Huseyin Bilgin Bilgic

Development of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bovis and Anaplasma marginale in cattle

Graphical abstract Highlights ► Novel multiplex PCR for Theileria annulata, Babesia bovis and Anaplasma marginale. ► Specific and sensitive tool which can be applied to epidemiological studies. ► Simple and efficient assay which has been validated using field samples.

Can Anaplasma ovis in small ruminants be neglected any longer

Anaplasma species are obligate intracellular rickettsial pathogens transmitted by ticks with an impact on human and animal health. Anaplasma ovis infects sheep and goats in many regions of the world, and it can be diagnosed by different methods like Giemsa staining, PCR or competitive ELISA. In this study, a PCR based on the gene coding for major surface protein 4 (MSP-4) was used to examine field samples collected from sheep in different countries. Altogether, 1161 blood samples from Turkey (n = 830), Iraq (n = 195), Sudan (n = 96) and Portugal (n = 40) were examined, of which 31.4%, 66.6% 41.6% and 82.5%, respectively, were positive. This indicates high prevalence of A. ovis in the countries under investigation, and it can be assumed that the situation in other areas of the world might be similar. Thus, A. ovis should be considered as an important constraint of livestock production, and further efforts are needed to better understand the epidemiology and to implement suitable control measures.

Evaluation of cytochrome b as a sensitive target for PCR based detection of T. annulata carrier animals

Bovine tropical theileriosis, caused by the tick-borne protozoan Theileria annulata, imposes a serious constraint upon breed improvement programmes and livestock production in tropical and sub-tropical regions of the world. Animals that recover from primary infection serve as carriers and play a critical role in the epidemiology of the disease, acting as reservoirs of infection. However, conclusive identification of carrier animals can be problematic. This study describes assessment of candidate target genes for PCR assay-based detection of T. annulata infected carrier animals. Following in silico screening and rejection of three major multi-copy gene families, an assay based on PCR amplification of a 312 bp segment of the T. annulata gene for cytochrome b (Cytob1 assay) was established. Sensitivity was evaluated using serial dilutions of blood obtained from experimentally infected calves, while specificity was confirmed by testing DNA representing twelve different T. annulata stocks and other Theileria and Babesia species. Direct comparison with other target genes and published data indicated that Cytob1 PCR-based assays provide the greatest level of sensitivity, combined with a high level of specificity and the ability to detect different T. annulata genotypes. It can be concluded that the cytochrome b gene is the optimal target for PCR amplification and its incorporation in a Reverse Line Blot Assay offers the most sensitive method yet devised to detect the parasite in carrier animals. The use of this assay will increase the accuracy of epidemiological studies aimed at improving disease control in endemically unstable regions.

Population genetic analysis and sub-structuring in Babesia bovis

The tick-borne protozoan parasite, Babesia bovis is one of the causes of bovine babesiosis, an economically important disease of cattle in tropical and sub-tropical countries. Using the recently published genome sequence of the parasite, we developed a panel of eight mini- and micro-satellite markers and used these to investigate the role of genetic exchange in the population structure and diversity of the parasite using isolates from Zambia and Turkey. This population genetic analysis showed that genetic exchange occurs and that there are high levels of genetic diversity, with geographical sub-structuring quantified using Wrights F Index. Linkage disequilibrium was observed when isolates from both countries were treated as one population, but when isolates from Zambia were analysed separately linkage equilibrium was observed. The Turkish isolates were sub-structured, containing two genetically distinct sub-groups, both of which appeared to be in linkage equilibrium. The results of the Zambian study suggest that a sub-set of the parasite population is responsible for the westward spread of babesiosis into the previously disease-free central region of the country. The Zambian isolates had a significantly higher number of genotypes per sample than those from Turkey and age was found to be a significant predictor of the multiplicity of infection. The high levels of diversity seen in the Zambian and Turkish B. bovis populations have implications in the development of subunit vaccines against the disease and the spread of drug resistance.

Prevalence of tick-borne haemoparasites in small ruminants in Turkey and diagnostic sensitivity of single-PCR and RLB

BackgroundTick-borne haemoparasitic diseases (TBHDs), caused by Theileria, Babesia, Anaplasma and Ehrlichia, are common in regions of the world where the distributions of host, pathogen and vector overlap. Many of these diseases threaten livestock production and some also represent a concern to human public health. The primary aim of this study was to determine the prevalence of the above-mentioned pathogens in a large number of blood samples (n = 1979) collected from sheep (n = 1727) and goats (n = 252) in Turkey. A secondary aim was to assess the diagnostic sensitivity of a number of species-specific polymerase chain reaction (PCR) tests and the reverse line blotting (RLB) assay. DNA samples were screened using species-specific PCR for the presence of Theileria ovis, Theileria sp. MK, T. lestoquardi, T. uilenbergi, T. luwenshuni, Babesia ovis, Anaplasma ovis and A. phagocytophilum while RLB was undertaken to test for the presence of all known Theileria, Babesia, Anaplasma and Ehrlichia species. The diagnostic sensitivity of these two approaches was then compared in terms of their ability to detect single species and mixed infections.ResultsOverall, 84 and 74.43% of the small ruminants sampled were identified as hosting one or more pathogen(s) by species-specific PCR and RLB respectively. The presence of Theileria sp. OT1, T. luwenshuni and T. uilenbergi in Turkey was revealed for the first time while the presence of Babesia motasi, B. crassa and T. separata in Turkish small ruminants was confirmed using molecular methods. A high prevalence of mixed infection was evident, with PCR and RLB approaches indicating that 52.24 and 35.42% of animals were co-infected with multiple species, respectively. More than 80% of the mixed infections contained T. ovis and/or A. ovis. The RLB approach was found to be capable of detecting mixed infections with species such as Theileria sp. OT1, Theileria sp. OT3, T. separata, B. crassa and Babesia spp.ConclusionThe results indicated that pathogens causing TBHDs are highly prevalent in sheep and goats in Turkey. The diagnostic sensitivity of species-specific single PCR was generally higher than that of RLB. However, the latter approach was still capable of identifying a high proportion of individuals containing mixed-species infections. The use of species-specific single PCR is recommended to accurately estimate pathogen prevalence and to identify co-infected hosts.

Molecular surveillance of Theileria parasites of livestock in Oman.

BACKGROUND Theileriosis is one of the most prevalent infectious diseases of livestock in the Arabian Peninsula, and causes high rates of mortality and morbidity in sheep and cattle. However, there is a paucity of information on the distribution of Theileria spp. over the whole region and their impact on different hosts. The present study carried out a country-wide molecular survey for Theileria spp. of livestock in Oman across four governorates. The aim of the survey was to define the prevalence of Theileria spp. in cattle, sheep and goats, highlight risk factors for infection and identify the main tick species involved in parasite transmission. MATERIAL AND METHODS A total of 2020 animals were examined in the survey consisting of sheep [n=592], goats [n=981] and cattle [n=447]. All three species were raised and co-grazed on the same farms. Theileria parasites were detected using PCR-RFLP and RLB of the 18S rRNA gene. Cloning and sequencing of the 18S rRNA was carried out on 11 T. lestoquardi isolates from Ash-Sharqiyah, and Ad-Dhahira governorates, and phylogenetic relationships were inferred using additional sequences of T. lestoquardi, T. annulata and T. ovis available in GenBank. RESULTS Theileria spp. prevalence was 72.3%, 36.7% and 2.7% among cattle, sheep and goats, respectively. Strong similarity in results was obtained using RLB and PCR-RFLP for detection of Theileria spp. however, RLB detected a higher rate of mixed infection than PCR-RFPL (P<0.001). Theileria annulata was the only parasite detected in cattle, while sheep and goats carried T. ovis, T. lestoquardi and T. annulata as well as Theileria spp. OT1. Of the four Theileria spp. detected in small ruminants, overall T. ovis was most prevalent (sheep [33.4%], goats [2.0%]), whereas T. lestoquardi was less prevalent (sheep [22.0%], goats [0.5%]). A large proportion of infected sheep (19%) carried mixed infection of T. ovis and T. lestoquardi. However, single T. lestoquardi infections (3.0%) were less prevalent than T. ovis infections (14.5%). Risk of Theileria spp. infection was significantly higher for exotic breeds, relative to native breeds, of cattle (p=0.00002) and sheep (p=0.005). Phylogenetic analysis placed T. lestoquardi in Oman in the same clade as other T. lestoquardi strains isolated from the same regional area (Iraq and Iran). The main tick species, identified on the examined animals, Hyalomma anatolicum, was widely distributed and was found in all of the surveyed governorates. CONCLUSION Theileria spp. are widespread in Oman with variable prevalence detected in different regions. Two economically important hosts, cattle and sheep are at high risk from virulent T. annulata and T. lestoquardi, respectively. The survey indicates extensive exposure to ticks and transmission of infection that has a significant economic impact. The higher prevalence of T. lestoquardi as mixed rather than single infection requires further investigation.

Identification and analysis of immunodominant antigens for ELISA-based detection of Theileria annulata

Tropical or Mediterranean theileriosis, caused by the protozoan parasite Theileria annulata, remains an economically important bovine disease in North Africa, Southern Europe, India, the Middle East and Asia. The disease affects mainly exotic cattle and imposes serious constraints upon livestock production and breed improvement programmes. While microscopic and molecular methods exist which are capable of detecting T. annulata during acute infection, the identification of animals in the carrier state is more challenging. Serological tests, which detect antibodies that react against parasite-encoded antigens, should ideally have the potential to identify carrier animals with very high levels of sensitivity and specificity. However, assays developed to date have suffered from a lack of sensitivity and/or specificity and it is, therefore, necessary to identify novel parasite antigens, which can be developed for this purpose. In the present study, genes encoding predicted antigens were bioinformatically identified in the T. annulata genome. These proteins, together with a panel of previously described antigens, were assessed by western blot analysis for immunoreactivity, and this revealed that four novel candidates and five previously described antigens were recognised by immune bovine serum. Using a combination of immunoprecipitation and mass spectrophotometric analysis, an immunodominant protein (encoded by TA15705) was identified as Ta9, a previously defined T cell antigen. Western blotting revealed another of the five proteins in the Ta9 family, TA15710, also to be an immunodominant protein. However, validation by Enzyme-Linked Immunosorbent Assay indicated that due to either allelic polymorphism or differential immune responses of individual hosts, none of the novel candidates can be considered ideal for routine detection of T. annulata-infected/carrier animals.

Identification of candidate transmission-blocking antigen genes in Theileria annulata and related vector-borne apicomplexan parasites

BackgroundVector-borne apicomplexan parasites are a major cause of mortality and morbidity to humans and livestock globally. The most important disease syndromes caused by these parasites are malaria, babesiosis and theileriosis. Strategies for control often target parasite stages in the mammalian host that cause disease, but this can result in reservoir infections that promote pathogen transmission and generate economic loss. Optimal control strategies should protect against clinical disease, block transmission and be applicable across related genera of parasites. We have used bioinformatics and transcriptomics to screen for transmission-blocking candidate antigens in the tick-borne apicomplexan parasite, Theileria annulata.ResultsA number of candidate antigen genes were identified which encoded amino acid domains that are conserved across vector-borne Apicomplexa (Babesia, Plasmodium and Theileria), including the Pfs48/45 6-cys domain and a novel cysteine-rich domain. Expression profiling confirmed that selected candidate genes are expressed by life cycle stages within infected ticks. Additionally, putative B cell epitopes were identified in the T. annulata gene sequences encoding the 6-cys and cysteine rich domains, in a gene encoding a putative papain-family cysteine peptidase, with similarity to the Plasmodium SERA family, and the gene encoding the T. annulata major merozoite/piroplasm surface antigen, Tams1.ConclusionsCandidate genes were identified that encode proteins with similarity to known transmission blocking candidates in related parasites, while one is a novel candidate conserved across vector-borne apicomplexans and has a potential role in the sexual phase of the life cycle. The results indicate that a ‘One Health’ approach could be utilised to develop a transmission-blocking strategy effective against vector-borne apicomplexan parasites of animals and humans.

[Detection of Anaplasma / Ehrlichia Species of Cattle and Ticks in Aydın Region].

OBJECTIVE The aim of this study is to detect the Anaplasma/Ehrlichia species of cattle and ticks and to provide knowledge on the prevalence of these species during sampling periods. METHODS A total of 679 blood and 186 tick samples were collected from the Osmanbükü, Akçaova, Dalama, and Söke districts of Aydın. The samples were screened with genus polymerase chain reaction (PCR) for Anaplasma/Ehrlichia spp., species-specific polymerase chain reaction for Anaplasma marginale and A. centrale, and nested PCR for A. bovis and A. phagocytophilum. RESULTS A. centrale was detected in Söke during September and in Dalama and Akçaova during March, June, September, and December. A. marginale was detected in Osmanbükü during June; in Söke during March and December; in Akçaova during June, September, and March; and in Dalama during the entire sampling period. A. phagocytophilum was detected in all regions during the entire sampling period. None of the samples were positive for A. bovis. Mixed infections were detected in 50 blood samples. A. marginale and A. phagocytophilum were detected in the tick samples. CONCLUSION In this study, A. phagocytophilum was abundantly detected compared with A. marginale and A. centrale. A. phagocytophilum and A. centrale were extensively found in Akçaova and A. marginale was mostly seen in Dalama. Parasites were extensively detected in September and March. The analysis indicated that collected ticks were infected with different Anaplasma/Ehrlichia species.

[Gerbils, As Experimental Animals (Meriones unguiculatus): Is A Good Role Model for Leishmania major?].

OBJECTIVE This study aimed to observation the possible visceralization tendency and dissemination of L. major amastigotes in gerbils (Meriones unguiculatus) using a classic smear technique, inoculated into enriched Novy-MacNeal-Nicolle (NNN) culture and polymerase chain reaction (PCR) assay for diagnosis of infection. METHODS In this study, L. major isolated from a man who 18 years old, living in Bitlis province of Turkey. This strain also was utilized to infect gerbils. A total of 1 × 10(8)/mL promastigotes were inoculated to 10 gerbils. Necropsy was performed on infected gerbils for monitoring the visceralization tendency of the parasites. Tissue samples were prepared from each animal and stained by Giemsa and inoculated into NNN culture. However, a real-time PCR assay was performed to confirm the infection the clinical material. RESULTS Examination of Giemsa-stained tissue smears showed that infected animals with L.major were positive for Leishmania amastigotes in all tissues at the first month post infection and Leishmania promastigotes were cultured at 26°C in culture flasks containing NNN. Melting curve analyses of ribozomal internal transcribed spacer 1 (ITS-1) PCR showed the peak concordant with L. major. CONCLUSION As a result, the present study confirmed by both Giemsa-stained smears and PCR, visceralization and dissemination of L. major amastigotes, the principal cause of zoonotic cutaneous leishmaniasis in gerbils.