Evaluation of Immunodiagnostic Performances of Neospora caninum Peroxiredoxin 2 (NcPrx2), Microneme 4 (NcMIC4), and Surface Antigen 1 (NcSAG1) Recombinant Proteins for Bovine Neosporosis.

Bovine neosporosis is among the main causes of abortion in cattle worldwide, causing serious economic losses in the beef and dairy industries. A highly sensitive and specific diagnostic method for the assessment of the epidemiology of the disease, as well as it surveillance and management, is imperative, due to the absence of an effective treatment or vaccine against neosporosis. In the present study, the immunodiagnostic performance of Neospora caninum peroxiredoxin 2 (NcPrx2), microneme 4 (NcMIC4), and surface antigen 1 (NcSAG1) to detect IgG antibodies against N. caninum in cattle were evaluated and compared with that of the indirect fluorescent antibody test (IFAT). The results revealed that NcSAG1 had the highest sensitivity and specificity, with values of 88.4% and 80.7%, respectively, followed by NcPrx2, with a high sensitivity of 87.0% but a low specificity of 67.0%, whereas NcMIC4 showed sensitivity and specificity of 84.1% and 78.9%, respectively, when compared with IFAT. A high degree of agreement was observed for NcSAG1 (k = 0.713) recombinant protein, showing the highest diagnostic capability, followed by NcMIC4 (k = 0.64) and NcPrx2 (k = 0.558). The present study demonstrates that NcSAG1 is helpful as an antigen marker and also demonstrates the potential immunodiagnostic capabilities of NcPrx2 and NcMIC4, which could serve as alternative diagnostic markers for detecting N. caninum infection in cattle. These markers may find utility in future treatment management, surveillance, and risk assessment of neosporosis in livestock or other animal host species. Further research should be directed toward understanding the in vivo immune response differences resulting from immunization with both recombinant proteins.

Recombinant Dense Granule Protein (NcGRA4) Is a Novel Serological Marker for Neospora caninum Infection in Goats.

Neospora caninum is widely recognised as one of the most significant causes of abortion in cattle, with infections also occurring in sheep and goats. To prevent and control animal neosporosis, it is crucial to develop sensitive and specific methods for detecting N. caninum infection. Recently, several recombinant proteins have been utilised in serological assays for the diagnosis of neosporosis. In this study, we used commercial gene synthesis to produce dense granular antigen 4 (NcGRA4) recombinant protein. NcGRA4 plasmids were expressed in the Escherichia coli system and then purified. The purified recombinant protein was analysed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis. To evaluate the diagnostic potential of recombinant NcGRA4 protein, we tested 214 serum samples from goat farms via indirect enzyme-linked immunosorbent assay (iELISA) and compared the results to those from the indirect fluorescent antibody test (IFAT). Western blotting analysis revealed a single NcGRA4 band with an expected molecular weight of 32 kDa. The specific IgG against N. caninum was detected in 34.1% and 35% of samples evaluated by NcGRA4 iELISA and IFAT, respectively. The sensitivity and specificity of the NcGRA4 iELISA were 71.6% and 86.3%, respectively, when compared with the results from IFAT. Our results demonstrate that a recombinant protein that can be used to detect animal neosporosis can be produced using a synthetic NcGRA4 gene. Overall, recombinant NcGRA4 shows promise as a sensitive and specific serological marker for identifying target IgG in goat samples.

Immunoproteomics to identify species-specific antigens in Neospora caninum recognised by infected bovine sera.

Bovine neosporosis is a disease of concern due to its global distribution and significant economic impact through massive losses in the dairy and meat industries. To date, there is no effective chemotherapeutic drug or vaccine to prevent neosporosis. Control of this disease is therefore dependent on efficient detection tests that may affect treatment management strategies. This study was conducted to identify the specific immunoreactive proteins of Neospora caninum tachyzoites recognised by sera from cattle infected with N. caninum, Toxoplasma gondii, Cryptosporidium parvum, Babesia bovis and B. bigemina, and by sera from uninfected cattle using two-DE dimensional gel electrophoresis (2-DE) combined with immunoblot and mass spectrometry (LC-MS/MS). Among 70 protein spots that reacted with all infected sera, 20 specific antigenic spots corresponding to 14 different antigenic proteins were recognised by N. caninum-positive sera. Of these immunoreactive antigens, proteins involved in cell proliferation and invasion process were highly immunogenic, including HSP90-like protein, putative microneme 4 (MIC4), actin, elongation factor 1-alpha and armadillo/beta-catenin-like repeat-containing protein. Interestingly, we discovered an unnamed protein product, rhoptry protein (ROP1), possessing strong immunoreactivity against N. caninum but with no data on function available. Moreover, we identified cross-reactive antigens among these apicomplexan parasites, especially N. caninum, T. gondii and C. parvum. Neospora caninum-specific immunodominant proteins were identified for immunodiagnosis and vaccine development. The cross-reactive antigens could be evaluated as potential common vaccine candidates or drug targets to control the diseases caused by these apicomplexan protozoan parasites.

Title: L'immunoprotéomique pour identifier chez Neospora caninum les antigènes spécifiques de l'espèce reconnus par les sérums de bovins infectés. Abstract: La néosporose bovine est une maladie préoccupante en raison de sa distribution mondiale et de son impact économique important par d'énormes pertes dans les industries laitières et de la viande. À ce jour, il n'existe aucun médicament chimiothérapeutique ou vaccin efficace pour prévenir la néosporose. Par conséquent, le contrôle de cette maladie dépend de tests de détection efficaces qui affecteraient les stratégies de gestion du traitement. Cette étude a été menée pour identifier les protéines immunoréactives spécifiques des tachyzoïtes de Neospora caninum reconnues par les sérums de bovins infectés par N. caninum, Toxoplasma gondii, Cryptosporidium parvum, Babesia bovis et B. bigemina et par les sérums de bovins non infectés, à l'aide d'un gel d'électrophorèse bidimensionnel (2DE) combiné à l'immunoblot et à la spectrométrie de masse (LC-MS/MS). Parmi 70 spots protéiques ayant réagi avec tous les sérums infectés, 20 spots antigéniques spécifiques correspondant à 14 protéines antigéniques différentes ont été reconnus par les sérums positifs à N. caninum. Parmi ces antigènes immunoréactifs, les protéines impliquées dans la prolifération cellulaire et le processus d'invasion étaient hautement immunogènes, notamment la protéine de type HSP90, le micronème putatif 4 (MIC4), l'actine, le facteur d'élongation 1-alpha et la protéine à répétition de type armadillo/bêta-caténine. Fait intéressant, nous avons découvert un produit protéique sans nom, la protéine de rhoptries (ROP1), possédant une forte immunoréactivité contre N. caninum mais sans données disponibles sur sa fonction. De plus, nous avons identifié des antigènes à réaction croisée parmi ces parasites apicomplexes, en particulier N. caninum, T. gondii et C. parvum. Des protéines immunodominantes spécifiques de Neospora caninum ont été identifiées pour l'immunodiagnostic et le développement de vaccins. Les antigènes à réaction croisée pourraient être évalués comme candidats vaccins communs potentiels ou comme cibles médicamenteuses pour contrôler les maladies causées par ces parasites protozoaires apicomplexes.

Molecular Detection and Genetic Diversity of Tick-Borne Pathogens in Goats from the Southern Part of Thailand.

Tick-borne hemoprotozoan and rickettsial diseases affect the health and productivity of small ruminants in tropical and subtropical regions. Despite the large population of goats in the southern part of Thailand, there is limited information on the prevalence of tick-borne pathogens. In this study, polymerase chain reaction was used to detect the presence of Theileria spp., T. ovis, T. orientalis, Babesia ovis, Anaplasma ovis, and A. marginale in 262 goats from three provinces in the southern part of Thailand. In this investigation, Theileria spp. and A. ovis were detected while T. ovis, B. ovis, and A. marginale were not detected. Overall infection rates of Theileria spp. and A. ovis were 10.3% and 1.5%, respectively. The co-infections of two parasites was observed in 1.5% of goats. Sequence analysis showed the presence of T. luwenshuni and T. orientalis in the goat samples. This study is the first to use the molecular detection of T. orientalis in Thai goats, and presents genetic characterization using the major piroplasm surface protein (MPSP) gene. In the phylogenetic analysis, the T. orientalis MPSP sequence was classified as type 7. The A. ovis major surface protein 4 (MSP4) gene sequences shared high identities and similarity with each other and clustered with isolates from other regions. This study provides information about the prevalence and genetic diversity of tick-borne pathogens in goats in the study area, and is expected to be valuable for the development of effective control measures to prevent disease in animals in Thailand.

Development and evaluation of recombinant GRA8 protein for the serodiagnosis of Toxoplasma gondii infection in goats.

BACKGROUND: The development of sensitive and specific methods for detecting Toxoplasma gondii infection is critical for preventing and controlling toxoplasmosis in humans and other animals. Recently, various recombinant proteins have been used in serological tests for diagnosing toxoplasmosis. The production of these antigens is associated with live tachyzoites obtained from cell cultures or laboratory animals for genomic extraction to amplify target genes. Synthetic genes have gained a key role in recombinant protein production. For the first time, we demonstrated the production of the recombinant protein of the T. gondii dense granular antigen 8 (TgGRA8) gene based on commercial gene synthesis. Recombinant TgGRA8 plasmids were successfully expressed in an Escherichia coli system. The recombinant protein was affinity-purified and characterized via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Furthermore, the diagnostic potential of the recombinant protein was assessed using 306 field serum samples from goats via indirect enzyme-linked immunosorbent assay (iELISA) and the latex agglutination test (LAT). RESULTS: Western blotting using known positive serum samples from goats identified a single antigen at the expected molecular weight of TgGRA8 (27 kDa). iELISA illustrated that 15.40% of goat samples were positive for T. gondii-specific IgG antibodies. In addition, TgGRA8 provided high sensitivity and specificity, with significant concordance (91.83) and kappa values (0.69) compared with the results obtained using LAT. CONCLUSION: Our findings highlight the production of a recombinant protein from a synthetic TgGRA8 gene and the ability to detect T. gondii infection in field samples. The sensitivity and specificity of TgGRA8 demonstrated that this protein could be a good serological marker for detecting specific IgG in goat sera.

Exposure to Toxoplasma gondii in Asian Elephants (Elephas maximus indicus) in Thailand.

Toxoplasma gondii is the causative agent of toxoplasmosis in humans and various animal species worldwide. In Thailand, seroprevalence studies on T. gondii have focused on domestic animals, and information on infections in Asian elephants (Elephas maximus indicus) is scarce. This study was conducted to determine the seroprevalence of T. gondii infection in archival sera collected from 268 elephants living in Thailand. The serum samples were analyzed for anti-T. gondii immunoglobulin G antibodies using the latex agglutination test (LAT) and indirect enzyme-linked immunosorbent assay (iELISA) based on T. gondii lysate antigen (TLA-iELISA) and recombinant T. gondii dense granular antigen 8 protein (TgGRA8-iELISA). The prevalence of antibodies against T. gondii was 45.1% (121/268), 40.7% (109/268), and 44.4% (119/268) using LAT, TLA-iELISA, and TgGRA8-iELISA, respectively. Young elephants had a higher seropositivity rate than elephants aged >40 years (odds ratio = 6.6; p < 0.001; 95% confidence interval: 2.9-15.4). When LAT was used as the reference, TLA-iELISA and TgGRA8-iELISA showed a substantial (κ = 0.69) and moderate (κ = 0.42) agreement, respectively. Although our findings suggest the widespread exposure of Asian elephants to T. gondii in Thailand, the source of infection was not investigated. Therefore, investigation of the predisposing factors associated with toxoplasmosis is necessary to identify the potential risk factors for infection.

Molecular detection and genetic characterisation of pathogenic Theileria, Anaplasma and Ehrlichia species among apparently healthy sheep in central and western Kenya.

Tick-borne diseases (TBDs) caused by Theileria, Babesia, Anaplasma and Ehrlichia species are common in tropical and subtropical regions. In this study, we investigated the presence and genetic diversity of Theileria spp., Anaplasma ovis, B. ovis, E. ruminantium and Anaplasma spp. in sheep from the Machakos and Homa Bay counties of Kenya. In order to improve the diagnosis and control of ovine TBDs, a total of 76 blood samples from apparently healthy sheep were screened using a polymerase chain reaction (PCR). The assays were conducted using primers based on Theileria spp. 18S rRNA, Anaplasma ovis Major surface protein-4 (AoMSP4), B. ovis 18S rRNA, E. ruminantium pCS20 and Anaplasma spp. 16S rRNA. The overall infection rates for Theileria spp., A. ovis, E. ruminantium and Anaplasma spp. were 39/76 (51.3%), 26/76 (34.2%), 6/76 (7.9%) and 31/76 (40.8%), respectively. The overall co-infection was 47/76 (61.8%). All Theileria spp. positive samples were confirmed to be of Theileria ovis on sequencing. A phylogenetic analysis of the 18S rRNA gene sequences of T. ovis revealed that all isolates of this study clustered with T. ovis sequences extracted from the GenBank suggesting this gene is highly conserved. E. ruminantium pCS20 sequences were in the same clade on the phylogenetic tree. However, three AoMSP4 sequences from this study appeared in the same clade, while one sequence formed a separate branch revealing genetic divergence. The 16S rRNA sequencing revealed uncharacterised Anaplasma spp. and A. ovis. The phylogenetic analyses of the uncharacterised Anaplasma spp. revealed that the two sequences from this study appear in an independent clade from other sequences extracted from the GenBank. This study provides important information regarding the occurrence of tick-borne pathogens and their degree of genetic diversity among sheep in Kenya, which is useful for the diagnosis and control of TBDs.

Genetic characterization of tick-borne pathogens in ticks infesting cattle and sheep from three South African provinces.

Ticks are involved in the transmission of many public health and veterinary important pathogens. Although tick-borne pathogens are widely distributed in South Africa, information on tick-pathogen relationship needs to be updated particularly using modern molecular techniques. This study used PCR and sequencing to confirm the identity of the tick species collected from cattle and sheep from KwaZulu-Natal, Free State and Eastern Cape. Furthermore, presence of Babesia spp., Theileria spp., Anaplasma marginale, Rickettsia spp., Ehrlichia ruminantium and Coxiella burnetii was detected from tick DNA using species-specific PCR or nested PCRs. The study samples consisted of 390 adult ticks (male and female) which were pooled according to species, host animal and sampling site (three ticks per pool) for DNA extraction. The PCR results revealed that out of 130 tick DNA pools, 30 (23.1%) were positive for at least one pathogen. The most frequent pathogen was C. burnetii (9.2%), followed by Rickettsia spp. (7.7%), A. marginale (3.8%), T. mutans (3.1%), T. taurotragi (2.3%) and E. ruminantium (1.5%). The highest prevalence of pathogens was observed in ticks collected from cattle in Eastern Cape (16/42) and the lowest was in ticks obtained from sheep in Free State (1/21). Infected ticks were identified as Rhipicephalus evertsi evertsi (n = 13), R. appendiculatus (n = 3), R. decoloratus (n = 7) and Amblyomma hebraeum (n = 7). Coinfection with two pathogens was found in 21% of pathogen-positive pools. Analysis of Theileria taurotragi 18S rRNA, T. mutans 18S rRNA, C. burnetii htpB, Rickettsia spp. gltA, Rickettsia spp. ompA, E. ruminantium pCS20 and A. marginale Msp5 sequences showed that the pathogens detected in this study were genetically related to isolates previously reported in Africa. These findings provide important information on distribution of ticks and tick-borne pathogens of ruminants and will contribute in the formulation of future control strategies in South Africa.

Prevalence, risk factors, and genetic diversity of veterinary important tick-borne pathogens in cattle from Rhipicephalus microplus-invaded and non-invaded areas of Benin.

Babesiosis, theileriosis, anaplasmosis, and heartwater are tick-borne diseases (TBD) that threaten livestock production in sub-Saharan Africa including Benin. This country has been faced with an invasion of Rhipicephalus microplus, a major vector for babesiosis, theileriosis, and anaplasmosis over the last decade. Yet, data on TBD and the impact of the invasive ticks are lacking, making risk level evaluation and disease control arduous. In this study, epidemiological features of Babesia bovis, B. bigemina, Theileria spp., Anaplasma marginale and Ehrlichia ruminantium infections in Benin cattle were investigated in R. microplus-invaded and non-invaded areas. Detection of pathogens was based on species-specific PCR assays and resulting data were used to identify risk factors. Genetic diversity and phylogenies were then evaluated using several markers. Out of 207 samples examined, 170 (82.1%), 109 (52.7%), 42 (20.3%) 24 (11.6%) and 1 (0.5%) were positive for T. mutans, A. marginale, B. bigemina, B. bovis and E. ruminantium, respectively. Animal gender (for B. bovis), exposure to R. microplus (for B. bigemina and A. marginale), animal age (for B. bigemina and A. marginale) and cattle breed and/or antiprotozoal treatment (for T. mutants) significantly modulated pathogen occurrence. In addition, R. microplus exposure was significantly related to co-infection patterns and cases of clinical theileriosis and/or anaplasmosis were recorded among cattle highly exposed to the tick. In the genetic characterization, Theileria spp. and E. ruminantium sequences were conserved. Babesia spp. and A. marginale, however, showed high sequence polymorphisms that indicate the presence of several strains and may be linked to R. microplus invasion. Taken together, these results ascertain the endemicity of tick-borne infections in Benin and suggest that the characteristics of Babesia spp. and A. marginale infections in R. microplus-invaded and non-invaded areas are different.

Molecular analysis of tick-borne protozoan and rickettsial pathogens in small ruminants from two South African provinces.

Tick-borne protozoan and rickettsial diseases are a major threat to livestock in tropical and sub-tropical regions of Africa. In this study we investigated the presence and distribution of Theileria spp., Babesia ovis, Anaplasma ovis, Anaplasma phagocytophilum, Ehrlichia ruminantium and SFG Rickettsia in sheep and goats from Free State and KwaZulu-Natal provinces. A total of 91 blood samples were screened in this study, 61 from goats and 30 from sheep. PCR assay was conducted using primers based on Theileria spp. 18S rRNA, Babesia ovis (BoSSU rRNA), Anaplasma ovis (AoMSP4), Anaplasma phagocytophilum epank1, Ehrlichia ruminantium pCS20 and SFG Rickettsia OmpA. Overall infection rates of Theileria spp., Anaplasma ovis and Ehrlichia ruminantium were 18 (19.8%), 33 (36.3%) and 13 (14.3%), respectively. The co-infection of two pathogens were detected in 17/91 (18.7%) of all samples, goats having higher rates of co-infection compared to sheep. Phylogenetic tree analysis sequence of pCS20 gene of E. ruminantium of this study was found to be in the same clade with Kumm2 and Riverside strains both from South Africa. The phylogram of SSU rRNA of Theileria ovis had longer branch length compared to all other sequences most of which were from Asia and Middle East. This study provides important data for understanding the tick-borne diseases occurrence in the study area and it is expected to improve the approach for the diagnosis and control of these diseases.

Genetic mutations in sodium channel domain II and carboxylesterase genes associated with phenotypic resistance against synthetic pyrethroids by Rhipicephalus (Boophilus) decoloratus ticks in Uganda.

We previously reported emergence of super synthetic pyrethroid (SP) resistant Rhipicephalus (Boophilus) decoloratus ticks in Uganda. This study investigated the genetic basis of phenotypic resistance against SP in R. (B.) decoloratus and sought to identify novel diagnostic mutations for rapid detection of SP resistance in the above tick species. Genomic DNA was extracted from pooled larvae of 20 tick populations (19 of known SP susceptibility and 1 unknown susceptibility). The voltage sensitive sodium channel (VSSC) domain II S4-5 linker (SP target) and partial carboxylesterase (SP metabolizing enzyme) genes were amplified by PCR, cloned and sequenced. The resultant sequences were analyzed to determine single nucleotide polymorphisms (SNPs) associated with phenotypic resistance in the various tick populations investigated. Novel SNPs that introduced Eco RI and Eco RII restriction sites in carboxylesterase gene were identified in silco and validated with restriction fragment length polymorphism (RFLP) against 18 tick populations of known SP susceptibility. The study identified a super knock down resistance (kdr) mutation T58C in R. (B.) decoloratus VSSC associated with stable SP resistance. We further identified multiple nonsynonymous mutations in carboxylesterase of SP resistant ticks; one of which conferred novel EcoRII (G195C) restriction site for PCR-RFLP detection of SP resistance. In conclusion, this study is the first to report super kdr mutation in sodium channel domain II and multiple mutations in carboxylesterase genes that may concurrently mediate stable resistance against synthetic pyrethroids in R. (B.) decoloratus ticks from Uganda. The Eco RII based PCR-RFLP is a useful tool for rapid detection of stable SP resistant R. (B.) decoloratus ticks.

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 diversity of bovine Babesia spp., Theileria orientalis, and Anaplasma marginale in beef cattle in Thailand.

Babesia spp., Theileria orientalis, and Anaplasma marginale are significant tick-borne pathogens that affect the health and productivity of cattle in tropical and subtropical areas. In this study, we used PCR to detect the presence of Babesia bovis, Babesia bigemina, and T. orientalis in 279 beef cattle from Western Thailand and A. marginale in 608 beef cattle from the north, northeastern, and western regions. The PCRs were performed using species-specific primers based on the B. bovis spherical body protein 2 (BboSBP2), B. bigemina rhoptry-associated protein 1a (BbiRAP-1a), T. orientalis major piroplasm surface protein (ToMPSP), and A. marginale major surface protein 4 (AmMSP4) genes. To determine the genetic diversity of the above parasites, amplicons of B. bovis and B. bigemina ITS1-5.8s rRNA gene-ITS2 regions (B. bovis ITS, B. bigemina ITS), ToMPSP, and AmMSP4 genes were sequenced for phylogenetic analysis. PCR results revealed that the prevalence of B. bovis, B. bigemina, T. orientalis, and A. marginale in the Western region was 11.1, 12.5, 7.8, and 39.1 %, respectively. Coinfections of two or three parasites were observed in 17.9 % of the animals sampled. The study revealed that the prevalence of A. marginale in the western region was higher than in the north and northeastern regions (7 %). Sequence analysis showed the BboSBP2 gene to be more conserved than B. bovis ITS in the different isolates and, similarly, the BbiRAP-1a was more conserved than B. bigemina ITS. In the phylogenetic analysis, T. orientalis MPSP sequences were classified into types 3, 5, and 7 as previously reported. A. marginale MSP4 gene sequences shared high identity and similarity with each other and clustered with isolates from other countries. This study provides information on the prevalence and genetic diversity of tick-borne pathogens in beef cattle and highlights the need for effective strategies to control these pathogens in Thailand.

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.

Expression of truncated Babesia microti apical membrane protein 1 and rhoptry neck protein 2 and evaluation of their protective efficacy.

In this study, we evaluated the protective effect of recombinant Babesia microti apical membrane protein 1 (rBmAMA1) and rhoptry neck protein 2 (rBmRON2) against B. microti infection using a hamster model. The genes encoding the predicted domains I and II of BmAMA1 and the gene encoding the predicted transmembrane regions 2 and 3 of BmRON2 were expressed as His fusion recombinant proteins in Escherichia coli. Three groups with 5 hamsters in each group were immunized with rBmAMA1, rBmRON2 and rBmAMA1+rBmRON2, then challenged with B. microti. The result showed that only the group immunized with rBmAMA1+rBmRON2 exhibited limited protection against B. microti challenge infection, characterized by significant decreased of parasitemia and higher hematocrit values from day 6-10 post challenge infection. However, there was no significant difference in the groups immunized with rBmAMA1 or rBmRON2 alone. The absence of a significant difference in the total amount of antibodies against rBmAMA1 and rBmRON2 between the group immunized with single and combined proteins. This result suggests that the protection cannot be solely attributed to the quantity of antibodies produced, but also to their ability to target important epitopes from both antigens. These results suggest that combined immunization with rBmAMA1 and rBmRON2 is a promising strategy against B. microti.

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.

Primary Babesia rodhaini infection followed by recovery confers protective immunity against B. rodhaini reinfection and Babesia microti challenge infection in mice.

In the present study, we investigated the protective immunity against challenge infections with Babesia rodhaini and Babesia microti in the mice recovered from B. rodhaini infection. Six groups with 5 test mice in each group were used in this study, and were intraperitoneally immunized with alive and dead B. rodhaini. The challenge infections with B. rodhaini or B. microti were performed using different time courses. Our results showed that the mice recovered from primary B. rodhaini infection exhibited low parasitemia and no mortalities after the challenge infections, whereas mock mice which had received no primary infection showed a rapid increase of parasitemia and died within 7 days after the challenge with B. rodhaini. Mice immunized with dead B. rodhaini were not protected against either B. rodhaini or B. microti challenge infections, although high titers of antibody response were induced. These results indicate that only mice immunized with alive B. rodhaini could acquire protective immunity against B. rodhaini or B. microti challenge infection. Moreover, the test mice produced high levels of antibody response and low levels of cytokines (INF-γ, IL-4, IL-12, IL-10) against B. rodhaini or B. microti after challenge infection. Mock mice, however, showed rapid increases of these cytokines, which means disordered cytokines secretion occurred during the acute stage of challenge infection. The above results proved that mice immunized with alive B. rodhaini could acquire protective immunity against B. rodhaini and B. microti infections.