Molecular identification of tick-borne pathogens infecting cattle in Mymensingh district of Bangladesh reveals emerging species of Anaplasma and Babesia.

Tick-borne diseases are considered a major hindrance to the health and productive performance of cattle in Bangladesh. To elucidate the epidemiology of tick-borne pathogens (TBPs) in local cattle, a cross-sectional study was performed in the 12 subdistricts (Upazilas) of Mymensingh district in Bangladesh. Blood samples and ticks were collected from 384 clinically healthy cattle kept by 135 farmers from 96 randomly selected villages. DNA extracted from the blood samples was subsequently screened by polymerase chain reaction (PCR) and a Reverse Line Blot (RLB) hybridization assay using an in-house prepared chemiluminescence solution for the presence of Anaplasma, Ehrlichia, Rickettsia, Babesia and Theileria spp. A total of 2,287 ticks were collected from 232 infested cattle (60.4%, 232/384) and identified morphologically as Rhipicephalus (Boophilus) microplus (n = 1,432, 62.6%) and Haemaphysalis bispinosa (n = 855; 37.4%). The RLB results demonstrated that the majority of the cattle (62.2%) were infected with at least one TBP. Theileria orientalis infections were most common (212/384, 55.2%) followed by infections with Anaplasma bovis (137/384, 35.67%), Anaplasma marginale (16/384, 4.17%), Babesia bigemina (4/384, 1.04%) and Babesia bovis (2/384, 0.52%). A previously uncharacterized Anaplasma sp. (Anaplasma sp. Mymensingh) and Babesia sp. (Babesia sp. Mymensingh), which are genetically closely related to Anaplasma platys and B. bigemina, were detected in 50 of 384 (13.0%) and 1 of 384 (0.3%) of the blood samples, respectively. Key risk factors for the occurrence of T. orientalis, A. marginale and Anaplasma sp. Mymensingh were identified. In conclusion, this study revealed that cattle in Mymensingh district are mainly infested with R. microplus and H. bispinosa ticks and may carry multiple TBPs. In addition, two previously uncharacterized pathogens were detected in the bovine blood samples. The pathogenicity of these species remains to be determined.

At least two genetically distinct large Babesia species infective to sheep and goats in China.

A fatal disease of sheep and goats in the northern part of China has been reported to be due to Babesia ovis. However, some characteristics of the causative agent in recent reports are not in accordance with the original attributes ascribed to this parasite. Therefore, the 18S small subunit ribosomal RNA (18S rRNA) genes of a number of Babesia isolates in China were sequenced and compared with that of other Babesia and Theileria species in an attempt to clarify their taxonomic position. In the present study, seven Babesia isolates were collected from distinct areas of northern China, and the 18S rRNA genes were amplified and sequenced. The phylogenetic trees were inferred based on 18S rRNA gene sequences of the Chinese ovine Babesia isolates and some of ovine Babesia and Theileria species available in GenBank. In the phylogenetic tree, Babesia sp. isolates from Madang, Tianzhu, Lintan, Ningxian, Hebei and Liaoning all grouped with B. motasi with 88.2-99.9% identity, while Babesia sp. Xinjiang grouped in a separate clade between B. ovis and B. crassa with 79.7-81.2% identity. The results indicated that there are at least two distinct Babesia species groups-B. motasi and Babesia sp. Xinjiang, the latter was distinctly different from other ovine Babesia isolates from China with less than 86.6% identity.

Serological diagnostic tools for the major tick-borne protozoan diseases of livestock.

Tick-borne protozoan diseases, babesiosis and theileriosis, are among the most important diseases affecting the productivity of livestock worldwide and resulting in high economic losses. A prerequisite for the control of these diseases is to study their epidemiology by mapping their distribution and seasonality. As clinical diagnostic and surveillance tools, serological tests such as the complement fixation test (CFT), the indirect fluorescent antibody test (IFAT) and the enzyme linked immunosorbent assay (ELISA) have been successfully used over decades. With the development in molecular biology, recombinantly expressed parasite molecules have emerged and substituted crude parasite antigen used in serology. A popular format of these tests is the antibody binding competitive inhibition and the indirect antibody detection ELISA. Under the precondition that these tests are correctly designed and validated, they provide a powerful tool for epidemiology, with greater advantages of affordability and amenability to standardization. This paper reviews the pathogenic tick-borne protozoan diseases and the respective diagnostic ELISA based serological tests currently available for serosurveillance.

The Emergence of Theileria parva in Jonglei State, South Sudan: Confirmation Using Molecular and Serological Diagnostic Tools.

A cross-sectional survey was carried out in four counties of Jonglei State, South Sudan, between May and June 2012 to determine the distribution and northern limit of Theileria parva, the causative agent of East Coast fever in cattle, and its tick vector Rhipicephalus appendiculatus, as a prerequisite to the deployment of relevant control strategies. A total of 1636 ticks, 386 serum samples and 399 blood samples were collected from indigenous, apparently healthy, cattle of different age groups. Tick species were identified morphologically, and the identity of R. appendiculatus was confirmed by DNA barcoding. Overall, the T. parva infection rate in R. appendiculatus was 25% as shown by nested PCR. ELISA was used to assess antibodies to T. parva, and the overall seroprevalence was 22.8%. PCR of the blood samples showed 55 (13.8%) were positive for T. parva. This is the first molecular confirmation of T. parva DNA in areas north of Juba, where it was previously known and established. The northern limit of T. parva was determined as N°06.17.792, about 242 Km north from Juba. Implication of this limit on the epidemiology and control of ECF is discussed.

Transferable suppression and intrinsic unresponsiveness in delayed-type hypersensitivity to sheep red blood cells of mice: two distinct mechanisms?

In mice rendered unresponsive in DTH by the i.v. infection of 10(9) SRBC, two forms of specific unresponsiveness could be distinguished, referred to as intrinsic unresponsiveness and suppression. Suppression could be transferred by splenic T cells. It was short-lived and sensitive to cyclophosphamide (Cy, 100 mg/kg i.v.) given after sensitization. On the other hand, intrinsic unresponsiveness was not transferable by either serum or spleen cells. It was long-lived and resistant to Cy (200 mg/kg i.v.) given after sensitization. Induction of both transferable suppression and intrinsic unresponsiveness depended on Cy-sensitive (200 mg/kg i.v.) precursors. Since no evidence for clonal deletion could be obtained, it is suggested that unresponsiveness in general is mediated by complex cellular interactions which are readily perturbed in transfer experiments. In this way, cell recipients would end up possessing incomplete regulatory circuits, intact circuits still being present in donor animals.

Variant specific opsonization of Trypanosoma evansi measured by luminol-dependent chemiluminescence.

Using luminol-dependent chemiluminescence (LCL), the specificity of antibodies to variable antigen type (VAT)-populations of Trypanosoma evansi was studied in four infected ponies. Trypanosomes of each wave of parasitemia were isolated and multiplied in irradiated mice. Their opsonization by serum collected during the infection was investigated with LCL and results for isolated VAT-populations are shown in the paper. Antibodies specific to each VAT-population were first found three days after the maximum of a parasitemic wave. There was no cross reactivity between different VAT-populations. LCL proved to be a rapid and automatic method for the demonstration of antibodies with specificity to variable antigen types of trypanosomes.

Proliferation and cytokine profile of T. annulata-infected ovine, caprine, and bovine lymphoblastoid cells.

T. annulata, the causative agent of tropical theileriosis in cattle, can also infect ovine and caprine leukocytes in vitro. In vivo studies showed that this parasite causes a mild infection in both these animal species, and in sheep merozoite stage development seems to be inhibited. Since the nature of T. annulata infected caprine and ovine cells is not known, all three cell lines were karyotyped and phenotypically characterized by flow cytometry. They all express mRNA of cytokines IL-1 alpha, IL-1 beta, IL-8, and TNF-alpha, but not of IFN-gamma, IL-2, and IL-4. In contrast, IL-6 mRNA was expressed in the cattle cell line only, while mRNA of IL-10 was exclusively produced by the sheep cell line. The observed differences in cytokine mRNA expression may be responsible for the different pathogenesis of T. annulata infection in cattle and sheep.

Detection and differentiation of Theileria annulata and Theileria parva using macroschizont-derived DNA probes.

A lamda gt11 expression library based on T.annulata-infected cells was screened with an antiserum raised in rabbits against partially purified schizonts of T.annulata. Two clones were detected, sequenced and designated as SA288 and SB288 (Shayan et al., submitted for publication). From the sequences of these two genes oligonucleotide primers were designed for specific amplification of parasite DNA by polymerase chain reaction (PCR). We could show that these genes are of parasitic origin and do occur in all T.annulata stocks tested in the present study. In addition, a target sequence for SA288 could also be identified in T.parva-schizonts. None of them reacted with genomic DNA of different Babesia spp. A third primer pair was designed from the DNA-sequence of a gene encoding for the T.parva-specific casein kinase II-alpha subunit. Using this primer pair, a target sequence could only be detected in T.parva. Taken together, the primers described here can be used as molecular tools in PCR for the detection of Theileria parasites and to distinguish T.annulata from T.parva.

Lymphocyte stimulatory capacity of Theileria annulata-infected ovine lymphoblastoid cells.

T. annulata, the causative agent of tropical theileriosis in cattle, is transmitted by ticks of the genus Hyalomma. Sporozoites of this parasite invade their target cells, where they differentiate to macroschizonts. T. annulata additionally invades and transforms ovine and caprine leukocytes. T. annulata infection in the ovine system is poorly studied, thus we used a mixed lymphocyte culture (MLC) to analyze the capacity of these cells to activate naïve uninfected ovine cells. The peak response was observed on day three or four and the response could not be induced by lysates of infected cells or their supernatants. The stimulated cells expressed IL-2 and secreted an IL-2-like growth factor.

Generation of cytotoxic T lymphocytes and cytostatic acting cells in T. annulata-immune cattle.

Cattle immunized against Theileria annulata with schizont containing autologous cell lines are immune to challenge with a homologous parasite strain. Two cell types have been detected in the peripheral blood of the immunized animals: cytotoxic T lymphocytes (CTL) and cytostatic acting cells (CAC). Killing the target cells by CTL is infection associated and is MHC class I restricted. Hence, no cytotoxicity was observed against target cells that were treated with the theilericidal drug buparvaquone or autologous Con A-blasts. The growth inhibition of CAC is MHC unrestricted, and not mediated by cytokine interferon gamma (IFN-gamma).

Phylogenetic analysis by rRNA comparison of the highly pathogenic sheep-infecting parasites Theileria lestoquardi and a Theileria species identified in China.

In the Northwestern part of China there have been reports of clinical cases in small ruminants of a haemoparasite with the characteristics of Theileria hirci (T. lestoquardi). However, some properties of this parasites argue against its classification as T. lestoquardi. In this paper, we present evidence that T. lestoquardi and the Chinese Theileria isolate are distinct parasite species. Phylogenetic analysis of determined nucleotide sequences of small subunit ribosomal RNA (srRNA) genes of T. lestoquardi and the Chinese Theileria parasite show that they belong to different clades within the phylogenetic tree of piroplasms. The srRNA sequence of the Chinese parasite was found to be most closely related to T. buffeli, which, with T. sergenti, belongs to an evolutionary lineage of non-lymphoproliferative Theileria species. On the other hand, it was clearly divergent to a lineage of lymphoproliferative Theileria species; T. annulata, T. parva, T. taurotragi, and T. lestoquardi, the latter being most closely related to T. annulata.

Application of chemi- and bioluminescence in studies of immunological reactions against protozoa.

The opsonization and lysis of different protozoa by antibodies and/or complement was followed using luminol-dependent chemiluminescence and bioluminescence. The addition of immune serum to variable antigen type populations of Trypanosoma evansi led to the specific opsonization of trypanosomes resulting in an intense metabolic activation and chemiluminescence response of phagocytic cells. In comparison to those of uninfected control mice, the phagocytosis of coccidia merozoites by spleen cells from mice infected with Eimeria falciformis was enhanced during the acute stage of a primary infection. Opsonizing activity was demonstrated in phosphate-buffered saline extracts of gut contents of mice infected for 10 days. The incubation of E. falciformis merozoites together with guinea-pig complement resulted in slow lysis of the cells. The addition of mouse serum collected greater than 6 days after an infection led to an accelerated lysis of the merozoites, indicating the appearance of complement-fixing antibodies in the serum. Heat-inactivated immune serum alone had no lysing activity on merozoites. In the presence of complement, bovine lymphoblastoid cells infected with Theileria annulata were lysed by anti-lymphoblastoid cell serum raised in mice but not by serum from cattle which had developed immunity to Theileria annulata.

The effect of halofuginone, Wellcome 993 C, oxytetracycline, and diminazene diaceturate on Babesia equi-infected lymphoblastoid cell cultures.

The efficacy of halofuginone (DL-trans-7-bromo-6-chloro-3,3-(3-hydroxy-2-piperidyl) acetonyl-4-(3H) quinazolinone), Wellcome 993 C (2-hydroxy-3-cyclohexyl-1,4-naphthoquinone), and oxytetracycline, all of which have been shown to have a schizonticidal effect in the treatment of bovine theileriosis, and the babesicidal drug diminazene diaceturate, were tested against the schizont stages of Babesia equi in cell culture. The in vitro test system measured DNA synthesis in treated and untreated cell lines. Halofuginone (0.02 microgram/ml) and Wellcome 993 C (5 micrograms/ml) suppressed the incorporation of tritiated thymidine by more than 80%. Oxytetracycline was less effective, while diminazene diaceturate showed no notable effect. The insufficient schizonticidal activity may explain the failure of diminazene diaceturate to cure B. equi infections. Schizonticidal drugs either alone, or in combination with known babesicidal drugs, should be tested in the chemotherapy of B. equi infections.

Identification and characterization of Theileria annulata heat-shock protein 90 (HSP90) isoforms.

Heat-shock proteins (HSPs) refer to a group of proteins whose synthesis is enhanced upon sudden increase in temperature or exposure to a variety of other stressors. In this study, Theileria annulata (T. annulata) HSP90 was identified and characterized as a first step to understand the function of this molecule in T. annulata-infected cells. Our results indicated the existence in the genome of T. annulata of two HSP90 genes: one located in chromosome one (TaHSP90-Chr1) and the other in chromosome four (TaHSP90-Chr4). The amino acid alignment between the two isoforms has shown identity and similarity values of 23.52% and 30.26%, respectively. Theileria annulata recombinant HSP90 proteins were expressed using a bacterial expression system and could be recognized in Western blots by rabbit anti-serum raised against an antigenic peptide derived from a unique sequence of TaHSP90-Chr1. On the other hand, bovine HSP90 was detected in T. annulata-infected cells using Western blot and immunocytostaining. To demonstrate the effect of the inhibition of HSP90 on the survival of T. annulata-infected cells, Geldanamycin (GA), a specific inhibitor for HSP90, was used. Upon GA treatment, p53 was observed to translocate into the host cell nucleus, a phenomenon that occurs in cells undergoing apoptosis. Using flowcytometry, a significant increase (P = 0.028) in cell death (%) was observed in T. annulata-infected cells treated with two different GA concentrations, 0.5 and 1 µm, and incubated for 24, 48 and 72 h.

Cytoplasmic sequestration of p53 promotes survival in leukocytes transformed by Theileria.

The function of the p53 protein as the central effector molecule of the p53 apoptotic pathway was investigated in a reversible model of epigenetic transformation. The infection of bovine leukocytes by the intracellular protozoan parasite Theileria annulata results in parasite-dependent transformation and proliferation of the host cells. We found p53 to be largely localized in the host cell cytoplasm and associated with the parasite membrane of isolated schizonts. Curing infected cells of the parasite with the theilericidal drug buparvaquone resulted in a time-dependent translocation of p53 into the host cell nucleus and the upregulation of the proapoptotic Bax and Apaf-1 and the downregulation of the anti-apoptotic Bcl-2 proteins. Although buparvaquone treatment led to apoptosis of the host cell, inhibition of either p53 or Bax significantly reduced buparvaquone-induced apoptosis of the transformed cells. Thus, the p53 apoptotic pathway of host cells is not induced by infection and transformation with Theileria by a mechanism involving cytoplasmic sequestration of p53. The close association of host cell p53 with the parasite membrane implies that the parasite either interacts directly with p53 or mediates cytoplasmic sequestration of p53 by interacting with other host cell proteins regulating p53 localization.

Development of a loop-mediated isothermal amplification (LAMP) assay for rapid diagnosis of Babesia canis infections.

Vector-borne diseases are rising in interest due to global warming, which is believed to impact on the distribution of vectors into new areas thus influencing the occurrence and epidemiology of vector-borne pathogens. Babesia canis belongs to the Piroplasmidae and there are three described subspecies, namely B. canis canis, B. canis rossi and B. canis vogeli. They are each transmitted by a different tick-species, Dermacentor reticulatus, Haemaphysalis leachi and Rhipicephalus sanguineus, respectively. There are also differences in the geographical distribution and pathogenicity to dogs of each subspecies. In this study, we aimed to establish a rapid and easy to perform DNA-based test using loop-mediated isothermal amplification to detect all three Babesia canis subspecies in one assay.

Animal health: harmonisation and distribution of pathogen detection and differentiation tools.

Quality and safe meat production and livestock husbandry are important foci for addressing the wider underlying economic and political challenges. In the last few years, an intense focus of the scientific community has been placed on breakouts of livestock diseases especially in Asia, which have spread into neighbouring countries including Europe. These outbreaks had a serious impact on the livelihood of the farmers as well as the economy of the affected countries. Given this, the establishment of a network of diagnostic facilities is a great demand both at the national and regional levels. In most of the cases, diagnostic assays are either not available or they are not validated. The aim of this collaborative network was to: 1 Distribute and harmonize diagnostic tools required for pathogen detection and differentiation. 2 Build the capacity to ensure the conduction of integrated disease control measures.

Evaluation of Theileria annulata recombinant immunodominant proteins for the development of ELISA.

A number of Theileria annulata genes have been cloned, sequenced and expressed, including TaSP, TaD, TaSE and TamtHSP70. Several recent publications document the suitability of the recombinant TaSP protein for use in the diagnosis of tropical theileriosis. To investigate whether TaD, TaSE or TamtHSP70 elicit a humoural immune response in the T. annulata-infected host and to assess the potential of these proteins for development of diagnostics, a total of 156 field sera from Sudan and 49 negative sera from Germany were investigated in ELISA for the presence of specific antibodies against these recombinant proteins in comparison to TaSP. Antibodies against TaD and TaSE were found to be present, whereas no antibody response could be detected against the recombinant TamtHSP70. Highest titres were found to be present against the TaSP protein, with antibody titres against TaD and TaSE being in general somewhat lower. Correlation analysis showed a significant correlation of TaSP and TaSE and of TaSE and TaD antibody titres, however not between TaSP and TaD. In conclusion, the infected bovine host was shown to produce antibodies against three of the four recombinant T. annulata proteins tested, all three having been described or predicted to be parasite membrane proteins. The outstanding performance of the TaSP protein for detection of T. annulata infection in indirect ELISA was confirmed.

Development of a competitive ELISA for detection of Theileria annulata infection.

In previous studies, Theileria annulata surface protein (TaSP) was identified as an immunodominant antigen and successfully used to develop and validate a recombinant-protein-based ELISA for the detection of circulating antibodies in serum of T. annulata-infected animals. In this study, the same antigen was used to develop a competitive ELISA (cELISA) using a monoclonal antibody that was found to bind to TaSP. The cELISA accurately differentiated T. annulata-infected from uninfected animals and demonstrated a satisfactory performance with a calculated sensitivity and specificity of 77.4% and 100%, respectively. Thus the test proved its suitability for the diagnosis of tropical theileriosis and has application for use in serological surveys to monitor the prevalence of the disease or identify carrier animals with high specificity.

Development and evaluation of a loop-mediated isothermal amplification method for diagnosis of tropical theileriosis.

A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for diagnosis of tropical theileriosis. A set of six primers was designed based on the unique gene of Theileria annulata (Theileria annulata strain Ankara hypothetical protein (GeneDB TA04795). The protocol for the reaction was setup and the specificity and sensitivity of the assay were established. The specificity experiment showed that LAMP primers amplified T. annulata DNA successfully, while no amplification was seen for Theileria parva, Theileria mutans, Theileria sergenti, Theileria sinensis, Babesia bovis as well as bovine genomic DNA and water control. When the sensitivity of LAMP assay was compared with that of conventional PCR a 10-fold higher sensitivity was found, with a detection limit of 10 pg/microl of genomic DNA isolated from a T. annulata-infected cell line. The LAMP product was confirmed by restriction digestion and staining with SYBR Green I. Furthermore, the LAMP assay was applied for the diagnosis of T. annulata in field samples and compared with reverse line blot (RLB), demonstrating that results of the LAMP assay corresponded to those of RLB. These results indicate that the LAMP assay is rapid and simple to run, cost-effective, sensitive and specific and has potential usefulness for application in epidemiological studies on T. annulata infection of cattle.