Safety and immunogenecity of a live attenuated Rift Valley fever vaccine (CL13T) in camels.

BACKGROUND: Rift Valley fever is an emerging zoonotic viral disease, enzootic and endemic in Africa and the Arabian Peninsula, which poses a significant threat to both human and animal health. The disease is most severe in ruminants causing abortions in pregnant animals, especially sheep animals and high mortality in young populations. High mortality rates and severe clinical manifestation have also been reported among camel populations in Africa, to attend however none of the currently available live vaccines against RVF have been tested for safety and efficacy in this species. In this study, the safety and efficacy (through a neutralizing antibody response) of the thermostable live attenuated RVF CL13T vaccine were evaluated in camels in two different preliminary experiments involving 16 camels, (that 12 camels and 4 pregnant camels). RESULTS: The study revealed that the CL13T vaccine was safe to use in camels and no abortions or teratogenic effects were observed. The single dose of the vaccine stimulated a strong and long-lasting neutralizing antibody response for up to 12 months. CONCLUSION: The presence of neutralization antibodies is likely to correlate with protection; however protection would need to be confirmed by challenge experiments using the virulent RVF virus.

Mechanical transmission of lumpy skin disease virus by Rhipicephalus appendiculatus male ticks.

Lumpy skin disease (LSD) is an economically important, acute or sub-acute, viral disease of cattle that occurs across Africa and in the Middle East. The aim of this study was to investigate if lumpy skin disease virus (LSDV) can be transmitted mechanically by African brown ear ticks (Rhipicephalus appendiculatus Neum.). Laboratory-bred R. appendiculatus males were fed on experimentally infected viraemic 'donor' cattle. Partially fed male ticks were then transferred to feed on an uninfected 'recipient' cow. The recipient animal became viraemic, showed mild clinical signs of LSD and seroconverted. Additionally, R. appendiculatus males were found to transmit LSDV through feeding on skin lacking visible lesions, demonstrating that viraemic animals without lesions at the feeding site of ticks may be a source of infection. This is the first time that transmission of poxviruses by a tick species has been demonstrated and the importance of this mode of transmission in the spread of LSDV in endemic settings is discussed.

African horse sickness in The Gambia: circulation of a live-attenuated vaccine-derived strain.

African horse sickness virus serotype 9 (AHSV-9) has been known for some time to be circulating amongst equids in West Africa without causing any clinical disease in indigenous horse populations. Whether this is due to local breeds of horses being resistant to disease or whether the AHSV-9 strains circulating are avirulent is currently unknown. This study shows that the majority (96%) of horses and donkeys sampled across The Gambia were seropositive for AHS, despite most being unvaccinated and having no previous history of showing clinical signs of AHS. Most young horses (<3 years) were seropositive with neutralizing antibodies specific to AHSV-9. Eight young equids (<3 years) were positive for AHSV-9 by serotype-specific RT-PCR and live AHSV-9 was isolated from two of these horses. Sequence analysis revealed the presence of an AHSV-9 strain showing 100% identity to Seg-2 of the AHSV-9 reference strain, indicating that the virus circulating in The Gambia was highly likely to have been derived from a live-attenuated AHSV-9 vaccine strain.

Equine encephalosis virus: evidence for circulation beyond southern Africa.

Prior to the recent outbreak of equine encephalosis in Israel in 2009, equine encephalosis virus (EEV) had only been isolated from equids in South Africa. In this study we show the first evidence for the circulation of EEV beyond South Africa in Ethiopia, Ghana and The Gambia, indicating that EEV is likely to be freely circulating and endemic in East and West Africa. Sequence analysis revealed that the EEV isolate circulating in The Gambia was closely related to an EEV isolate that was isolated from a horse from Israel during the EEV outbreak in 2009, indicating that the two viruses have a common ancestry. Interestingly horses in Morocco tested negative for EEV antibodies indicating that the Sahara desert may be acting as a geographical barrier to the spread to the virus to North African countries. This evidence for EEV circulation in countries in East and West Africa sheds light on how the virus may have reached Israel to cause the recent outbreak in 2009.

Midge-transmitted bluetongue in domestic dogs.

The role of domestic dogs in the long-distance spread of bluetongue virus (BTV) remains unproven. It is currently known that dogs are capable of being infected with BTV, can mount an antibody response to the virus and in some cases die showing severe clinical signs of disease. Infection of dogs is currently thought to be by oral ingestion of infected meat or meat products rather than through vector feeding. In this study we show that a high percentage of domestic dogs in Morocco (21%) were seropositive for BTV and, as these dogs were fed tinned commercial food only, and had no access to other meat products, the most likely source of infection was through Culicoides midges. This finding increases the chances of dogs being infected with BTV during an outbreak but their role in the onward transmission of BTV remains unproven.

Fine structure of Pacinian corpuscles in the mesentery of the cat.

Pacinian corpuscles in the mesentery of adult cats were fixed with either glutaraldehyde, osmium tetroxide or permanganate solutions by close intra-arterial injection through the mesenteric artery, and were processed, after electron staining and Epon embedding, for electron microscopy. Better resolution of the corpuscle's ultrastructure was obtained than available heretofore. The myelinated segment of the corpuscle contains blood vessels separated from the axon by collagen fibers and 3 to 4 layers of lamellae. No blood vessels are found in the central core, though access from the vessels is afforded by diffusion through the "cleft" of the inner core. Two cell types are discernible in the inner core hemilamellae; the "clear cells" in which pinocytotic vesicles and organelles abound and reflect the greater metabolic activity of these cells, in contrast to the "dark cells." The ultraterminal is ellipsoidal in form with projections into the "cleft" which give this portion an irregular appearance in section. The terminal and ultraterminal are packed with mitochondria, and "synaptic" vesicles are seen in the ultraterminal. The innermost laminae of the inner core cells are in close apposition to the terminal and break their regular pattern of hemilamellation to surround the small ultraterminal projections at the apical part of the corpuscle.

Bluetongue virus: European Community proficiency test (2007) to evaluate ELISA and RT-PCR detection methods with special reference to pooling of samples.

Bluetongue virus European Community national reference laboratories (BTV-EC-NRLs) participated in an inter-laboratory proficiency test in 2007. The aim of the inter-laboratory proficiency test was to determine the ability of laboratories to detect antibodies to a series of BTV serotypes by cELISA and to detect viral RNA in animals infected with the European strain of BTV-8 by RT-PCR. Both serum and EDTA blood sample were diluted in order to determine the sensitivity of the assays. All the cELISAs were 'fit-for purpose' to detect antibodies to the common BTV serotypes circulating in Europe and the real time RT-PCR assays were all capable of detecting BTV-8 RNA albeit with varying sensitivities. There were however inconsistencies in the ability of the gel-based PCR assays to detect BTV RNA. In addition, samples taken on the first day of viraemia and at the peak of viraemia from animals experimentally infected with BTV-8, were diluted to determine if the diluting of samples affected the ability of the Shaw et al. (Shaw, A.E., M., P., Alpar, H.O., Anthony, S., Darpel, K.E., Batten, C.A., Carpenter, S., Jones, H., Oura, C.A.L., King, D.P., Elliott, H., Mellor, P.S., Mertens, P.P.C., 2007. Development and validation of a real-time RT-PCR assay to detect genome bluetongue virus segment 1. Journal of Virological Methods) RT-PCR assay to detect BTV-RNA at these time-points. Results indicated that, if samples were taken at the onset of viraemia, diluting at 1/5 resulted in a reduced ability of the assay to detect BTV RNA in the diluted compared to the neat samples. Diluting samples taken at the peak of viraemia at 1/10 however resulted in no loss in sensitivity.

Prevalence and Genetic Diversity of Toxoplasma gondii in Free-Ranging Chickens from the Caribbean.

PURPOSE: Toxoplasma gondii is a zoonotic parasite capable of infecting a wide range of hosts. Free-range chickens are important sentinels in the epidemiology of this parasite as they feed from the ground and are likely to ingest oocysts shed in the faeces of infected cats. Atypical strains of T. gondii are known to dominate in South America where they are associated with more severe disease in humans, yet relatively little is known about the strains circulating in neighbouring Caribbean islands. METHODS: In this study, hearts and brains were collected from free-range chickens in Antigua and Barbuda (n = 45), Dominica (n = 76) and Trinidad (n = 41), and DNA was extracted for nested ITS1 PCR and PCR-RFLP. Sera were collected and screened for antibodies using the modified agglutination test (MAT). RESULTS: Antibodies to T. gondii were detected in 20.5, 38.2 and 17.1% of chickens in Antigua and Barbuda, Dominica and Trinidad, respectively. Toxoplasma gondii DNA was also detected by PCR in 24.4, 17.1 and 17.1% of chickens, respectively, giving an overall prevalence of 31.1, 42.1, and 29.3% for each of the 3 island nations. Results of PCR-RFLP revealed 2 new atypical genotypes (designated ToxoDB #281 and #282) and one Type III (ToxoDB #2) in chickens from Antigua. Partial genotyping of a further 8 isolates (7 from Antigua and one from Trinidad) revealed different allele-types at five or more markers for 7 of the isolates, suggesting atypical genotypes. CONCLUSIONS: This is the first study to report the prevalence of T. gondii in free-range chickens in Antigua and Barbuda, Dominica and Trinidad and Tobago. It is also the first to report the presence of atypical genotypes in Antigua and Barbuda and Trinidad and Tobago.

Bluetongue virus: European Community inter-laboratory comparison tests to evaluate ELISA and RT-PCR detection methods.

European Community national reference laboratories participated in two inter-laboratory comparison tests in 2006 to evaluate the sensitivity and specificity of their 'in-house' ELISA and RT-PCR assays for the detection of bluetongue virus (BTV) antibodies and RNA. The first ring trial determined the ability of laboratories to detect antibodies to all 24 serotypes of BTV. The second ring trial, which included both antisera and EDTA blood samples from animals experimentally infected with the northern European strain of BTV-8, determined the ability of laboratories to detect BTV-8 antibodies and RNA, as well as the diagnostic sensitivity of the assays. A total of six C-ELISAs, six real-time RT-PCR and three conventional RT-PCR assays were used. All C-ELISAs were capable of detecting the BTV serotypes currently circulating in Europe (BTV-1, 2, 4, 8, 9 and 16), however some assays displayed inconsistencies in the detection of other serotypes, particularly BTV-19. All C-ELISAs detected BTV-8 antibodies in cattle and sheep by 21 dpi, while the majority of assays detected antibodies by 9 dpi in cattle and 8 dpi in sheep. All the RT-PCR assays were able to detect BTV-8, although the real-time assays were more sensitive compared to the conventional assays. The majority of real-time RT-PCR assays detected BTV RNA as early as 2 dpi in cattle and 3 dpi in sheep. These two ring trails provide evidence that national reference laboratories within the EC are capable of detecting BTV antibodies and RNA and provide specificity and sensitivity information on the detection methods currently available.

Evidence for transplacental and contact transmission of bluetongue virus in cattle.

This paper presents evidence that a field strain of bluetongue virus serotype 8 (BTV-8) was transmitted transplacentally and that it was also spread by a direct contact route. Twenty pregnant heifers were imported from the Netherlands into Northern Ireland during the midge-free season. Tests before and after the animals were imported showed that eight of them had antibodies to bluetongue virus, but no viral RNA was detected in any of them by reverse transcriptase-PCR (RT-PCR). Two of the seropositive heifers gave birth to three calves that showed evidence of bluetongue virus infection (RT-PCR-positive), and one of the calves was viraemic. Two further viraemic animals (one newly calved Dutch heifer, and one milking cow originally from Scotland) were also found to have been infected with BTV-8 and evidence is presented that these two animals may have been infected by direct contact, possibly through the ingestion of placentas infected with BTV-8.

Development and initial evaluation of a real-time RT-PCR assay to detect bluetongue virus genome segment 1.

Since 1998, multiple strains of bluetongue virus (BTV), belonging to six different serotypes (types 1, 2, 4, 8, 9 and 16) have caused outbreaks of disease in Europe, causing one of the largest epizootics of bluetongue ever recorded, with the deaths of >1.8 million animals (mainly sheep). The persistence and continuing spread of BTV in Europe and elsewhere highlights the importance of sensitive and reliable diagnostic assay systems that can be used to rapidly identify infected animals, helping to combat spread of the virus and disease. BTV has a genome composed of 10 linear segments of dsRNA. We describe a real-time RT-PCR assay that targets the highly conserved genome segment 1 (encoding the viral polymerase--VP1) that can be used to detect all of the 24 serotypes, as well as geographic variants (different topotypes) within individual serotypes of BTV. After an initial evaluation using 132 BTV samples including representatives of all 24 BTV serotypes, this assay was used by the European Community Reference Laboratory (CRL) at IAH Pirbright to confirm the negative status of 2,255 animals imported to the UK from regions that were considered to be at risk during the 2006 outbreak of BTV-8 in Northern Europe. All of these animals were also negative by competition ELISA to detect BTV specific antibodies and none of them developed clinical signs of infection. These studies have demonstrated the value of the assay for the rapid screening of field samples.

Clinical signs and pathology shown by British sheep and cattle infected with bluetongue virus serotype 8 derived from the 2006 outbreak in northern Europe.

Four poll Dorset sheep and four Holstein-Friesian cattle were infected with the northern European strain of bluetongue virus (BTV), BTV-8, to assess its pathogenicity in UK breeds. The time course of infection was monitored in both species by using real-time reverse transcriptase-PCR (RT-PCR), conventional RT-PCR and serology. Two of the sheep developed severe clinical signs that would have been fatal in the field; the other two were moderately and mildly ill, respectively. The cattle were clinically unaffected, but had high levels of viral RNA in their bloodstream. Real-time RT-PCR detected viral RNA as early as one day after infection in the cattle and three days after infection in the sheep. Antibodies against BTV were detected by six days after infection in the sheep and eight days after infection in the cattle. Postmortem examinations revealed pathology in the cattle that was more severe than suggested by the mild clinical signs, but the pathological and clinical findings in the sheep were more consistent.

Bluetongue virus infection in naïve cattle: Identification of circulating serotypes and associated Culicoides biting midge species in Trinidad.

To better understand risks associated with trading cattle, it is important to know which serotypes of Bluetongue virus (BTV) are circulating within the exporting and importing country. Hence, this study was conducted to identify the circulating serotypes of BTV in Trinidad. Blood samples were collected monthly from sixty BTV- naïve imported cattle over a six month period after their arrival in the country. Virological (PCR and virus isolation) and serological (ELISA) analyses were carried out on the samples and CDC light traps were placed near the cattle enclosure to trap and identify the species of Culicoides biting midges that were present. All of the cattle seroconverted for BTV antibodies within three months of their arrival in the country and real-time reverse transcription PCR (rRT-PCR) detected BTV-RNA in the samples throughout the remainder of the study. The patterns of infection observed in the cattle indicated serial infections with multiple serotypes. A combination of BTV serotype-specific rRT-PCR on the original blood samples and virus isolation followed by serotype-specific rRT-PCR on selected samples, confirmed the presence of BTV serotypes 1, 2, 3, 5, 12 and 17. This is the first report of BTV-2 and BTV-5 in Trinidad. Light-suction traps placed in close proximity to the cattle predominantly trapped Culicoides insignis Lutz 1913 species (96%), with a further six Culicoides species making up the remaining 4% of trapped samples. The circulation of multiple BTV serotypes in Trinidad underlines the need for regular surveillance, which will contribute to the development of risk assessments for trade in livestock.

Linkage disequilibrium between alleles at highly polymorphic mini- and micro-satellite loci of Theileria parva isolated from cattle in three regions of Kenya.

Theileria parva schizont-infected lymphocyte culture isolates from western, central and coastal Kenya were analysed for size polymorphism at 30 T. parva-specific variable number tandem repeat (VNTR) loci using a panel of mini- and micro-satellite markers. The mean number of alleles ranged from 3 to 11 at individual loci and 183 distinct alleles were observed in total, indicating high genetic diversity within the T. parva gene pool in Kenyan cattle. The frequency distribution of the length variation of specific alleles among isolates ranged from normal to markedly discontinuous. Genetic relationships between isolates were analysed using standard indices of genetic distance. Genetic distances and dendrograms derived from these using neighbour-joining algorithms did not indicate significant clustering on a geographical basis. Analysis of molecular variance demonstrated that the genetic variation between individual isolates was 72%, but only 2.3% when isolates from different regions were pooled. Both these observations suggest minimal genetic sub-structuring relative to geographical origin. Linkage disequilibrium was observed between pairs of loci within populations, as in certain Ugandan T. parva populations. A novel observation was that disequilibrium was also detected between alleles at three individual pairs of VNTR loci when isolates from the three regional meta-populations were pooled for analysis.

Population genetic analysis and sub-structuring of Theileria parva in Uganda.

In recent years the population structures of many apicomplexan parasites including Plasmodium spp., Toxoplasma gondii and Cryptospordium parvum have been elucidated. These species show a considerable diversity of population structure suggesting different strategies for transmission and survival in mammalian hosts. We have undertaken a population genetic analysis of another apicomplexan species (Theileria parva) to investigate the levels of diversity of this parasite and the role of genetic exchange in three geographically separate populations. The principal hindrance to carrying out such a study on field isolates was the high proportion of blood samples that contain multiple genotypes, making it impossible to determine the genotypes of the parasites directly. This problem was overcome by sampling only young indigenous calves between 3 and 9 months of age in which approximately 60% of the T. parva infected calves contained a single/predominant allele at each locus, making it possible to undertake population genetic analyses. Blood samples were collected from calves in three geographically distinct regions of Uganda and were analysed using 12 polymorphic mini and microsatellite markers that were evenly dispersed across the four chromosomes. We have identified 84 multilocus genotypes (MLG) from these samples, indicating high levels of diversity in the parasite. Analysis of linkage disequilibrium between pairs of loci provides evidence that the population in Lira district had an epidemic structure. The population in Mbarara was substructured containing two genetically distinct sub-groups and the larger sub-group also had an epidemic population structure. The population from Kayunga was in linkage disequilibrium. Genetic distances and Wrights fixation index (F(ST)) indicate that there is evidence for geographical sub-structuring between the Lira and the Kayunga populations.

Identification of a 40S Ribosomal protein (S17) that is differentially expressed between the macroschizont and piroplasm stages of Theileria annulata.

The nucleotide and protein sequence of the 40S ribosomal protein S17 (RibS17) of the protozoan parasite Theileria annulata has been determined. Southern blot analysis showed the gene was single copy and comparative sequence analysis revealed that the predicted polypeptide had high sequence homology with the RibS17 from other organisms. Northern blot analysis showed that there was a 3-fold increase in the level of RibS17 RNA between the macroschizont and the piroplasm stage of the lifecycle, whereas, there was no difference in expression between the sporozoite and the macroschizont stages. Antisera to the purified fusion protein, corresponding to the terminal 50 amino acids of the protein sequence, were raised in rabbits. Western analysis detected a polypeptide of the predicted size that was more abundant in the piroplasm stage compared with the macroschizont stage. Immunofluorescence analysis with the same antisera revealed a strong signal in the macroschizont and piroplasm stages, but the antiserum did not cross-react with the bovine host cells. The antisera did, however, cross-react with Toxoplasma gondii tachyzoites and Plasmodium falciparum merozoites. The possible functional significance of the stage related increase in abundance of a ribosomal protein is discussed.

Application of a reverse line blot assay to the study of haemoparasites in cattle in Uganda.

Recent advances in genomic technology have focused many veterinary researchers on the possibility of producing one multivalent recombinant vaccine against all the haemoparasites that infect cattle in the tropics. Before such a vaccine is developed it is essential to define target cattle populations as well as the range of anti-pathogen vaccines required in order to control disease. To further this objective, we have evaluated a reverse line blot (RLB) assay, which simultaneously detects the principal tick transmitted protozoan and rickettsial cattle pathogens, in different epidemiological scenarios in Uganda. A critical question is the sensitivity, particularly in relation to detecting carrier animals. As Theileria parva is considered to be the most important pathogen in the region, we assessed the sensitivity of the RLB assay for T. parva and showed that 1-2 x 10(3) parasites per ml of blood could be detected-a level comparable with previously developed PCR methods and well below conventional microscopic detection. We applied the RLB assay to evaluate the differences in pathogen profiles between crossbred and indigenous cattle and show that there were different profiles, with a low prevalence of T. parva and Theileria taurotragi in the indigenous cattle compared to a high prevalence in the crossbred cattle. In contrast, we show higher prevalences of Theileria mutans and Theileria velifera in the indigenous compared to the crossbred cattle. Interestingly Anaplasma marginale, Babesia bovis and Babesia bigemina were of low prevalence but a high prevalence of Ehrlichia bovis was seen, raising the question of whether this rickettsial species could be pathogenic in cattle. Analysis of animals with clinical symptoms of East Coast Fever showed that, while T. parva is a major cause of these symptoms, T. mutans and possibly T. taurotragi and T. velifera, may also cause clinical disease. Overall, the results presented here highlight the complexity of tick-borne pathogen infections in cattle in Uganda.

A panel of microsatellite and minisatellite markers for the characterisation of field isolates of Theileria parva.

Mini- and microsatellite sequences show high levels of variation and therefore provide excellent tools for both the genotyping and population genetic analysis of parasites. Herein we describe the identification of a panel of 11 polymorphic microsatellites and 49 polymorphic minisatellites of the protozoan haemoparasite Theileria parva. The PCR products were run on high resolution Spreadex gels on which the alleles were identified and sized. The sequences of the mini- and microsatellites were distributed across the four chromosomes with 16 on chromosome 1, 12 on chromosome 2, 14 on chromosome 3 and 18 on chromosome 4. The primers from the 60 sequences were tested against all the Theileria species that co-infect cattle in East and Southern Africa and were found to be specific for T. parva. In order to demonstrate the utility of these markers, we characterised eight tissue culture isolates of T. parva isolated from cattle in widely separated regions of Eastern and Southern Africa (one from Zambia, one from Uganda, two from Zimbabwe, four from Kenya) and one Kenyan tissue culture isolate from Cape buffalo (Syncerus caffer). The numbers of alleles per locus range from three to eight indicating a high level of diversity between these geographically distinct isolates. We also analysed five isolates from cattle on a single farm at Kakuzi in the central highlands of Kenya and identified a range of one to four alleles per locus. Four of the Kakuzi isolates represented distinct multilocus genotypes while two exhibited identical multilocus genotypes. This indicates a high level of diversity in a single population of T. parva. Cluster analysis of multilocus genotypes from the 14 isolates (using a neighbour joining algorithm) revealed that genetic similarity between isolates was not obviously related to their geographical origin.

Detection of African swine fever virus in infected pig tissues by immunocytochemistry and in sity hybridisation.

The techniques for determining cellular sites of establishment and persistence of African swine fever virus (ASFV) were established in susceptible domestic pigs and the resistant African reservoir hosts, the warthog and bushpig. Detection, both in vitro and in vivo, was achieved by in situ hybridisation and immunocytochemistry, focusing principally on specific probes for vp73, a major capsid protein. Hybridisation of radio-labelled probes for DNA and RNA was relatively insensitive and time consuming whereas hybridisation of non-radioactive DNA probes was quicker and more sensitive. Detection of vp73 protein by immunocytochemistry was as sensitive as non-radioactive DNA hybridisation, offering in addition improved speed, ease and morphology. Both non-radioactive DNA hybridisation and immunocytochemistry were therefore used to detect ASFV DNA and protein in a range of porcine cells infected in vitro with different ASFV isolates. Malta and Malawi isolates infected both alveolar and bone marrow macrophages, but infected negligible numbers of endothelial (< 1%) and kidney cells (IBRS2 cells). Attenuated Uganda isolate, however, infected a high percentage of endothelial cells and IBRS2 cells as well as alveolar and bone marrow macrophages. When used to investigate the cell tropism of ASFV in tissues from pigs infected with the highly virulent Malawi isolate of ASFV, both techniques identified virus principally in cells of the mononuclear phagocytic system. In the lung, double staining revealed that pulmonary intravascular macrophages, but not alveolar macrophages, were infected.

The distribution of atrial natriuretic polypeptide (ANP)-containing cells in the adult rat heart.

The present study was performed to clarify the distribution of ANP-containing cells in the adult rat heart by immunostaining for ANP using antiserum against alpha-human ANP. ANP-immunoreactive cells were generally present in the atrial walls except for the sinoatrial node. In the ventricular walls, they were distributed in the impulse conducting system, particularly the left bundle branch, Purkinje fibers on the left side of the interventricular septum, and those in the false tendons in the left ventricle, while they were sporadically seen in the atrioventricular node and bundle of His. The immunoreactive cells contained specific granules that were positive for ANP. These findings demonstrate that ANP-containing cells are present in the atrial and ventricular walls.