Assessment of the prevalence of Theileria lestoquardi in sheep from the Sudan using serological and molecular methods.

Malignant theileriosis of sheep and goats caused by Theileria lestoquardi is considered to be among the most important tick borne diseases in the Sudan. Information on the prevalence of the disease in different parts of the Sudan is limited. The purpose of this study was to estimate the prevalence of the disease in five states of the Sudan using molecular and serological assays. A total of 393 blood and serum samples from clinically asymptomatic sheep were analysed using nested reverse line blot (nRLB) and loop mediated isothermal amplification (LAMP), as well as an enzyme-linked immunosorbent assay (ELISA). The results indicated a sero-prevalence of 33.8% while RLB and LAMP assays revealed molecular prevalences of 29.5 and 22.6% respectively. The prevalence of Theileria lestoquardi varied significantly according to the geographical origin of the infected animals, whereas age and gender did not have a significant effect. RLB data indicated that T. lestoquardi usually occurred as a co-infection with the non-pathogenic Theileria ovis. Using RLB as a gold standard, a sensitivity of 68.1% and a specificity of 96.4% were recorded for LAMP and a sensitivity of 75.9% and a specificity of 83.8% for ELISA. The Kappa coefficient between nRLB and LAMP indicated a significant level of agreement (0.692), but only moderate concordance (0.572) between nRLB and ELISA. The results of the present study confirm and extend earlier findings regarding the widespread of T. lestoquardi infections in sheep in the Sudan. The data provide evidence that should enable the veterinary authorities to deploy appropriate control measures.

Phylogenetic analysis of some Newcastle disease virus isolates from the Sudan.

A reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify 1412 bp of the fusion protein gene (F gene) of four Newcastle disease virus (NDV) isolates; two velogenic (TY-1/90 and DIK-90) and two lentogenic isolates (Dongla 88/1 and GD.S.1). Following sequencing, nucleotide sequences were annotated and 894 bp were compared phylogenetically with those from strains previously reported in the Sudan and the virus strains published on the GenBank. It could be demonstrated that TY-1/90 and DIK-90 strains belong to the genotype VI of NDV and are in close genetic relationship to sub- genotype VIb. TY-1/90 and DIK-90 strains were observed to be genetically unrelated to the earlier Sudanese isolates of 1970/80s and the late of 2000s suggesting a different origin. The close genetic relationship to the European and African pigeon paramyxovirus type 1 (PPMV-1) suggests a common ancestor. Dongola, GD.S.1 strains were classified into genotype II that comprises non-pathogenic lentogenic NDV strains. The present genetic classification of NDV isolates of the Sudan provides valuable information on genotypes of NDV. Further molecular epidemiological investigations of the recent outbreaks of Newcastle disease in the Sudan are needed in order to improve the efficiency of control strategies and vaccine development.

Identification and partial characterization of a Salp15 homolog from Ixodes ricinus.

The immunomodulatory molecule Salp15 is originally described in Ixodes scapularis and has been shown to inhibit CD4 T cell activation. Many Salp15 homologs have been described from Ixodes species, and all were well conserved at C-terminal residues that seem to be essential for the function of the protein. In this study, a gene sequence was amplified from cDNA isolated from engorged female I. ricinus ticks, which was predicted to generate a protein of 12.3 kDa. The protein displayed distinct amino acid differences from previously described I. ricinus Salp15 homologs, with amino acid identity ranging between 46.6% and 93.9%. It was referred to as I. ricinus Salp15-like protein. The protein showed 48.1% sequence identity to I. scapularis Salp15. We analyzed the effect of the recombinant I. ricinus Salp15-like protein on the production of cytokines from human peripheral blood mononuclear cells stimulated with LPS. The recombinant protein exerted no effect on the production of TNF-α and IL-6, but the production of IL-10 was dose-dependently reduced. It can be concluded that I. ricinus Salp15-like protein exerts an immunomodulatory effect on the host. The inhibition of IL-10 production may possibly lead to a retardation of B cell activity.

Coinfection of sheep with Anaplasma, Theileria and Babesia species in the Kurdistan Region, Iraq.

Infections of small ruminants with Anaplasma, Theileria and Babesia species are widely distributed in the old world and are of great economic impact. In Iraq, data on disease occurrence in sheep caused by above-mentioned infectious agents are scarce. This study provides information on various haemoparasitic agents infecting sheep in the Kurdistan Region, Iraq, using molecular diagnostic tools. Altogether, 195 samples originating from three governorates in the Kurdistan Region, namely Duhok, Erbil and Sulaimaniya, were analysed. The following pathogens were identified: Anaplasma ovis (62.6%), Theileria ovis (14.35%), T. lestoquardi (7.7%), T. uilenbergi (5.6%) and Babesia ovis (1.5%). T. uilenbergi is detected for the first time in Iraq. Coinfection of sheep with different pathogens could be observed in this study, and it was found that 45 of 195 (23%) of the samples contained more than one pathogen. Even triple-positive samples were identified in 3% of the investigated animals. In conclusion, we confirm the coinfection of sheep with various haemoparasitic pathogen species in the Kurdistan Region of Iraq. Further investigations are needed to reveal the epidemiology of the diseases, the respective tick vectors, and, in the case of coinfection, pathogens' interaction and possible cross-protection.

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.

Whole antigenic lysates of Ixodes ricinus, but not Der-p2 allergen-like protein, are potent inducers of basophil activation in previously tick-exposed human hosts.

The clinical suspicion of tick anaphylaxis is based on a history of the bite and occurs often during the warm season. Further arguments are the presence of natural hosts in the immediate environment and, eventually, the identification of the tick. The diagnosis is confirmed when immediate-type sensitization is shown by positive skin prick tests performed with specific tick extracts or the demonstration of specific IgE in vitro. In the current study, we hypothesize that hard tick-derived material contains potent inducers being able to promote basophil stimulation, which correlates with a sensitization immunological response following tick bites. To this end, biological material from two hard tick cell lines (IRE11 and IDE8 - derived from Ixodes ricinus and I. scapularis, respectively) as well as I. ricinus salivary gland and body lysates were used in a human basophil activation test (BAT) to analyse binding and cross-linking capacity of membrane-bound IgE, because basophils are one of the main effector cells of allergic reactions. Additionally, Der-p2 allergen-like gene from I. ricinus was recombinantly expressed as a 15-kDa histidine-tagged fusion protein, purified and included as a stimulus within the setup. Blood was drawn and submitted to BAT screening from a pool of 36 individuals, both bitten and who served solely as negative controls. We have found that seven subjects (19%), all of whom were at least two times tick-bitten, positively reacted to the aforementioned stimuli, whereas the reactivity level of the ones bearing single bites proved to be within the normal range. Moreover, no significant upregulation of the assessed basophil activation marker was detected in the case of Der-p2, except a faint reaction at high dosages. We conclude that at least two tick bites of the human host must occur in order to induce significant basophil activation.

Prevalence of variations of the cystic artery in the Sudanese.

The anatomy of the cystic artery is very variable, creating potential problems during surgery. This study documents variations in the origin of the cystic artery and its location in relation to the biliary ducts among 106 Sudanese people and compared the variations between the sexes and races. The cystic artery originated from the right hepatic artery in 78% of cases, the common hepatic artery in 17%, the left hepatic artery in 2% and the gastroduodenal artery in 3% (other arteries 0%). No differences were found between the sexes. Statistically significant variations in the origin and position of the cystic artery were found comparing these data with previous studies in Caucasians and Asians.

Prevalence of variations of the cystic artery in the Sudanese

The anatomy of the cystic artery is very variable, creating potential problems during surgery. This study documents variations in the origin of the cystic artery and its location in relation to the biliary ducts among 106 Sudanese people and compared the variations between the sexes and races. The cystic artery originated from the right hepatic artery in 78% of cases, the common hepatic artery in 17%, the left hepatic artery in 2% and the gastroduodenal artery in 3% [other arteries 0%]. No differences were found between the sexes. Statistically significant variations in the origin and position of the cystic artery were found comparing these data with previous studies in Caucasians and Asians

Prevalence of Theileria equi and Babesia caballi infections in horses belonging to resource-poor farmers in the north-eastern Free State Province, South Africa.

The prevalence of Theileria equi and Babesia caballi infections in the north-eastern Free State Province of South Africa was determined by examination of thin and thick Giemsa-stained blood smears, IFAT and PCR. No parasites were detected by microscopy from any blood samples collected at five study sites, Qwaqwa, Kestell, Harrismith, Vrede and Warden. Of the tested serum samples, 28/29 (96.5%), 20/21 (95.2%) and 42/42 (100%) were positive by IFAT for T. equi infections in Harrismith, Kestell and Qwaqwa, respectively, and 5/29 (17.2%), 13/21 (61.9%) and 30/42 (71.4%) were sero-positive for B. caballi infections in Harrismith, Kestell and Qwaqwa, respectively. All DNA samples from the study sites were negative for B. caballi infections by PCR, but five samples, two from each of Kestell and Warden and one from Vrede, were PCR positive for T. equi infections. The high prevalence of antibodies against T. equi and B. caballi in the sampled horses indicates that the animals had been exposed to T. equi and B. caballi infections but the absence of parasitaemia and very low number of positive PCR samples, however, imply that T. equi and B. caballi are endemically stable in the north-eastern Free State Province.

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.

Diagnosis of Babesia caballi and Theileria equi infections in horses in Sudan using ELISA and PCR.

The purpose of this study was to estimate the prevalence of equine piroplasmosis in Sudan. The presence of antibodies against Babesia caballi and Theileria equi was determined in serum samples obtained from 158 horses raised in different locations in Sudan by enzyme-linked immunosorbent assay (ELISA). The B. caballi 48-kDa and the T. equi EMA-2 purified recombinant proteins were used as antigens in the ELISA test. Results showed that seven (4.4%) were positive for B. caballi and 80 (63.5%) were positive for T. equi. Polymerase chain reaction (PCR) assays have been applied using primers targeting the B. caballi 48-kDa merozoite antigen, the T. equi SSUrRNA and the T. equi EMA-1 genes. PCR performed on 131 blood spots in filter paper revealed that 33 (25.2%) samples were positive for T. equi but no positives were found for B. caballi. It is concluded that equine piroplasmosis is endemic in the country. This is the first study on serological and molecular epidemiological diagnosis on equine piroplasmosis in Sudan.

First report of Cryptosporidium deer-like genotype in Malaysian cattle.

Fifty faecal samples from diarrheic calves between 1 and 6 months old were collected per rectum from 5 farms around Petaling District in Selangor, Malaysia for Cryptosporidium species detection and genotyping investigation. Oocysts were purified using sedimentation and gradient centrifugation, then examined by immunofluorescence assay (IFAT). Genomic DNA was extracted from all samples and nested PCR was performed to amplify the SSU rRNA gene. Eighteen samples (36%) were positive for Cryptosporidium species by PCR. The sequence and phylogenetic analysis of 14 isolates indicated that Cryptosporidium parvum was most common (11 isolates) followed by Cryptosporidium deer-like genotype (3 isolates). The present work reports the first data on Cryptosporidium genotyping from cattle in Malaysia.

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.

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.