Improved molecular detection of Ehrlichia and Anaplasma species applied to Amblyomma ticks collected from cattle and sheep in Ethiopia.

Detection of Ehrlichia and Anaplasma species in animals and tick vectors is crucial for an understanding of the epidemiology of diseases caused by these pathogens. In this study, a pair of primers designated EBR2 and EBR3 was designed from the Anaplasma 16S rDNA sequence and was used along with a previously described primer EHR 16SD for the simultaneous detection of Ehrlichia and Anaplasma species by nested PCR. The primers were used to amplify 925bp of DNA from known species of Ehrlichia and Anaplasma. Restriction with MboII and MspI enzymes allowed Ehrlichia and Anaplasma speciation. Restriction with MboII differentiated between An. marginale, Anaplasma (formerly Ehrlichia) sp. Omatjenne, and An. centrale with An. marginale and Anaplasma (formerly Ehrlichia) sp. Omatjenne yielding 2 distinct fragments each while An. centrale produced 3 distinct bands. Ehrlichia ruminantium and An. phagocytophylum remained undigested. Subsequent restriction with MspI differentiated E. ruminantium from An. phagocytophylum with 2 and 4 fragments, respectively. When used on tick samples from the field, 63 ticks (16.4%) out of 384 collected from cattle and sheep were positive for one or more species of Ehrlichia and Anaplasma. The positivity ranged from 6.3% at Andasa to 36.7% at Habernosa. Higher overall infection rates were found in Amblyomma lepidum than in Amblyomma variegatum ticks (p=0.009). Amblyomma lepidum from Habernosa were more often infected with all detected species of Anaplasma and Ehrlichia than Am. variegatum. At Bako, however, Anaplasma (formerly Ehrlichia) sp. Omatjenne was detected only in Am. variegatum. A significantly higher proportion of ticks collected from cattle (20.6%) was found positive than in those collected from sheep (3.3%) (p=0.003). Simultaneous detection of Ehrlichia and Anaplasma species and correct identification of mixed infections was possible. Since the ticks were collected from animals, the occurrence of the major species of Ehrlichia and Anaplasma in ruminants in the area is confirmed.

Detection of Schmallenberg virus in different Culicoides spp. by real-time RT-PCR.

To identify possible vectors of Schmallenberg virus (SBV), we tested pools containing heads of biting midges (Culicoides) that were caught during the summer and early autumn of 2011 at several places in Belgium by real-time RT-PCR. Pools of heads originating from following species: C. obsoletus complex, C. dewulfi and C. chiopterus were found positive, strongly indicating that these species are relevant vectors for SBV.

A new tool for the molecular identification of Culicoides species of the Obsoletus group: the glass slide microarray approach.

Culicoides species of the Obsoletus group (Diptera: Ceratopogonidae) are potential vectors of bluetongue virus serotype 8 (BTV 8), which was introduced into central Western Europe in 2006. Correct morphological species identification of Obsoletus group females is especially difficult and molecular identification is the method of choice. In this study we present a new molecular tool based on probe hybridization using a DNA microarray format to identify Culicoides species of the Obsoletus group. The internal transcribed spacer 1 (ITS1) gene sequences of 55 Culicoides belonging to 13 different species were determined and used, together with 19 Culicoides ITS1 sequences sourced from GenBank, to design species-specific probes for the microarray test. This test was evaluated using the amplified ITS1 sequences of another 85 Culicoides specimens, belonging to 11 species. The microarray test successfully identified all samples (100%) of the Obsoletus group, identifying each specimen to species level within the group. This test has several advantages over existing polymerase chain reaction (PCR)-based molecular tools, including possible capability for parallel analysis of many species, high sensitivity and specificity, and low background signal noise. Hand-spotting of the microarray slide and the use of detection chemistry make this alternative technique affordable and feasible for any diagnostic laboratory with PCR facilities.

An outbreak of haemolytic anaemia associated with infection of Theileria orientalis in naive cattle.

CASE HISTORY: An outbreak of haemolytic anaemia occurred when 87 cattle were introduced from a presumed non-infected herd from south Otago to a herd in Northland (n=580 cows), New Zealand, where theileriosis is endemic. CLINICAL FINDINGS: Clinical signs associated with Theileria spp. infection included lethargy, anorexia, inappetance, pale mucous membranes, and varying severity of anaemia. In the naive imported cattle, 11/29 (38%) of those tested showed haematological signs of anaemia (haematocrit (HCT) <0.25 L/L). A negative association was present between the HCT and the number of Theileria spp. organisms counted using light microscopy (correlation coefficient=-0.4; p<0.05). Haemoparasites consistent with Theileria spp. were observed on examination of a blood smear. Theileria orientalis group (Theileria buffeli/orientalis) species was confirmed using PCR and DNA sequencing, and other causes for anaemia were excluded in the most clinically severely affected cow. The 18S sequence data and phylogenetic analysis of the CoxIII sequences showed samples had the greatest similarity to T. orientalis Chitose from Japan. DIAGNOSIS: Haemolytic anaemia associated with infection of T. orientalis. CLINICAL RELEVANCE: Previous reports have suggested that T. orientalis group species may be non-pathogenic in healthy cattle, and an incidental finding in blood samples. However, this investigation provided evidence that in New Zealand, this pathogen is capable of causing clinical disease in cattle not necessarily debilitated by another disease. The potential for disease should be considered when naive cattle are brought in from non-endemic to endemic regions, for instance cattle from the South Island moved to regions where the vector for T. orientalis group species, Haemaphysalis longicornis, is active, and T. orientalis is present.

Demonstration of differences in virulence between two Theileria parva isolates.

In areas with a low incidence of infection due to unimodal presence of ticks, Theileria parva has been observed to induce a disease with relatively low pathology. This is followed by a carrier state, rather than death and therefore provides a better chance of transmission of the parasite back to the tick vector since in unimodal conditions, the different tick stages occur at different times. One isolate from such an area in Zambia, T. parva Chitongo, was compared for virulence with T. parva Muguga, isolated from an area exhibiting a continuous presence of all vector stages in East Africa. To reduce any variation due to infection dose, an in vitro standardized dose was used to initiate infection of groups of three local zebu cattle with each isolate. Parameters of virulence measured were prepatent period, fever, survival (based on ECF index), parasitosis, piroplasm parasitaemia and hematological parameters. Our results suggest that T. parva Chitongo developed a slightly later onset (1-2 days) and lower levels of parasitosis in the lymph node, causing less and later mortality. Comparison of the in vitro rate of transformation confirmed that the time needed to transform an infected lymphocyte took 4 days longer for T. parva Chitongo than T. parva Muguga. Elucidating the mechanism responsible for the lower virulence of T. parva Chitongo could be useful for designing an attenuated vaccine.

Endemic type of animal trypanosomiasis is not associated with lower genotype variability of Trypanosoma congolense isolates circulating in livestock.

In order to verify whether the low impact on livestock production in endemic areas is related to a low number of trypanosome strains circulating in livestock, 37 Trypanosoma congolense isolates collected from cattle in 11 sites in an endemic trypanosomiasis area in Eastern Zambia were characterised for genotype variability using a modified amplified fragment length polymorphism technique (AFLP). Isolates were further cloned to evaluate the occurrence of mixed infections in individuals. The results obtained revealed a high genotype diversity (94.6%) among these isolates. Apart from one site, all isolates gave different AFLP profiles in each of the sites. When clones were compared, three (8%) of the 37 isolates had mixed infections. These results indicate the circulation of a high number of strains in this trypanosomiasis endemic area despite the low impact the disease has on livestock production.

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.

The comparative role of cattle, goats and pigs in the epidemiology of livestock trypanosomiasis on the plateau of eastern Zambia.

To determine and compare the prevalence of trypanosome infections in different livestock species (cattle, pigs and goats) in areas where game animals are scarce and livestock constitute the main food source of tsetse, a survey was conducted on the plateau of the Eastern Province of Zambia in Katete and Petauke districts where Glossina morsitans morsitans is the only tsetse species present. Blood was collected from a total of 734 cattle, 333 goats and 324 pigs originating from 59 villages in both districts and was examined using the buffy coat method and the PCR-RFLP as diagnostic tools. The prevalence of trypanosome infections differed substantially between livestock species. Using microscopic diagnostic methods, trypanosome infections were detected in 13.5% of the cattle and 0.9% of the pigs. All goats were parasitologically negative. The PCR-RFLP analyses increased the trypanosomiasis prevalence to 33.5, 6.5 and 3.3% in cattle, pigs and goats respectively. The majority of the infections (91.2%) were due to Trypanosoma congolense. The presence of a trypanosome infection in cattle and pigs resulted in a significant decline in the packed cell volume. The outcome of the study clearly shows that despite the availability of goats and pigs, cattle seem to be the major livestock species affected by the disease in trypanosomiasis endemic areas. The high proportion of infections in cattle could be partly attributed to their higher availability and attractiveness to tsetse.

Molecular detection of Ehrlichia ruminantium infection in Amblyomma variegatum ticks in The Gambia.

In West Africa, losses due to heartwater disease are not known because the incidence/prevalence has not been well studied or documented. To develop a diagnostic tool for molecular epidemiology, three PCR-based diagnostic assays, a nested pCS20 PCR, a nested map1 PCR and a nested reverse line blot (RLB) hybridization assay, were evaluated to determine their ability to detect infection in vector ticks, by applying them simultaneously to A. variegatum field ticks to detect Ehrlichia ruminantium, the causative agent of heartwater. The nested pCS20 PCR assay which amplified the pCS20 gene fragment showed the highest detection performance with a detection rate of 16.6%; the nested map1 PCR, which amplified the gene encoding the major antigenic protein1 (map1 gene) showed a detection rate of 11% and the RLB, based on the 16S rDNA sequence of anaplasma and ehrlichial species, detected 6.2%. The RLB, in addition, demonstrated molecular evidence of Ehrlichia ovina, Anaplasma marginale and Anaplasma ovis infections in The Gambia. Subsequently, the pCS20 assay was applied to study the prevalence and distribution of E. ruminantium tick infection rates at different sites in five divisions of The Gambia. The rates of infection in the country ranged from 1.6% to 15.1% with higher prevalences detected at sites in the westerly divisions (Western, Lower River and North Bank; range 8.3-15.1%) than in the easterly divisions (Central River and Upper River; range 1.6-7.5%). This study demonstrated a gradient in the distribution of heartwater disease risk for susceptible livestock in The Gambia which factor must be considered in the overall design of future upgrading programmes.

An outbreak of East Coast Fever on the Comoros: a consequence of the import of immunised cattle from Tanzania?

In 2003 and 2004, a severe epidemic decimated the cattle population on Grand Comore, the largest island of the Union of Comoros. Fatalities started soon after the import of cattle from Tanzania. Theileria parva and its vector, Rhipicephalus appendiculatus, could be identified as the main culprits of the epidemic. Characterisation by multilocus genotyping revealed that the T. parva parasites isolated on the Comoros were identical to the components of the Muguga cocktail vaccine used in Tanzania to immunise cattle. Therefore, it is believed that East Coast Fever reached the Comoros while some of the imported livestock got infected in Tanzania by ticks of which the immature stadia fed on Muguga cocktail vaccinated animals. Since the Comorian government neither has the financial means nor the competent staff to pursue an adequate epidemiosurveillance, the danger exists that without external assistance and in a context of continuing globalisation more transboundary diseases will affect the Comorian livestock sector in the future.

Epidemiological studies on Theileriosis and the dynamics of Theileria parva infections in Rwanda.

An epidemiological analysis based on three country wide surveys was carried out to determine the prevalence of infections with Theileria spp. in Rwanda. In the 1998 dry season, a total of 264 blood samples were submitted to Theileria spp. characterisation using the 18S species-specific PCR-RFLP assay. The same samples together with 634 samples (317 samples/season) collected during the 2002 dry season and the 2003 wet season were further analysed using the p104 Theileria parva specific PCR. The results from the 18S characterisation showed the presence of four Theileria spp., namely T. parva, T. mutans, T. taurotragi and T. velifera in the field. Half of the animals had multiple Theileria spp. infections. T. parva was the most prevalent and a high correlation (94%) was found between the prevalence results using the 18S and the p104 PCR assays. The prevalence of T. parva infections was stable over time and over season but decreased significantly from the high land to the low land areas. This unexpected trend cannot be explained alone by ecology or the dynamics of the tick population in the different zones, many other components such as breed type, tick control practices and grazing system are likely to play a role. Another important finding was the fact that young animals are infected early in life in all regions except in the high land zone indicating the existence of a particular epidemiological situation in this part of the country.

SSCP analysis of the P2 purine transporter TcoAT1 gene of Trypanosoma congolense leads to a simple PCR-RFLP test allowing the rapid identification of diminazene resistant stocks.

Analyses were made on a Trypanosoma congolense contig coding a putative P2-like nucleoside transporter (the contig was named in this study TcoAT1). The sequence includes a start and stop codon and presents a high similarity with the gene TbAT1 of T. brucei (Smallest Sum Probability 2.8e-136). To investigate a possible link between point mutations and diminazene aceturate (DA) resistance in mice, the TcoAT1 putative genes of 26 T. congolense strains, characterised for DA sensitivity in the single dose mouse test, were screened by means of the Single Strand Conformation Polymorphism technique (SSCP). Results showed that the SSCP profiles of 23 out of 26 (88.5%) T. congolense strains were confirmed by the sensitivity test in mice with the commonly accepted criterion for sensitivity to diminazene being a CD80 of 20mg/kg in the mouse test. The remaining T. congolense strains showed a resistant SSCP profile and relapsed in mice after treatment at doses lower than 20mg/kg indicating that the SSCP is more sensitive than the single dose mouse test for the detection of resistance to diminazene. However, none of the strains used in this study showed a sensitive SSCP profile while they were resistant in the single dose mouse test. The sequencing of the TcoAT1 gene of two sensitive, two intermediate and two resistant strains allowed the set up of a PCR-RFLP test for the discrimination between sensitive and resistant strains confirming the SSCP results for the 26 strains of this study.

Trypanosomosis prevalence in cattle on Mafia Island (Tanzania).

During two consecutive surveys (February and August/Sept 2002), a total of 970 cattle from the cattle population of Mafia Island (United Republic of Tanzania) were blood-sampled. All blood samples were microscopically screened for the presence of trypanosomes and a portion of these were checked for antibodies with an Ab-ELISA and for the presence of trypanosomal DNA with PCR. Microscopic evidence of trypanosomes of the congolense group (sub-genus Nannomonas) was found in 0.8% of the animals (8/970) and in two cases the species identified was confirmed by PCR as Trypanosoma congolense savannah type. Non-pathogenic Trypanosoma theileri were detected in 3.2% (31/970) of the samples using the Dark Ground-Buffy Coat (DG-BC) technique. For survey 1 (S1), detection of antibodies (Ab-ELISA) against pathogenic trypanosomes indicated a seroprevalence of 14.2% (68/480). Of the samples, either DG positive or with a PCV lower then 25, examined by PCR, a total of 8.4% (5/59) (selected from 970 samples), were found positive for T. congolense. The low prevalence of pathogenic trypanosomes on Mafia Island is intriguing, especially in view of the omnipresence of the tsetse fly Glossina brevipalpis. Although the presence of detected trypanosomal antibodies does not necessarily indicate a current infection, the combination of serological/parasitological examinations and the results of the PCR do support this low prevalence of trypanosomosis in cattle. Despite the low prevalence, pathogenic trypanosomes are present on Mafia Island and possible reasons for this low infection rate, taking account of the relation between Glossina species present, transmission risk and trypanosomes found in cattle, are discussed also in view of a future appropriate intervention strategy.

Comparison of the virulence of Trypanosoma congolense strains isolated from cattle in a trypanosomiasis endemic area of eastern Zambia.

The virulence of 31 genetically different Trypanosoma congolense strains belonging to the Savannah subgroup and isolated from cattle at 11 sites in a trypanosomiasis endemic area of eastern Zambia was compared. Virulence testing, done in OF1 mice, revealed three virulence categories. Strains were considered extremely virulent when the median survival time ranged between 5 and 9 days. Moderately virulent strains had a median survival time between 10 and 30 days and low virulence, more than 30 days. For each strain, the prepatent period was determined and the PCV of the infected animals was measured at regular intervals. A total of six (19.4%) strains belonged to the extremely virulent category with a short prepatent period (mean 2.3+/-0.3 days), high parasitaemia, decline in PCV of 15.6+/-1.1% during the first 7 days p.i. and a short median survival time (mean 6 days). The remainder of the strains belonged to the moderate (13 strains) or low (12 strains) virulence categories with median survival times of 13 and 60 days, respectively. They had longer prepatent periods (means 3.2+/-1.6 days and 3.5+/-1.6 days for moderately virulent and strains with low virulence, respectively) and the decline in PCV was less steep (decline of 14.2+/-0.6 and 9.7+/-0.6% during the first 7 days of infection with moderately virulent strains and strains with low virulence, respectively). Extremely virulent strains were isolated from cattle at four sampling sites with 60% of the cattle from one sampling site harbouring such extremely virulent strains. Results from this study demonstrated substantial differences in the virulence of T. congolense strains of the Savannah subgroup, isolated in one geographic area from a single host species. On the assumption that information on virulence obtained from tests in mice can be extrapolated to cattle, the high proportion of strains with low to moderate virulence is thought to be attributed to the important role of susceptible cattle as reservoirs of trypanosomes in the study area and the ensuing selection against extremely virulent strains.

A modified AFLP for Trypanosoma congolense isolate characterisation.

The amplified fragment length polymorphism (AFLP) technique is a reliable and powerful DNA fingerprint tool for genetic characterisation and analysis. In this paper, we described a modified AFLP with high resolution for Trypanosoma congolense using one enzyme and agarose or Elchrom gel electrophoresis. Eleven allopatric and fourteen sympatric isolates of T. congolense savannah were used to assess the resolution of the method and its ability to characterise T. congolense isolates. Two enzymes (Eco RI or Bgl II) and corresponding non-selective and selective primers were used to identify the most appropriate combination. Patterns generated by Bgl II enzyme and a single selective primer A, C, G or T produced clear profiles. Each of the four selective primers produced different profiles for all the 25 T. congolense isolates. Due to the reduction in the number of bands, profiles could be analysed using agarose or Elchrom gels. Although comparison of a great number of samples could benefit from software help, this technique did not require flurochrome detection methods. The results of the present study demonstrated that this modified AFLP makes the characterisation of T. congolense easier while maintaining high resolution.

Effect of improving flue gas cleaning on characteristics and immobilisation of APC residues from MSW incineration.

The flue gas cleaning system of a MSW incinerator with a capacity of 350 kt/year was changed to improve the HCl elimination efficiency. Instead of the semi-wet operating spray reactor and subsequent baghouse, a two-step wet flue gas cleaning was added behind the baghouse. Elemental composition, X-ray powder diffraction patterns and TGA measurements showed that the resulting APC residue was totally different from the former residue. As a consequence, leaching characteristics of both residues also differed and another treatment was required prior to disposal. For the former residue, mainly leaching of Pb (>100 mg/l), necessitated treatment prior to landfilling. The lower alkalinity of the new residue resulted in a leachate pH of 9.7 and a Pb concentration of 0.8 mg/l. The leachate pH of the former residue was 12.4. The leaching of Pb and Zn increased above 100 mg/l when immobilising the new residue with cement. Better results were obtained when immobilising with micro silica. The high CaCl2 x 2H2O content of the new residue brought along clogging of the bag filter system. Adding 1.4% of CaO (or 1.9% of Ca(OH)2) to the residue already improved these inconveniences but again significantly changed the leaching behaviour of the residue.

A survey for Mycobacterium avium subspecies paratuberculosis in the Royal Zoological Society of Antwerp.

Paratuberculosis is a chronic intestinal disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (Map). Very little is known about the status of paratuberculosis in European zoos. In this study, the presence of Map in the animal collection of the Royal Zoological Society of Antwerp (RZSA) was investigated. Faecal and post mortem samples from 48 ruminants were used to set up cultures. DNA from faeces, tissue and positive cultures were tested by IS900 polymerase chain reaction (PCR). Additionally, 448 serum samples were tested with an ELISA kit. All culture samples were negative whereas PCR gave three positives on biopsy samples and one positive on faecal samples. With the ELISA, 21 sera could be classified as positive. There is evidence that Map is present in the RZSA but no high level faecal shedders could be detected. Further investigations are required in other European Zoos in order to complete the picture of Map infections.

Identification of a genetic marker for isometamidium chloride resistance in Trypanosoma congolense.

Isometamidium chloride has remained a very important prophylactic and therapeutic drug against trypanosomosis in cattle since its introduction into the market in the 1950s with, unfortunately, a concomitant development of resistance in trypanosomosis endemic areas. Amplified Fragment Length Polymorphism (AFLP) was used to compare two isogenic clones of Trypanosoma congolense. The parent clone, sensitive to isometamidium, has a CD50 (the curative dose that gives complete cure in 50% of the animals) in the mouse of 0.018 mg/kg and its derivative exposed to increasing doses of isometamidium, has a CD50 that is 94-fold higher. Sixty-four combinations of eight Eco RI and eight Mse I primers were used in comparative AFLP analysis to detect subtle genetic differences between the two clones. Thirty-five polymorphic fragments of DNA that were observed only in the resistant clone were purified and then sequenced. The nucleotide sequences were used in searching the GeneDB T. congolense database to find surrounding sequences upstream of an open reading frame and downstream to a stop codon. The sequences of the open reading frames were subsequently compared to the sequences in the genomic databases. A predicted gene coding for an 854 amino acids protein was thus identified. The protein contains a putative ATP binding site, Walker B and LSGG motifs and eight predicted trans-membrane domains. The gene in the resistant strain of T. congolense has a triplet insertion coding for an extra lysine. Using polymerase chain reaction-restriction fragment length polymorphism, the insertion was sought in the genomes of 35 T. congolense strains isolated from different geographic origins and whose response to isometamidium chloride had been determined through single dose mouse tests. The presence of the insertion, specifying an extra codon was found to always be present in the genomes of T. congolense clones that were resistant to isometamidium chloride.

Management of incinerator residues in Flanders (Belgium) and in neighbouring countries. A comparison.

This paper covers the Flemish legislative tools concerning the management of bottom ash, fly ash and APC residue from municipal waste incinerators, with respect to their contamination with heavy metals. The situation in Flanders is compared to the one in the Walloon region, The Netherlands, Germany and France. Waste management in the countries considered differs on the level of available management options, of leaching tests and of limit values. To make an indicative comparison of leaching tests and limit values in the different countries, leaching tests were carried out on bottom ash and fly ash, and the results are compared to the relevant limit values for recycling and landfilling of the different countries. The comparison of legislations as well as the leaching results show that discrepancies in waste management between the different regions and countries exist. Recently, European limit values for landfilling became available. European legislation on recycling, however, has not been developed and urgently needs to be considered and drafted as the market for recycling can be expanding rapidly.