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.

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.

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.

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.

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.

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.

Improved diagnosis for nine viral diseases considered as notifiable by the world organization for animal health.

Nine viral diseases included in the World Organization for Animal Health list of notifiable diseases (former list A) were chosen for their contagiousness and high capacity of spreading to improve their diagnosis using new and emerging technologies. All the selected diseases--foot-and-mouth disease, swine vesicular disease, vesicular stomatitis, classical swine fever, African swine fever, bluetongue, African horse sickness, Newcastle disease and highly pathogenic avian influenza--are considered as transboundary diseases, which detection causes the prohibition of livestock exportation, and, thus, it leads to high economical losses. The applied diagnostic techniques can fall into two categories: (i) nucleic-acid detection, including padlock probes, real-time PCR with TaqMan, minor groove binding probes and fluorescence energy transfer reaction probes, isothermal amplification like the Cleavase/Invader assay or the loop-mediated amplification technology and the development of rapid kits for 'mobile' PCR and (ii) antigen-antibody detection systems like simplified and more sensitive ELISA tests. Besides, internal controls have been improved for nucleic acid-detecting methods by using an RNA plant virus--Cowpea Mosaic Virus--to ensure the stability of the RNA used as a positive control in diagnostic real-time RT-PCR assays. The development of these diagnosis techniques has required the joint efforts of a European consortium in which nine diagnostic laboratories and an SME who have collaborated since 2004 within the European Union-funded Lab-on-site project. The results obtained are shown in this paper.

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.

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.

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.

Determination of potential risk factors associated with Theileria annulata and Theileria parva infections of cattle in the Sudan.

A multi-variate logistic regression analysis was performed on two sets of data on the prevalence of Theileria annulata in Northern Sudan and Theileria parva in Southern Sudan, to determine the potential risk factors that might affect the distribution of the infections in those regions. The logistic regression model was fit with the tested risk factors for each disease, separately. The results indicated that locations, management systems and age could be held as risk factors for T. annulata infection in Northern Sudan, while for T. parva locations and seasons could be held as risk factors in Southern Sudan. The results of this study will assist in the development of more effective control strategies for smallholder dairy farms in the country.

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.

Epidemiological studies on tick-borne diseases of cattle in Central Equatoria State, Southern Sudan.

A herd-based study was carried out in Central Equatoria State, Southern Sudan, to study epidemiological aspects of tick-borne diseases. Six herds of cattle situated in three different locations were selected and investigated every 3 months during the year 2005. Blood smears for Giemsa staining and blood spots on filter paper for deoxyribonucleic acid extraction were collected from 600 apparently healthy indigenous cattle. A total of 69 (11.5%) samples showed the presence of piroplasms in Giemsa-stained blood smears, and polymerase chain reaction increased the detection limit to 297 (49.5%). Using reverse line blot, it was possible to detect and differentiate eight different piroplasms namely, Theileria parva (71.2%), Theileria mutans (73%), Theileria velifera (45.3%), Theileria taurotragi (2.7%), Theileria buffeli (0.5%), Theileria annulata (0.2%), Babesia bovis (1.7%), and Babesia bigemina (0.3%). Mixed infections were detected in 406 samples (67.7%) accounting for 17 different combinations. High infection of Theileria parva was reported among young calves compared to older cattle. The highest prevalence of Theileria parva was reported in the rainy season (October). The implications of these results on the epidemiology of tick-borne diseases are discussed with emphasis on East Coast fever.

Molecular characterization of a Theileria lestoquardi gene encoding for immunogenic protein splice variants.

A Theileria lestoquardi schizont cDNA library was screened using sera collected from sheep recovering from a natural malignant theileriosis infection. An immunogenic clone (clone-5) was isolated and its full sequence was obtained using rapid amplification of cDNA ends polymerase chain reaction (PCR) technique. PCR experiments and sequencing demonstrated the presence of two transcript forms of the gene, resulting from splicing variation at the single intron found in the gene. Both gene products, clone-5 long and clone-5 short variants with calculated molecular weights of 99.9 and 72.7 kDa, respectively, were expressed in a T. lestoquardi-infected cell line. BLAST searches suggested the presence of homologues of the gene in both the Theileria parva and Theileria annulata genomes, with identities of 53 and 62% on the DNA level, respectively. The intron was preserved in size, sequence, and location within the gene in these parasites. Analysis of the subcellular localization of the clone-5 proteins showed a predominant parasite membrane association in T. lestoquardi-infected cells. Both recombinantly produced forms were found to be reactive with sera from infected animals. Bioinformatic analyses were employed to address the possible function of the gene products in the biology of T. lestoquardi.

Development of a recombinant indirect ELISA for the diagnosis of Theileria sp. (China) infection in small ruminants.

Theileria sp. (China) causes severe limitations on the development of the livestock industry in the north-west of China. An enzyme-linked immunosorbent assay (ELISA) based on merozoite homogenate of the parasite for diagnosis of infection has been established; however, cross-reactivity with other small ruminant-infecting piroplasms could not be excluded. Thus, a prerequisite for epidemiological surveys and diagnosis was the establishment of a recombinant protein-based ELISA. To this end, serum from Theileria sp. (China)-infected sheep was used to screen a Theileria lestoquardi expression library, resulting in the identification of a specifically reacting clone with a high identity to the heat shock protein 70 (HSP70) of Theileria parva and Theileria annulata and thus named TlHSP70. An HSP70 homologue was also confirmed to be expressed by Theileria sp. (China) merozoites (TcHSP70). A part of the TlHSP70 protein, found to be conserved in TcHSP70, was recombinantly expressed and used to establish an ELISA. A total of 260 field serum samples tested resulted in a sensitivity and specificity of 94.3 and 89.5%, respectively, in comparison with the merozoite homogenate ELISA. The potentials of the application of the test in epidemiological surveys to map out the prevalence of the disease and for routine diagnostics are described.

A new recombinant protein-based ELISA for the diagnosis of malignant theileriosis of sheep and goats.

Tick-borne diseases of small ruminants are of highly economic importance in many countries. Malignant theileriosis of sheep and goats caused by Theileria lestoquardi is considered among the most important of these diseases and constitutes an obstacle to the sheep industry in countries like the Sudan. Here the application of a newly discovered surface protein of T. lestoquardi (Clone-5) in an enzyme-linked immunosorbent assay (ELISA) and the potentials of the application of the test in epidemiological surveys and diagnosis are described. Clone-5 contains a predicted number of 20 antigenic determinant sites and two polypeptides derived from the protein were recombinantly produced, purified and tested with control serum samples in both ELISA and Western Blot. One of the polypeptides was further used in validation experiments that involved the testing of negative and positive field serum samples collected from an area that had witnessed an outbreak of malignant theileriosis in Northern Sudan. ELISA, based on this recombinant protein, demonstrated a satisfactory performance with a calculated sensitivity and specificity of 94.6 and 88%, respectively, when countertested with a standard indirect fluorescent antibody test (IFAT). Moreover, no cross-reactions could be demonstrated against Theileria species (China) nor Cowdria spp. This test is recommended for further field validation experiments.

Is the 10th and 11th intercostal space a safe approach for percutaneous nephrostomy and nephrolithotomy?

The aim of this study was to determine the rate of complications in percutaneous nephrostomy (PCN) and nephrolithotomy (PCNL) performed through the 11th and 10th intercostal spaces using our monitoring technique and to discuss the safety of the procedure. Out of 398 PCNs and PCNLs carried out during a 3-year period, 56 patients had 57 such procedures performed using an intercostal approach. The 11th intercostal route was used in 42 and the 10th in 15 cases. One patient had two separate nephrostomies performed through the 10th and 11th intercostal spaces. The technique utilizes bi-planar fluoroscopy with a combination of a conventional angiographic machine to provide anterior-posterior fluoroscopy and a C-arm mobile fluoroscopy machine to give a lateral view, displayed on two separate monitors. None of the patients had clinically significant thoracic or abdominal complications. Two patients had minor chest complications. Only one developed changes (plate atelectasis, elevation of the hemi-diaphragm) directly related to the nephrostomy (2%). The second patient had bilateral plate atelectasis and unilateral congestive lung changes after PCNL. These changes were not necessarily related to the procedure but rather to general anesthesia during nephrolithotomy. The authors consider PCN or PCNL through the intercostal approach a safe procedure with a negligible complication rate, provided that it is performed under bi-planar fluoroscopy, which allows determination of the skin entry point just below the level of pleural reflection and provides three-dimensional monitoring of advancement of the puncturing needle toward the target entry point.

Application of the recombinant Theileria annulata surface protein in an indirect ELISA for the diagnosis of tropical theileriosis.

The recombinant surface protein of Theileria annulata (TaSP) was used in the standardization and validation of an enzyme linked immunosorbent assay (ELISA) for the detection of circulating antibodies against tropical theileriosis. ELISA data were expressed as the percentage positivity (PP) of the reactivity of an internal positive control. A total of 50 sera samples from a disease-free area were used for the calculation of the cut-off value which served as a threshold between the positive and the negative sera samples. This was determined as the mean PP plus two standard deviations or the twice the mean PP of the results obtained with these negative samples. The obtained thresholds were 17.8% and 18.3%, respectively. Accordingly, the reactivity of 140 field sera samples collected at random from an area known to be endemic for tropical theileriosis in Sudan was determined as PP values which were then compared to the results obtained using the indirect fluorescence antibody test (IFAT) from the same samples. Both tests showed a high degree of correlation. The TaSP-ELISA had a sensitivity of 99.1% and specificity of 90.47% when taking the IFAT as a reference test. Our test has proved its suitability for the diagnosis of tropical theileriosis and could be used in serological surveys to map out the prevalence of the disease or to monitor vaccination efficiencies in disease-free populations.