated that A. ovis MSP4, A. phagocytophilum epank1, A. bovis 16S rRNA, B. motasi-like rap1-b, B. motasi-like Xinjiang rap1-a, T. luwenshuni 18S rRNA, and T. uilenbergi 18S rRNA from border regions, northwestern China, showed 99-100% identity with documented isolates from other countries. To the best of the authors' knowledge, this is the first report of T. uilenbergi and T. luwenshuni infections of sheep in border regions, northwestern China. Furthermore, these findings provide important data for understanding the distribution of Babesia, Theileria, and Anaplasma in sheep between border countries and China.

Development and evaluation of an enzyme-linked immunosorbent assay based on recombinant TgSRS2 for serodiagnosis of Toxoplasma gondii infection in cats.

An enzyme-linked immunosorbent assay (ELISA) based on recombinant SAG1-related sequence 2 of Toxoplasma gondii (rTgSRS2) was developed to detect toxoplasmosis in cats. The specificity and sensitivity of rTgSRS2 ELISA were confirmed using a series of serum samples from T. gondii-experimentally infected mice. A total of 76 field samples from cats were examined by the developed ELISA. The rTgSRS2 ELISA showed a good diagnostic performance characterized by high concordance (88.16) and kappa value (0.76) with latex agglutination test (LAT). The sensitivity and specificity of the test were 92.68% and 82.86%, respectively. These results suggest that the ELISA based on rTgSRS2 could be a useful tool for serodiagnosis of T. gondii infection in cats.

First molecular detection and identification of Trypanosoma evansi in goats from Cebu, Philippines using a PCR-based assay.

The increasing number and severity of surra outbreaks in the Philippines led the government to consider it as the second most important disease of livestock in the country. It is one of the most economically important animal parasitic diseases and has been reported in several animal species, including water buffaloes, cattle, and horses in different regions of the Philippines. However, it has not yet been reported in Cebu, the usual gateway of livestock trade in the area that raises 6% of the 3.75 million goats in the country. In the current study, a PCR-based assay was conducted for the molecular detection and characterization of Trypanosoma evansi in goats in Cebu. A total of 251 goats were randomly sampled from four farms. DNA was extracted and ITS1-PCR was applied to detect different trypanosomes in goats. Eighty-five out of the 251 (33.9%) samples tested positive for T. evansi, two of which were also positive for T. theileri-like trypanosome. The detection rate of T. evansi was slightly higher in male goats (38.3%) than in females (32.5%), and in younger goats (34.5%) than in adults (33.5%). The findings, however, did not differ significantly to suggest any association between sex and age with T. evansi infection in goats. The detection of T. evansi and T. theileri-like trypanosome in goats was confirmed by sequence analysis of ITS1 region. To our knowledge, this is the first report on the molecular detection and identification of caprine T. evansi infection in Cebu, Philippines.

Subolesin vaccination inhibits blood feeding and reproduction of Haemaphysalis longicornis in rabbits.

BACKGROUND: Ticks can transmit numerous tick-borne pathogens and cause a huge economic loss to the livestock industry. Tick vaccines can contribute to the prevention of tick-borne diseases by inhibiting tick infestation or reproduction. Subolesin is an antigenic molecule proven to be a potential tick vaccine against different tick species and even some tick-borne pathogens. However, its effectivity has not been verified in Haemaphysalis longicornis, which is a widely distributed tick species, especially in East Asian countries. Therefore, the purpose of this study was to evaluate the effectivity of subolesin vaccination against H. longicornis in a rabbit model. METHODS: Haemaphysalis longicornis (Okayama strain, female, adult, parthenogenetic strain) and Japanese white rabbits were used as the model tick and animal, respectively. The whole open reading frame of H. longicornis subolesin (HlSu) was identified and expressed as a recombinant protein using E. coli. The expression was verified using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the immunogenicity of rHlSu against anti-H. longicornis rabbit serum was confirmed using Western blotting. After vaccination of rHlSu in rabbits, experimental infestation of H. longicornis was performed. Variables related to blood-feeding periods, pre-oviposition periods, body weight at engorgement, egg mass, egg mass to body weight ratio, and egg-hatching periods were measured to evaluate the effectiveness of subolesin vaccination. RESULTS: The whole open reading frame of HlSu was 540 bp, and it was expressed as a recombinant protein. Vaccination with rHlSu stimulated an immune response in rabbits. In the rHlSu-vaccinated group, body weight at engorgement, egg mass, and egg mass to body weight ratio were statistically significantly lower than those in the control group. Besides, egg-hatching periods were extended significantly. Blood-feeding periods and pre-oviposition periods were not different between the two groups. In total, the calculated vaccine efficacy was 37.4%. CONCLUSIONS: Vaccination of rabbits with rHlSu significantly affected the blood-feeding and reproduction in H. longicornis. Combined with findings from previous studies, our findings suggest subolesin has the potential to be used as a universal tick vaccine.

Molecular detection of tick-borne pathogens harbored by ticks collected from livestock in the Xinjiang Uygur Autonomous Region, China.

Ticks carry and transmit a wide range of pathogens (bacteria, viruses, and protozoa) that are of importance to humans and animals globally. However, information about the tick-borne pathogens harbored by ticks in the Xinjiang Uygur Autonomous Region (XUAR), northwestern China, is scarce. This study investigated the occurrence of tick species of domestic animals and tick-borne pathogens by using morphological molecular identification and sequence analysis in Turpan, Qitai, Altay, Hejing, Nileke, and Zhaosu counties (XUAR). A total of 5822 adult ticks (females and males) from 12 tick species were identified from 5 animal species (cattle, goats, sheep, camels, and horses) in 6 counties in the XUAR. Collected tick species included Dermacentor marginatus (24.7 %), Dermacentor nuttalli (20.8 %), Hyalomma anatolicum (13.7 %), Dermacentor niveus (13.1 %), Haemaphysalis punctata (10.7 %), Dermacentor silvarum (7.1 %), Dermacentor pavlovskyi (3.9 %), Hyalomma asiaticum (2.2 %), Rhipicephalus pumilio (1.9 %), Rhipicephalus sanguineus sensu lato (0.7 %), Rhipicephalus turanicus (0.6 %), and Hyalomma asiaticum kozlovi (0.6 %). Furthermore, 750 partially engorged adult ticks (females and males), including H. anatolicum (250), D. nuttalli (250), and D. marginatus (250), were individually separated according to species and sampling site, used for DNA extraction, and then screened for tick-borne pathogens. The most common pathogen was Rickettsia raoultii (36.80 %), followed by Brucella sp. (26.2 %), Anaplasma ovis (22.4 %), Babesia caballi (14.8 %), Theileria equi (8.7 %), and Theileria ovis (8.5 %). The sequencing of 6 genes showed a 96-100 % nucleotide identity between the sequences in this study and those deposited in GenBank. This study provides a scientific reference for the prevention and control of tick-borne diseases in the XUAR.

Babesia microti Confers Macrophage-Based Cross-Protective Immunity Against Murine Malaria.

Malaria and babesiosis, the two primary intraerythrocytic protozoan diseases of humans, have been reported in multiple cases of co-infection in endemic regions. As the geographic range and incidence of arthropod-borne infectious diseases is being affected by climate change, co-infection cases with Plasmodium and Babesia are likely to increase. The two parasites have been used in experimental settings, where prior infection with Babesia microti has been shown to protect against fatal malarial infections in mice and primates. However, the immunological mechanisms behind such phenomena of cross-protection remain unknown. Here, we investigated the effect of a primary B. microti infection on the outcome of a lethal P. chabaudi challenge infection using a murine model. Simultaneous infection with both pathogens led to high mortality rates in immunocompetent BALB/c mice, similar to control mice infected with P. chabaudi alone. On the other hand, mice with various stages of B. microti primary infection were thoroughly immune to a subsequent P. chabaudi challenge. Protected mice exhibited decreased levels of serum antibodies and pro-inflammatory cytokines during early stages of challenge infection. Mice repeatedly immunized with dead B. microti quickly succumbed to P. chabaudi infection, despite induction of high antibody responses. Notably, cross-protection was observed in mice lacking functional B and T lymphocytes. When the role of other innate immune effector cells was examined, NK cell-depleted mice with chronic B. microti infection were also found to be protected against P. chabaudi. Conversely, in vivo macrophage depletion rendered the mice vulnerable to P. chabaudi. The above results show that the mechanism of cross-protection conferred by B. microti against P. chabaudi is innate immunity-based, and suggest that it relies predominantly upon the function of macrophages. Further research is needed for elucidating the malaria-suppressing effects of babesiosis, with a vision toward development of novel tools to control malaria.

Drug screening of food and drug administration-approved compounds against Babesia bovis in vitro.

Babesia (B.) bovis is one of the main etiological agents of bovine babesiosis, causes serious economic losses to the cattle industry. Control of bovine babesiosis has been hindered by the limited treatment selection for B. bovis, thus, new options are urgently needed. We explored the drug library and unbiasedly screened 640 food and drug administration (FDA) approved drug compounds for their inhibitory activities against B. bovis in vitro. The initial screening identified 13 potentially effective compounds. Four potent compounds, namely mycophenolic acid (MPA), pentamidine (PTD), doxorubicin hydrochloride (DBH) and vorinostat (SAHA) exhibited the lowest IC50 and then selected for further evaluation of their in vitro efficacies using viability, combination inhibitory and cytotoxicity assays. The half-maximal inhibitory concentration (IC50) values of MPA, PTD, DBH, SAHA were 11.38 ± 1.66, 13.12 ± 4.29, 1.79 ± 0.15 and 45.18 ± 7.37 µM, respectively. Of note, DBH exhibited IC50 lower than that calculated for the commonly used antibabesial drug, diminazene aceturate (DA). The viability result revealed the ability of MPA, PTD, DBH, SAHA to prevent the regrowth of treated parasite at 4 × and 2 × of IC50. Antagonistic interactions against B. bovis were observed after treatment with either MPA, PTD, DBH or SAHA in combination with DA. Our findings indicate the richness of FDA approved compounds by novel potent antibabesial candidates and the identified potent compounds especially DBH might be used for the treatment of animal babesiosis caused by B. bovis.

Molecular investigation of tick-borne infections in cattle from Xinjiang Uygur Autonomous Region, China.

Tick-borne diseases cause significant losses to livestock production in tropical and subtropical regions. However, information about the tick-borne infections in cattle in Xinjiang Uygur Autonomous Region (XUAR), northwestern China, is scarce. In this study, nested polymerase chain reaction (PCR) assays and gene sequencing were used to detect and analyze epidemiological features of Babesia bovis, B. bigemina, Coxiella burnetii and Anaplasma bovis infections in XUAR. Out of 195 samples tested, 24 (12.3%), 67 (34.4%), 40 (20.5%) and 10 (5.1%) were positive for B. bovis, B. bigemina, C. burnetii and A. bovis, respectively. Sequencing analysis indicated that B. bovis SBP-4, B. bigemina Rap1a, C. burnetii htpB and A. bovis 16S rRNA genes from XUAR showed 99%-100% identity with documented isolates from other countries. Phylogenetic analyses revealed that B. bovis SBP-4, B. bigemina Rap1a, C. burnetii htpB and A. bovis 16S rRNA gene sequences clustered in the same clade with isolates from other countries. To the best of our knowledge, this is the first report of C. burnetii infection of cattle in XUAR. Furthermore, this study provides important data for understanding the distribution of tick-borne pathogens, and is expected to improve the approach for prevention and control of tick-borne diseases in China.

First description of Coxiella burnetii and Rickettsia spp. infection and molecular detection of piroplasma co-infecting horses in Xinjiang Uygur Autonomous Region, China.

Q fever, spotted fever rickettsioses and equine piroplasmosis, are some of the most serious equine tick-borne diseases caused by Coxiella burnetii, Rickettsia spp., Babesia caballi and/or Theileria equi. This study surveyed and molecularly characterized these pathogens infecting horses in ten ranches from XUAR, China using molecular technology. Among 200 horse blood samples, 163 (81.5%) were infected with at least one of the pathogens. Rickettsia spp. was the most prevalent pathogen (n = 114, 57.0%), followed by C. burnetii (n = 79, 39.5%), T. equi (n = 79, 39.5%) and B. caballi (n = 49, 24.5%). Co-infections were observed in 61.3% of positive samples in this study. Statistically significant differences were observed between the sampling regions for C. burnetii, B. caballi and T. equi, and also in different age group for C. burnetii and T. equi. The genotype analysis indicated that C. burnetii htpB, Rickettsia spp. ompA, B. caballi rap-1, B. caballi 18S rRNA, T. equi EMA-1 and T. equi 18S rRNA gene sequences from horses in XUAR were variable. To the best of our knowledge, this study is the first report of C. burnetii and Rickettsia spp. infection and co-infected with piroplasma in horses in China. Overall, this study revealed the high infection rate of the pathogens in horses in XUAR, China. The current findings are expected to provide a basis for better tick-borne disease control in the region.

Characterization of strain-specific phenotypes associated with knockout of dense granule protein 9 in Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of mammals and cause toxoplasmosis. Dense granule proteins play major structural functions within the parasitophorous vacuole (PV) and the cyst wall of T. gondii. Moreover, their particular location within the PV allows them to be involved in various interactions between parasites and the host cells. Dense granule protein 9 (GRA9) gene has been identified in T. gondii, although its role in the lytic cycle remains unclear. In the current study, the function of GRA9 in type I and type II Toxoplasma parasites was characterized. T. gondii GRA9 sequence and its expression were analyzed and derivatives of T. gondii RH and PLK strains with a null mutation in GRA9 were generated using CRISPR/Cas9 system. The phenotypes of GRA9 in wild types, knockout and complemented strains were analyzed in vitro and in vivo using Vero cells and BALB/c mice, respectively. Alignment of the amino acid sequence indicated that RH strain GRA9 contained one amino acid substitution when compared with PLK strain. Western blot analysis revealed that PLK strain had a higher expression level of GRA9 than RH strain. The phenotype analysis revealed that knockout of GRA9 in PLK parasites inhibited the plaque formation and egress from PV. Both the plaque formation and egress ability of PLKΔGRA9 strain were restored by complementation with a synonymous allele of PLK strain GRA9. Mouse experiments demonstrated that loss of GRA9 in PLK strain significantly reduced the pathogenicity of T. gondii. However, there was no phenotypic diferences between RH and RHΔGRA9 strains except the defect in host cell invasion. Overall, T. gondii GRA9 knockout only influenced the growth and virulence of PLK strain. These results indicate that GRA9 may be involved in parasite egress and virulence in mice in a strain-specific manner.

Differential diagnosis and molecular characterization of Theileria spp. in sika deer (Cervus nippon) in Hokkaido, Japan.

Sika deer (Cervus nippon) is widely distributed in Asian countries and is one of the most common wildlife animals in Hokkaido, Japan. Previous studies identified Theileria spp. in sika deer in Japan including Theileria sp. Thrivae belonging to T. cervi group and Theileria sp. sola belonging to T. capreoli group. However, the studies failed to differentiate these two species without sequencing. Therefore, epidemiological information on cervine theileriosis in Hokkaido, Japan is limited. This study differentiated the two Theileria spp. using restriction fragments length polymorphism (RFLP). Based on the PCR-RFLP, Theileria spp. were identified in 103 (88.0%) of 117 samples, and the prevalence of each parasites were 86.3% (n = 101) and 57.3% (n = 67) for Theileria sp. Thrivae and T. capreoli-like, respectively. Phylogenetic analysis based on the 18S rRNA showed a close relationship between Theileria sp. Thrivae and T. cervi in China. In addition, phylogenetic analysis of internal transcribed spacer regions also showed a close relationship between Theileria sp. Thrivae and T. cervi.

Evaluation of the protective effect of a prime-boost strategy with plasmid DNA followed by recombinant adenovirus expressing BmAMA1 as vaccines against Babesia microti infection in hamster.

In the present study, we have investigated the protective effect of a heterologous prime-boost strategy with priming plasmid DNA followed by recombinant adenovirus, both expressing BmAMA1, against Babesia microti infection. Four groups consisting of 3 hamsters per group were immunized with pBmAMA1/Ad5BmAMA1, pNull/Ad5BmAMA1, pBmAMA1/Ad5Null and pNull/Ad5Null, followed by challenge infection with B. microti. Our results showed that hamsters immunized with plasmid and adenovirus expressing BmAMA1 developed a robust IgG and IgG2a antibody response against BmAMA1, suggesting the DNA vaccine or viral vector vaccine tend to induce a Th1-biased response. Compared to the control hamsters, the hamsters vaccinated either with the prime-boost strategy or one of the two "vaccines" exhibited no significant protection against B. microti challenge. Although a slight difference in terms of parasitemia and hematocrit values at days 14-16 post challenge infection was observed, no other statistical difference was detected. Our results indicate that the prime-boost vaccination strategy of injection of plasmid and adenovirus expressing BmAMA1 is not efficient in protecting against B. microti infection.

A PCR survey of vector-borne pathogens in different dog populations from Turkey.

In the present study, a total of 192 blood samples were collected from pet dogs, kennel dogs and shepherd dogs in Konya district, Turkey, and tested by specific PCR for the presence of vector-borne pathogens. Several pathogens were identified, most of which can cause substantial morbidity in dogs. PCR results revealed that 54 (28.1%) dogs were infected with one or more pathogens. Positive results were obtained for Babesia spp. in 4 dogs (2.1%), Hepatozoon spp. in 8 dogs (4.2%) and Mycoplasma spp. in 46 dogs (24%). Three dogs (1.6%) were infected with two or three pathogens. The sequence analysis of the positive DNA samples revealed the presence of Babesia canis vogeli, Hepatozoon canis, Hepatozoon sp. MF, Mycoplasma haemocanis and Candidatus Mycoplasma haematoparvum. Ehrlichia canis and Anaplasma platys were not detected. Regardless of ownership status, vector-borne diseases were common in these dog populations. There was significant difference of pathogen prevalence among the different dog populations. Mycoplasma spp. was more frequent in the kennel dogs (31.9%) than in the pet (21.4%) and shepherd dogs (13.8%). Additionally, the frequency of Babesia spp. and Hepatozoon spp. was higher in the shepherd dogs which account for three quarters and half of the total number of Babesia spp. and Hepatozoon spp., respectively. To our knowledge, this is the first report of Mycoplasma infection in dogs in Turkey. The results of the present study provide a foundation for understanding the epidemiology of canine vector-borne diseases (CVBDs), and for strategies to control these diseases in Turkey.

Molecular detection and genetic characterization of Babesia, Theileria and Anaplasma amongst apparently healthy sheep and goats in the central region of Turkey.

Babesia spp., Theileria spp. and Anaplasma spp. are significant tick-borne pathogens of livestock globally. In this study, we investigated the presence and distribution of Babesia ovis, Theileria ovis and Anaplasma ovis in 343 small ruminants (249 sheep and 94 goats) from 13 towns in the Central Anatolia region of Turkey using species-specific PCR assays. The PCR were conducted using the primers based on the B. ovis ssu rRNA (BoSSUrRNA), T. ovis ssu rRNA (ToSSUrRNA) and A. ovis major surface protein 4 (AoMSP4) genes, respectively. Fragments of these genes were sequenced for phylogenetic analysis. PCR results revealed that the overall infections of A. ovis, T. ovis and B. ovis were 60.0%, 35.9% and 5.2%, respectively. Co-infection of the animals with two or three pathogens was detected in 105/343 (30.6%) of the ovine samples. The results of sequence analysis indicated that AoMSP4 were conserved among the Turkish samples, with 100% sequence identity values. In contrast, the BoSSUrRNA and ToSSUrRNA gene sequences were relatively diverse with identity values of 98.54%-99.82% and 99.23%-99.81%, respectively. Phylograms were inferred based on the BoSSUrRNA, ToSSUrRNA and AoMSP4 sequences obtained in this study and those from previous studies. B. ovis isolates from Turkey were found in the same clade as the isolates from other countries in phylogenetic analysis. On the other hand, the Turkish T. ovis isolates in the present study formed a monophyletic grouping with the isolates from other countries in a phylogeny based on ToSSUrRNA sequences. Furthermore, phylogenetic analysis using AoMSP4 sequences showed the presence of three genotypes of A. ovis. This study provides important data for understanding the epidemiology of tick-borne diseases in small ruminants and the degree of genetic heterogeneities among these pathogens in Turkey. To our knowledge, this is the first study on the co-infection of Babesia, Theileria and Anaplasma in sheep and goats in Turkey.

First molecular detection of tick-borne pathogens in dogs from Jiangxi, China.

In this study, blood samples obtained from 162 dogs in Jiangxi, China, were employed in molecular screening of canine tick-borne pathogens by PCR and sequencing. Babesia spp. gene fragment was detected in 12 (7.41%) dogs. All samples were negative for Hepatozoon spp., Ehrlichia canis, Coxiella spp., Borrelia spp., Rickettsia spp. and Anaplasma platys. Species-specific PCR analysis further confirmed that 8 (4.94%) and 4 (2.47%) dogs were infected by Babesia canis vogeli and Babesia gibsoni, respectively. Based on our analyses, Babesia spp. infection in Jiangxi appeared not related to age, gender, breed, usage, activity and health status or tick infestation history of the dogs. This is the first molecular report of Babesia canis vogeli and Babesia gibsoni in dogs from Jiangxi, China.

Enzyme-linked immunosorbent assays using recombinant TgSAG2 and NcSAG1 to detect Toxoplasma gondii and Neospora caninum-specific antibodies in domestic animals in Turkey.

Considering the scarce information on occurrences of Toxoplasma gondii and Neospora caninum in domestic animals from Turkey, the aim of this study was to investigate the seroprevalence of these parasite infections in cattle, horses, sheep, goats and dogs in Turkey. The specific antibodies against T. gondii and N. caninum were detected by iELISAs based on the recombinant TgSAG2 or NcSAG1 in a total of 2,039 serum samples from eleven provinces. The seroprevalence of T. gondii infections was 46.3%, 4.0%, 20.0%, 12.9% and 19.8%, that of N. caninum infections was 0.3%, 7.4%, 2.1%, 3.2% and 16.6% in the horses, cattle, sheep, goats and dogs, respectively. These results indicated that T. gondii and N. caninum infections are prevalent in Turkish domestic animals.

First report on Babesia vogeli infection in dogs in the Philippines.

Babesia vogeli is a tick-borne protozoal pathogen that infects erythrocytes. In Southeast Asia, this pathogen has only been reported in Thailand. In this study, nine dogs presented at three different veterinary clinics in Cebu City, Philippines were found positive for B. vogeli. DNA was extracted from blood samples and tested using a PCR for genus Babesia and a PCR specific for B. vogeli (both based on the 18S rRNA gene). Blood smears (triplicate) from each sample were found negative. All positive amplicons were sequenced and were found to be 99.4% identical to registered B. vogeli sequences at Genbank. Phylogenetic analysis revealed monophyletic grouping of Philippine sequences with the registered A. platys Genbank sequences. This is the first report of B. vogeli infection in dogs in the Philippines.

WITHDRAWN:First report on Babesia canis vogeli infection in dogs in the Philippines.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, http://dx.doi.org/10.1016/j.parint.2016.10.001.The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

Molecular identification and antigenic characterization of a merozoite surface antigen and a secreted antigen of Babesia canis (BcMSA1 and BcSA1).

BACKGROUND: Babesia canis is an apicomplexan tick-transmitted hemoprotozoan responsible for causing canine babesiosis in Europe and west Asia. Despite its importance, there is no known rapid diagnostic kit detection of B. canis infection in dogs. The present study identified two novel antigens of B. canis and used the recombinant antigens to establish a rapid, specific and sensitive serodiagnostic technique for detection of B. canis infection. METHODS: A complementary DNA (cDNA) expression library was constructed from the mRNA of B. canis and immunoscreened using B. canis-infected dog sera. The cDNAs encoding a merozoite surface antigen and a secreted antigen protein were identified and designated as BcMSA1 and BcSA1, respectively. The recombinant BcMSA1 and BcSA1 (rBcMSA1 and rBcSA1) expressed in Escherichia coli were purified and injected into mice for production of anti-sera. The native proteins were characterized by Western blot analysis and immunofluorescence. Furthermore, indirect enzyme-linked immunosorbent assays (iELISA) and rapid immunochromatographic tests (ICT) based on rBcMSA1 or rBcSA1 were established and evaluated to test specific antibodies in consecutive plasma samples from two B. canis-infected dogs. RESULTS: Antiserum raised against rBcMSA1 and rBcSA1 recognized the 39 kDa and 44 kDa native proteins by Western blot analysis, respectively. In addition, immunofluorescence and confocal microscopic observations revealed that BcMSA1 was found on the surface of parasites. However, BcSA1 localized in the matrix of the merozoites. The ELISA and ICT based on rBcMSA1 or rBcSA1 could detect specific antibodies in consecutive plasma samples from two B. canis-infected dogs. They showed no cross-reactions against the serum samples collected from dogs experimentally infected with closely related parasites. CONCLUSION: Taken together, the current results indicated that the rBcMSA1 and rBcSA1 are promising serodiagnostic antigens for developing iELISA and ICT to detect B. canis infection. To our knowledge, this study is the first to report BcMSA1 and BcSA1 as potential antigenic proteins for serodiagnosis of B. canis infection in dogs.