Artemis Efstratiou

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.

Transient transfection of intraerythrocytic Babesia gibsoni using elongation factor-1 alpha promoter

The development of gene manipulation techniques has been reported in many protozoan parasites over the past few years. However, these techniques have not yet been established for Babesia gibsoni. Here, we report for the first time, the successful transient transfection of B. gibsoni. The plasmid containing the firefly luciferase reporter gene (pBS-ELA) was transfected into B. gibsoni by an AMAXA 4D Nucleofector™ device. Transfection using program FA113 and Lonza buffer SF showed the highest luciferase expression. Twenty micrograms of plasmid produced the highest relative transfection efficiency. The fluorescent protein-expressing parasites were determined by GFP-containing plasmid (pBS-EGA) at 48 and 72h post transfection. This finding is the first step towards a stable transfection method for B. gibsoni, which may contribute to a better understanding of the biology of the parasite.

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.

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.

Detection and molecular characterization of tick-borne pathogens infecting sheep and goats in Blue Nile and West Kordofan states in Sudan

Tick-borne pathogens (TBPs) are common in livestock of sub-Saharan Africa. However, information regarding TBPs in sheep and goats in Sudan is limited. In this study, 178 blood samples of sheep and goats in Blue Nile and West Kordofan states were investigated for TBPs using PCR. Overall, 110 (61.8%) samples were found to be infected with at least one of the following pathogens: Anaplasma ovis, Theileria ovis, and Ehrlichia ruminantium. Babesia ovis and T. lestoquardi were not identified. A. ovis was the most prevalent pathogen (n = 107, 60.1%), followed by T. ovis (n = 23, 12.9%) and E. ruminantium (n = 1, 0.6%). The prevalence rates of A. ovis and T. ovis were significantly higher in sheep than in goats. Phylogenetic analysis of T. ovis 18S rRNA and A. ovis msp4, groEL, and 16S rRNA, revealed that the pathogens identified in this study are clustered together, indicating similar molecular characteristics. Additionally, phylogenetic analysis of E. ruminantium pCS20 revealed that E. ruminantium in this study belong to the West Africa group, and different to E. ruminantium previously identified in ticks from Sudan. We concluded that TBPs are highly prevalent in the study area and continuous monitoring of TBPs in sheep and goats in Sudan is highly required.

Molecular detection and characterization of tick-borne protozoan and rickettsial pathogens isolated from cattle on Pemba Island, Tanzania

Tick-borne diseases cause significant losses to livestock production in tropical and subtropical regions. In Tanzania, detailed studies on tick-borne pathogens in cattle using sensitive molecular detection methods are scarce. In this study, we investigated the occurrence of Theileria spp., Babesia spp., Anaplasma spp. and Ehrlichia spp. in 245 blood samples collected from cattle on Pemba Island, Tanzania. We used polymerase chain reaction (PCR) and gene sequencing to detect and identify pathogens. PCR screening revealed overall infection rates of 62.4% for Theileria spp., 17.6% for Babesia bigemina, 15.9% for Anaplasma marginale, 7.4% for Ehrlichia ruminantium and 4.5% for Babesia bovis. Further analysis using sequences of Theileria spp. 18S rRNA revealed infection of cattle with Theileria mutans (68.6%), Theileria taurotragi (48.4%), Theileria parva (41.2%), and Theileria ovis (1.9%). Co-infections of cattle, with up to six tick-borne pathogens, were revealed in 46.9% of the samples. Sequence analysis indicated that T. parva p104, E. ruminantium pCS20 and A. marginale MSP-5 genes are conserved among cattle blood samples in Pemba, with 99.3%-100%, 99.6%-100% and 100% sequence identity values, respectively. In contrast, the B. bigemina RAP-1a and B. bovis SBP-2 gene sequences were relatively diverse with 99.5%-99.9% and 66.4%-98.7% sequence identity values respectively. The phylogenetic analyses revealed that T. parva p104, E. ruminantium pCS20 and A. marginale MSP-5 gene sequences clustered in the same clade with other isolates from other countries. In contrast, the B. bigemina RAP-1 and B. bovis SBP-2 gene sequences showed significant differences in the genotypes, as they appeared in separate clades. This study provides important data for understanding the epidemiology of tick-borne diseases, and is expected to improve the approach for diagnosis and control of tick-borne diseases in Tanzania.

Molecular survey and characterization of Theileria annulata and Ehrlichia ruminantium in cattle from Northwest China

Theileriosis and ehrlichiosis are two important tick-borne diseases affecting cattle farming in China. However, limited information is available regarding prevalence and molecular characterization of Theileria annulata and Ehrlichia ruminantium in cattle in Xinjiang Uygur Autonomous Region (XUAR), northwestern China. In this study, a total of 176 blood samples of cattle from three rural areas of XUAR were collected in June 2017 and were tested by nested-PCR. A total of 34 (19.3%) samples were found to be infected with one or two pathogens. The overall prevalence rates of T. annulata and E. ruminantium were 18.2% and 1.7%, respectively. Phylogenetic analyses revealed that the E. ruminantium isolates from XUAR were located in the same clade but diverged from the isolates from African countries using pCS20 gene while T. annulata isolates from XUAR revealed differences in the genotypes using Tams1 sequences. To our knowledge, this is the first report of E. ruminantium infection in cattle in China. It also provides the first genetic characterization of T. annulata in cattle in XUAR. The current findings are important for understanding the distribution of agents of theileriosis and ehrlichiosis and in designing measures for the prevention and control of tick-borne diseases in cattle, other animals, and humans.

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.

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.