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1.  Diagnosis of foot-and mouth disease by real time reverse transcription polymerase chain reaction under field conditions in Brazil 
Background
Foot-and-mouth disease (FMD) is an economically important and highly contagious viral disease that affects cloven-hoofed domestic and wild animals. Virus isolation and enzyme-linked immunosorbent assay (ELISA) are the gold standard tests for diagnosis of FMD. As these methods are time consuming, assays based on viral nucleic acid amplification have been developed.
Results
A previously described real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay with high sensitivity and specificity under laboratorial and experimental conditions was used in the current study. To verify the applicability of this assay under field conditions in Brazil, 460 oral swabs from cattle were collected in areas free of FMD (n = 200) and from areas with outbreaks of FMD (n = 260). Three samples from areas with outbreaks of FMD were positive by real-time RT-PCR, and 2 of those samples were positive by virus isolation and ELISA. Four other samples were considered inconclusive by real-time RT-PCR (threshold cycle [Ct] > 40); whereas all 200 samples from an area free of FMD were real-time RT-PCR negative.
Conclusion
real-time RT-PCR is a powerful technique for reliable detection of FMDV in a fraction of the time required for virus isolation and ELISA. However, it is noteworthy that lack of infrastructure in certain areas with high risk of FMD may be a limiting factor for using real-time RT-PCR as a routine diagnostic tool.
doi:10.1186/1746-6148-4-53
PMCID: PMC2631516  PMID: 19117507
2.  Comparative assessment of passive surveillance in disease-free and endemic situation: Example of Brucella melitensis surveillance in Switzerland and in Bosnia and Herzegovina 
Background
Globalization and subsequent growth in international trade in animals and animal products has increased the importance of international disease reporting. Efficient and reliable surveillance systems are needed in order to document the disease status of a population at a given time. In this context, passive surveillance plays an important role in early warning systems. However, it is not yet routinely integrated in the assessment of disease surveillance systems because different factors like the disease awareness (DA) of people reporting suspect cases influence the detection performance of passive surveillance. In this paper, we used scenario tree methodology in order to evaluate and compare the quality and benefit of abortion testing (ABT) for Brucella melitensis (Bm) between the disease free situation in Switzerland (CH) and a hypothetical disease free situation in Bosnia and Herzegovina (BH), taking into account DA levels assumed for the current endemic situation in BH.
Results
The structure and input parameters of the scenario tree were identical for CH and BH with the exception of population data in small ruminants and the DA in farmers and veterinarians. The sensitivity analysis of the stochastic scenario tree model showed that the small ruminant population structure and the DA of farmers were important influential parameters with regard to the unit sensitivity of ABT in both CH and BH. The DA of both farmers and veterinarians was assumed to be higher in BH than in CH due to the current endemic situation in BH. Although the same DA cannot necessarily be assumed for the modelled hypothetical disease free situation as for the actual endemic situation, it shows the importance of the higher vigilance of people reporting suspect cases on the probability that an average unit processed in the ABT-component would test positive.
Conclusion
The actual sensitivity of passive surveillance approaches heavily depends on the context in which they are applied. Scenario tree modelling allows for the evaluation of such passive surveillance system components under assumed disease free situation. Despite data gaps, this is a real opportunity to compare different situations and to explore consequences of changes that could be made.
doi:10.1186/1746-6148-4-52
PMCID: PMC2626581  PMID: 19099610
3.  Intraperitoneal implantation of life-long telemetry transmitters in otariids 
Background
Pinnipeds, including many endangered and declining species, are inaccessible and difficult to monitor for extended periods using externally attached telemetry devices that are shed during the annual molt. Archival satellite transmitters were implanted intraperitoneally into four rehabilitated California sea lions (Zalophus californianus) and 15 wild juvenile Steller sea lions (Eumetopias jubatus) to determine the viability of this surgical technique for the deployment of long-term telemetry devices in otariids. The life history transmitters record information throughout the life of the host and transmit data to orbiting satellites after extrusion following death of the host.
Results
Surgeries were performed under isoflurane anesthesia and single (n = 4) or dual (n = 15) transmitters were inserted into the ventrocaudal abdominal cavity via an 8.5 to 12 cm incision along the ventral midline between the umbilicus and pubic symphysis or preputial opening. Surgeries lasted 90 minutes (SD = 8) for the 19 sea lions. All animals recovered well and were released into the wild after extended monitoring periods from 27 to 69 days at two captive animal facilities. Minimum post-implant survival was determined via post-release tracking using externally attached satellite transmitters or via opportunistic re-sighting for mean durations of 73.7 days (SE = 9.0, Z. californianus) and 223.6 days (SE = 71.5, E. jubatus).
Conclusion
The low morbidity and zero mortality encountered during captive observation and post-release tracking periods confirm the viability of this surgical technique for the implantation of long-term telemetry devices in otariids.
doi:10.1186/1746-6148-4-51
PMCID: PMC2621145  PMID: 19077193
4.  First case of highly pathogenic H5N1 avian influenza virus in Spain 
Background
The H5N1 strain of avian influenza virus has been involved in severe mortality in domestic poultry, and has also been found in different species of wildlife in Europe. The Basque Country avian influenza surveillance program began sample collection and processing the fall of 2005.
Results
Here we report the first confirmation of the presence of highly pathogenic H5N1 strain in a Great Crested Grebe (Podiceps cristatus) found dead in a pond near Vitoria in the Basque Country on the North of Spain. Regarding the survey for generic influenza type A virus, we have obtained positive results in about 8% of more that 3500 birds examined.
Conclusion
We think that the self-limiting nature of our finding and others proves that certain regions have ecological, geographical and climatological features that make it difficult for the H5N1 virus to spread [1] and cause disease at least in the large scale scenario that has been worrying human and animal health authorities during the last years.
doi:10.1186/1746-6148-4-50
PMCID: PMC2621144  PMID: 19077185
5.  Parasites of importance for human health in Nigerian dogs: high prevalence and limited knowledge of pet owners 
Background
Dogs are the most common pet animals worldwide. They may harbour a wide range of parasites with zoonotic potential, thus causing a health risk to humans. In Nigeria, epidemiological knowledge on these parasites is limited.
Methods
In a community-based study, we examined 396 dogs in urban and rural areas of Ilorin (Kwara State, Central Nigeria) for ectoparasites and intestinal helminths. In addition, a questionnaire regarding knowledge and practices was applied to pet owners.
Results
Nine ectoparasite species belonging to four taxa and six intestinal helminth species were identified: fleas (Ctenocephalides canis, Pulex irritans, Tunga penetrans), mites (Demodex canis, Otodectes sp., Sarcoptes scabiei var. canis), ticks (Rhipicephalus sanguineus, Ixodes sp.), and lice (Trichodectes canis); and Toxocara canis, Ancylostoma sp., Trichuris vulpis, Dipylidium caninum, Taenidae and Strongyloides sp. Overall prevalence of ectoparasites was 60.4% and of intestinal helminths 68.4%. The occurrence of C. canis, R. sanguineus, T. canis, Ancylostoma sp. and T. vulpis was most common (prevalence 14.4% to 41.7%). Prevalence patterns in helminths were age-dependent, with T. canis showing a decreasing prevalence with age of host, and a reverse trend in other parasite species. Knowledge regarding zoonoses was very limited and the diseases not considered a major health problem. Treatment with antiparasitic drugs was more frequent in urban areas.
Conclusion
Parasites of importance for human health were highly prevalent in Nigerian dogs. Interventions should include health education provided to dog owners and the establishment of a program focusing on zoonotic diseases.
doi:10.1186/1746-6148-4-49
PMCID: PMC2615757  PMID: 19068110
6.  Porcine reproductive and respiratory syndrome virus (PRRSV) in GB pig herds: farm characteristics associated with heterogeneity in seroprevalence 
Background
The between- and within-herd variability of porcine reproductive and respiratory syndrome virus (PRRSV) antibodies were investigated in a cross-sectional study of 103 British pig herds conducted 2003–2004. Fifty pigs from each farm were tested for anti-PRRSV antibodies using ELISA. A binomial logistic model was used to investigate management risks for farms with and without pigs with PRRSV antibodies and multilevel statistical models were used to investigate variability in pigs' log ELISA IRPC (relative index × 100) in positive herds.
Results
Thirty-five herds (34.0%) were seronegative, 41 (39.8%) were seropositive and 27 (26.2%) were vaccinated. Herds were more likely to be seronegative if they had < 250 sows (OR 3.86 (95% CI 1.46, 10.19)) and if the nearest pig herd was ≥ 2 miles away (OR 3.42 (95% CI 1.29, 9.12)). The mean log IRPC in seropositive herds was 3.02 (range, 0.83 – 5.58). Sixteen seropositive herds had only seropositive adult pigs. In these herds, pigs had -0.06 (95% CI -0.10, -0.01) lower log IRPC for every mile increase in distance to the nearest pig unit, and -0.56 (95% CI -1.02, -0.10) lower log IRPC when quarantine facilities were present. For 25 herds with seropositive young stock and adults, lower log IRPC were associated with isolating purchased stock for ≥ 6 days (coefficient -0.46, 95% CI -0.81, -0.11), requesting ≥ 48 hours 'pig-free time' from humans (coefficient -0.44, 95% CI -0.79, -0.10) and purchasing gilts (coefficient -0.61, 95% CI -0.92, -0.29).
Conclusion
These patterns are consistent with PRRSV failing to persist indefinitely on some infected farms, with fadeout more likely in smaller herds with little/no reintroduction of infectious stock. Persistence of infection may be associated with large herds in pig-dense regions with repeated reintroduction.
doi:10.1186/1746-6148-4-48
PMCID: PMC2614980  PMID: 19040719
7.  Quantification of deficits in lateral paw positioning after spinal cord injury in dogs 
Background
Previous analysis of the behavioural effects of spinal cord injury has focussed on coordination in the sagittal plane of movement between joints, limb girdle pairs or thoracic and pelvic limb pairs. In this study we extend the functional analysis of the consequences of clinical thoracolumbar spinal cord injury in dogs to quantify the well-recognised deficits in lateral stability during locomotion. Dogs have a high centre of mass thereby facilitating recognition of lateral instability.
Results
We confirm that errors in lateral positioning of the pelvic limb paws can be quantified and that there is a highly significant difference in variability of foot placement between normal and spinal cord injured dogs. In this study there was no detectable difference in lateral paw positioning variability between complete and incomplete injuries, but it appears that intergirdle limb coordination and appropriate lateral paw placement recover independently from one another.
Conclusion
Analysis of lateral paw position in the dog provides an additional tier of analysis of outcome after spinal cord injury that will be of great value in interpreting the effects of putative therapeutic interventions.
doi:10.1186/1746-6148-4-47
PMCID: PMC2631515  PMID: 19032742
8.  Molecular characterisation of a bovine-like rotavirus detected from a giraffe 
Background
Rotavirus (RV), is a member of the Reoviridae family and an important etiological agent of acute viral gastroenteritis in the young. Rotaviruses have a wide host range infecting a broad range of animal species, however little is known about rotavirus infection in exotic animals. In this paper we report the first characterisation of a RV strain from a giraffe calf.
Results
This report describes the identification and detailed molecular characterisation of a rotavirus strain detected from a 14-day-old Giraffe (Giraffa camelopardalis), presenting with acute diarrhea. The RV strain detected from the giraffe was characterized molecularly as G10P[11]. Detailed sequence analysis of VP4 and VP7 revealed significant identity at the amino acid sequence level to Bovine RV (BoRV).
Conclusion
This study demonstrates the need for continuous surveillance of RV strains in various animal populations, which will facilitate the identification of rotavirus hosts not previously reported. Furthermore, extending typical epidemiology studies to a broader host range will contribute to the timely identification of new emerging strain types.
doi:10.1186/1746-6148-4-46
PMCID: PMC2632637  PMID: 19014526
9.  Transmission potential, skin inflammatory response, and parasitism of symptomatic and asymptomatic dogs with visceral leishmaniasis 
Background
Visceral leishmaniasis in Brazil is caused by the protozoan Leishmania (Leishmania) chagasi and it is transmitted by sandfly of the genus Lutzomyia. Dogs are an important domestic reservoir, and control of the transmission of visceral leishmaniasis (VL) to humans includes the elimination of infected dogs. However, though dogs are considered to be an important element in the transmission cycle of Leishmania, the identification of infected dogs representing an immediate risk for transmission has not been properly evaluated. Since it is not possible to treat infected dogs, they are sacrificed when a diagnosis of VL is established, a measure that is difficult to accomplish in highly endemic areas. In such areas, parameters that allow for easy identification of reservoirs that represents an immediate risk for transmission is of great importance for the control of VL transmission. In this study we aimed to identify clinical parameters, reinforced by pathological parameters that characterize dogs with potential to transmit the parasite to the vector.
Results
The major clinical manifestations of visceral leishmaniasis in dogs from an endemic area were onicogriphosis, skin lesions, conjunctivitis, lymphadenopathy, and weight loss. The transmission potential of these dogs was assessed by xenodiagnosis using Lutzomyia longipalpis. Six of nine symptomatic dogs were infective to Lutzomyia longipalpis while none of the five asymptomatic dogs were infective to the sandfly. Leishmania amastigotes were present in the skin of all clinically symptomatic dogs, but absent in asymptomatic dogs. Higher parasite loads were observed in the ear and ungueal region, and lower in abdomen. The inflammatory infiltrate was more intense in the ears and ungueal regions of both symptomatic and asymptomatic dogs. In clinically affected dogs in which few or none Leishmania amastigotes were observed, the inflammatory infiltrate was constituted mainly of lymphocytes and macrophages. When many parasites were present, the infiltrate was also comprised of lymphocytes and macrophages, as well as a larger quantity of polymorphonuclear neutrophils (PMNs).
Conclusion
Dogs that represent an immediate risk for transmission of Leishmania in endemic areas present clinical manifestations that include onicogriphosis, skin lesions, conjunctivitis, lymphadenopathy, and weight loss. Lymphadenopathy in particular was a positive clinical hallmark since it was closely related to the positive xenodiagnosis.
doi:10.1186/1746-6148-4-45
PMCID: PMC2613136  PMID: 18990238
10.  Molecular and geographic analyses of vampire bat-transmitted cattle rabies in central Brazil 
Background
Vampire bats are important rabies virus vectors, causing critical problems in both the livestock industry and public health sector in Latin America. In order to assess the epidemiological characteristics of vampire bat-transmitted rabies, the authors conducted phylogenetic and geographical analyses using sequence data of a large number of cattle rabies isolates collected from a wide geographical area in Brazil.
Methods
Partial nucleoprotein genes of rabies viruses isolated from 666 cattle and 18 vampire bats between 1987 and 2006 were sequenced and used for phylogenetic analysis. The genetic variants were plotted on topographical maps of Brazil.
Results
In this study, 593 samples consisting of 24 genetic variants were analyzed. Regional localization of variants was observed, with the distribution of several variants found to be delimited by mountain ranges which served as geographic boundaries. The geographical distributions of vampire-bat and cattle isolates that were classified as the identical phylogenetic group were found to overlap with high certainty. Most of the samples analyzed in this study were isolated from adjacent areas linked by rivers.
Conclusion
This study revealed the existence of several dozen regional variants associated with vampire bats in Brazil, with the distribution patterns of these variants found to be affected by mountain ranges and rivers. These results suggest that epidemiological characteristics of vampire bat-related rabies appear to be associated with the topographical and geographical characteristics of areas where cattle are maintained, and the factors affecting vampire bat ecology.
doi:10.1186/1746-6148-4-44
PMCID: PMC2613875  PMID: 18983685
11.  Development and validation of an enzyme-linked immunosorbent assay for antibodies against Mycobacterium bovis in european wild boar 
Background
Bovine tuberculosis (bTB) remains a significant problem in some parts of Spain largely because of contacts between cattle and wildlife reservoirs in extensive grazing systems. European Wild boar (Sus scrofa) is one of the species involved in the transmission of the disease to other species. Fast and simple detection methods would be critical for assessing infection prevalence, study the mechanisms of pathogen transmission and monitoring the effects of TB control measures.
Results
An enzyme-linked immunosorbent assay (ELISA) to detect antibodies against Mycobacterium bovis in wild boar serum was developed and validated on 185 sera from TB positive and negative wild boar. Based on antigen inoculation of captive animals as well as tuberculosis compatible lesions, culture results and molecular analysis of hunted individuals, animals were allocated into two groups: tuberculosis positive group and tuberculosis negative group. After optimization of the positive to negative ratio using different combinations of serum dilutions and conjugate concentrations, the test yielded a sensitivity of 72.60% and a specificity of 96.43% for the best cut-off.
Conclusion
Although some negative group animals showed an ELISA positive reaction (< 3%), this assay showed a high potential for accurate diagnosis of TB in wild boar, as its large dynamic range supported a good discriminatory power and a satisfactory balance between sensitivity and specificity.
doi:10.1186/1746-6148-4-43
PMCID: PMC2606677  PMID: 18976491
12.  Estimating the temporal and spatial risk of bluetongue related to the incursion of infected vectors into Switzerland 
Background
The design of veterinary and public health surveillance systems has been improved by the ability to combine Geographical Information Systems (GIS), mathematical models and up to date epidemiological knowledge. In Switzerland, an early warning system was developed for detecting the incursion of the bluetongue disease virus (BT) and to monitor the frequency of its vectors. Based on data generated by this surveillance system, GIS and transmission models were used in order to determine suitable seasonal vector habitat locations and risk periods for a larger and more targeted surveillance program.
Results
Combined thematic maps of temperature, humidity and altitude were created to visualize the association with Culicoides vector habitat locations. Additional monthly maps of estimated basic reproduction number transmission rates (R0) were created in order to highlight areas of Switzerland prone to higher BT outbreaks in relation to both vector activity and transmission levels. The maps revealed several foci of higher risk areas, especially in northern parts of Switzerland, suitable for both vector presence and vector activity for 2006.
Results showed a variation of R0 values comparing 2005 and 2006 yet suggested that Switzerland was at risk of an outbreak of BT, especially if the incursion arrived in a suitable vector activity period. Since the time of conducting these analyses, this suitability has proved to be the case with the recent outbreaks of BT in northern Switzerland.
Conclusion
Our results stress the importance of environmental factors and their effect on the dynamics of a vector-borne disease. In this case, results of this model were used as input parameters for creating a national targeted surveillance program tailored to both the spatial and the temporal aspect of the disease and its vectors. In this manner, financial and logistic resources can be used in an optimal way through seasonally and geographically adjusted surveillance efforts. This model can serve as a tool for other vector-borne diseases including human zoonotic vectors which are likely to spread into Europe.
doi:10.1186/1746-6148-4-42
PMCID: PMC2575192  PMID: 18922155
13.  Recognition of lameness and decisions to catch for inspection among sheep farmers and specialists in GB 
Background
Epidemiological studies have used farmer estimates of the prevalence of lameness in their flocks. This assumes that farmers can identify lame sheep. Eight movie clips of sheep with locomotion from sound to moderately lame were used to investigate the ability of farmers and sheep specialists to recognise lame sheep. Each participant was asked to complete a form and indicate, for each movie clip, whether they thought the sheep was lame and whether they would catch it if it was the only lame sheep or if 2 – 5, 6 – 10 or > 10 sheep were equally lame. The farmers' responses were compared with their estimates of flock lameness prevalence and the interval between observing a lame sheep and catching it.
Results
178 farmers and 54 sheep specialists participated. Participants could identify even mildly lame sheep but made a separate decision on whether to catch them. This decision was dependent on the severity of lameness and the number of sheep lame in a group. Those who said they would catch the first lame sheep in a group were significantly more likely to catch mildly lame sheep (farmer-reported median prevalence of lameness 5% (IQR: 2%–6%)). In contrast, farmers who waited for several sheep to be lame indicated that they would only catch more severely lame sheep (farmer reported median flock lameness 11% (IQR: 9%–15%)). Approximately 15% of farmers did not catch individual lame sheep (farmer reported median flock lameness 15% (IQR: 10%–15%)). The flock prevalence of lameness increased as time to treatment increased and time to treatment was positively correlated with only catching more severely lame sheep.
Conclusion
If movie-clips are similar to the flock situation, farmers and specialists can recognise even mildly lame sheep but vary in their management from prompt treatment of the first lame sheep in a group to no individual sheep treatments. The former practices would be appropriate to minimise transmission of footrot, a common, infectious cause of lameness and so reduce its incidence. The analysis also suggests that farmers estimate lameness prevalence relatively accurately because farmers who treated the first mildly lame sheep in a group also reported the lowest prevalence of lameness in their flock.
doi:10.1186/1746-6148-4-41
PMCID: PMC2588441  PMID: 18854014
14.  Geographic and topographic determinants of local FMD transmission applied to the 2001 UK FMD epidemic 
Background
Models of Foot and Mouth Disease (FMD) transmission have assumed a homogeneous landscape across which Euclidean distance is a suitable measure of the spatial dependency of transmission. This paper investigated features of the landscape and their impact on transmission during the period of predominantly local spread which followed the implementation of the national movement ban during the 2001 UK FMD epidemic. In this study 113 farms diagnosed with FMD which had a known source of infection within 3 km (cases) were matched to 188 control farms which were either uninfected or infected at a later timepoint. Cases were matched to controls by Euclidean distance to the source of infection and farm size. Intervening geographical features and connectivity between the source of infection and case and controls were compared.
Results
Road distance between holdings, access to holdings, presence of forest, elevation change between holdings and the presence of intervening roads had no impact on the risk of local FMD transmission (p > 0.2). However the presence of linear features in the form of rivers and railways acted as barriers to FMD transmission (odds ratio = 0.507, 95% CIs = 0.297,0.887, p = 0.018).
Conclusion
This paper demonstrated that although FMD spread can generally be modelled using Euclidean distance and numbers of animals on susceptible holdings, the presence of rivers and railways has an additional protective effect reducing the probability of transmission between holdings.
doi:10.1186/1746-6148-4-40
PMCID: PMC2573875  PMID: 18834510
15.  Syndrome of arachnomelia in Simmental cattle 
Background
The syndrome of arachnomelia is an inherited malformation mainly of limbs, back and head in cattle. At present the arachnomelia syndrome has been well known mainly in Brown Swiss cattle. Nevertheless, the arachnomelia syndrome had been observed in the Hessian Simmental population during the decade 1964–1974. Recently, stillborn Simmental calves were observed having a morphology similar to the arachnomelia syndrome. The goal of this work was the characterization of the morphology and genealogy of the syndrome in Simmental to establish the basis for an effective management of the disease.
Results
The first pathologically confirmed arachnomelia syndrome-cases in the current Simmental population appeared in the year 2005. By 2007, an additional 140 calves with the arachnomelia syndrome were identified. The major pathological findings were malformed bones affecting the head, long bones of the legs and the vertebral column. It could be shown that, with the exception of two cases that were considered as phenocopies, all of the paternal and about two-third of the maternal pedigrees of the affected calves could be traced back to one common founder. Together with the data from experimental matings, the pedigree data support an autosomal recessive mutation being the etiology of the arachnomelia syndrome. The frequency of the mutation in the current population was estimated to be 3.32%.
Conclusion
We describe the repeated occurrence of the arachnomelia syndrome in Simmental calves. It resembles completely the same defect occurring in the Brown Swiss breed. The mutation became relatively widespread amongst the current population. Therefore, a control system has to be established and it is highly desirable to map the disease and develop a genetic test system.
doi:10.1186/1746-6148-4-39
PMCID: PMC2567314  PMID: 18828914
16.  Idiopathic Brainstem Neuronal Chromatolysis (IBNC): a novel prion protein related disorder of cattle? 
Background
The epidemic form of Bovine Spongiform Encephalopathy (BSE) is generally considered to have been caused by a single prion strain but at least two strain variants of cattle prion disorders have recently been recognized. An additional neurodegenerative condition, idiopathic brainstem neuronal chromatolysis and hippocampal sclerosis (IBNC), a rare neurological disease of adult cattle, was also recognised in a sub-set of cattle submitted under the BSE Orders in which lesions of BSE were absent. Between the years of 1988 and 1991 IBNC occurred in Scotland with an incidence of 7 cases per 100,000 beef suckler cows over the age of 6 years.
Results
When the brains of 15 IBNC cases were each tested by immunohistochemistry, all showed abnormal labelling for prion protein (PrP). Immunohistological labelling for PrP was also present in the retina of a single case available for examination. The pattern of PrP labelling in brain is distinct from that seen in other ruminant prion diseases and is absent from brains with other inflammatory conditions and from normal control brains. Brains of IBNC cattle do not reveal abnormal PrP isoforms when tested by the commercial BioRad or Idexx test kits and do not reveal PrPres when tested by Western blotting using stringent proteinase digestion methods. However, some weakly protease resistant isoforms of PrP may be detected when tissues are examined using mild proteinase digestion techniques.
Conclusion
The study shows that a distinctive neurological disorder of cattle, which has some clinical similarities to BSE, is associated with abnormal PrP labelling in brain but the pathology and biochemistry of IBNC are distinct from BSE. The study is important either because it raises the possibility of a significant increase in the scope of prion disease or because it demonstrates that widespread and consistent PrP alterations may not be confined to prion diseases. Further studies, including transmission experiments, are needed to establish whether IBNC is a condition in which prion protein is abnormally regulated or it is yet a further example of an infectious cattle prion disease.
doi:10.1186/1746-6148-4-38
PMCID: PMC2569918  PMID: 18826563
17.  Expressed sequence tags (ESTs) from immune tissues of turbot (Scophthalmus maximus) challenged with pathogens 
Background
The turbot (Scophthalmus maximus; Scophthalmidae; Pleuronectiformes) is a flatfish species of great relevance for marine aquaculture in Europe. In contrast to other cultured flatfish, very few genomic resources are available in this species. Aeromonas salmonicida and Philasterides dicentrarchi are two pathogens that affect turbot culture causing serious economic losses to the turbot industry. Little is known about the molecular mechanisms for disease resistance and host-pathogen interactions in this species. In this work, thousands of ESTs for functional genomic studies and potential markers linked to ESTs for mapping (microsatellites and single nucleotide polymorphisms (SNPs)) are provided. This information enabled us to obtain a preliminary view of regulated genes in response to these pathogens and it constitutes the basis for subsequent and more accurate microarray analysis.
Results
A total of 12584 cDNAs partially sequenced from three different cDNA libraries of turbot (Scophthalmus maximus) infected with Aeromonas salmonicida, Philasterides dicentrarchi and from healthy fish were analyzed. Three immune-relevant tissues (liver, spleen and head kidney) were sampled at several time points in the infection process for library construction. The sequences were processed into 9256 high-quality sequences, which constituted the source for the turbot EST database. Clustering and assembly of these sequences, revealed 3482 different putative transcripts, 1073 contigs and 2409 singletons. BLAST searches with public databases detected significant similarity (e-value ≤ 1e-5) in 1766 (50.7%) sequences and 816 of them (23.4%) could be functionally annotated. Two hundred three of these genes (24.9%), encoding for defence/immune-related proteins, were mostly identified for the first time in turbot. Some ESTs showed significant differences in the number of transcripts when comparing the three libraries, suggesting regulation in response to these pathogens. A total of 191 microsatellites, with 104 having sufficient flanking sequences for primer design, and 1158 putative SNPs were identified from these EST resources in turbot.
Conclusion
A collection of 9256 high-quality ESTs was generated representing 3482 unique turbot sequences. A large proportion of defence/immune-related genes were identified, many of them regulated in response to specific pathogens. Putative microsatellites and SNPs were identified. These genome resources constitute the basis to develop a microarray for functional genomics studies and marker validation for genetic linkage and QTL analysis in turbot.
doi:10.1186/1746-6148-4-37
PMCID: PMC2569028  PMID: 18817567
18.  Frequencies of polymorphisms associated with BSE resistance differ significantly between Bos taurus, Bos indicus, and composite cattle 
Background
Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases that affect several mammalian species. At least three factors related to the host prion protein are known to modulate susceptibility or resistance to a TSE: amino acid sequence, atypical number of octapeptide repeats, and expression level. These factors have been extensively studied in breeds of Bos taurus cattle in relation to classical bovine spongiform encephalopathy (BSE). However, little is currently known about these factors in Bos indicus purebred or B. indicus × B. taurus composite cattle. The goal of our study was to establish the frequency of markers associated with enhanced susceptibility or resistance to classical BSE in B. indicus purebred and composite cattle.
Results
No novel or TSE-associated PRNP-encoded amino acid polymorphisms were observed for B. indicus purebred and composite cattle, and all had the typical number of octapeptide repeats. However, differences were observed in the frequencies of the 23-bp and 12-bp insertion/deletion (indel) polymorphisms associated with two bovine PRNP transcription regulatory sites. Compared to B. taurus, B. indicus purebred and composite cattle had a significantly lower frequency of 23-bp insertion alleles and homozygous genotypes. Conversely, B. indicus purebred cattle had a significantly higher frequency of 12-bp insertion alleles and homozygous genotypes in relation to both B. taurus and composite cattle. The origin of these disparities can be attributed to a significantly different haplotype structure within each species.
Conclusion
The frequencies of the 23-bp and 12-bp indels were significantly different between B. indicus and B. taurus cattle. No other known or potential risk factors were detected for the B. indicus purebred and composite cattle. To date, no consensus exists regarding which bovine PRNP indel region is more influential with respect to classical BSE. Should one particular indel region and associated genotypes prove more influential with respect to the incidence of classical BSE, differences regarding overall susceptibility and resistance for B. indicus and B. taurus cattle may be elucidated.
doi:10.1186/1746-6148-4-36
PMCID: PMC2569919  PMID: 18808703
19.  A four year longitudinal sero-epidemiology study of Neospora caninum in adult cattle from 114 cattle herds in south west England: Associations with age, herd and dam-offspring pairs 
Background
Neosporosis caused by the protozoan parasite Neospora caninum, is an economically important cause of abortion, stillbirth, low milk yield, reduced weight gain and premature culling in cattle. Consequently, a seroepidemiological study of N. caninum antibodies was conducted in England with 29,782 samples of blood taken from 15,736 cattle from 114 herds visited on three occasions at yearly intervals. Herds were categorised into lower (< 10%) and higher (≥ 10%) median herd seroprevalence. Hierarchical models were run to investigate associations between the sample to positive (S/P) ratio and herd and cattle factors.
Results
Ninety-four percent of herds had at least one seropositive cow; 12.9% of adult cattle had at least one seropositive test. Approximately 90% of herds were seropositive at all visits; 9 herds (8%) changed serological status between visits. The median N. caninum seroprevalence in positive herds was 10% (range 0.4% to 58.8%). There was a positive association between the serostatus of offspring and dams that were ever seropositive. In the hierarchical model of low seroprevalence herds there was no significant association between S/P ratio and cattle age. There was a significantly lower S/P ratio in cattle in herds that were totally restocked after the foot-and-mouth epidemic of 2001 compared with those from continuously stocked herds and cattle purchased into these herds had a higher S/P ratio than homebred cattle. In the model of high seroprevalence herds the S/P ratio increased with cattle age, but was not associated with restocking or cattle origin.
Conclusion
There were no strong temporal changes in herd seroprevalence of N. caninum but 90% of herds had some seropositive cattle over this time period. Vertical transmission from seropositive dams appeared to occur in all herds. In herds with a high seroprevalence the increasing S/P ratio in 2–4 year old cattle is suggestive of exposure to N. caninum: horizontal transmission between adult cattle, infection from a local source or recrudescence and abortions. Between-herd movements of infected cattle enhance the spread of N. caninum, particularly into low seroprevalence herds. Some restocked herds had little exposure to N. caninum, while in others infection had spread in the time since restocking.
doi:10.1186/1746-6148-4-35
PMCID: PMC2556655  PMID: 18793402
20.  Peste des Petits Ruminants (PPR) in Ethiopia: Analysis of a national serological survey 
Background
Peste des petits ruminants (PPR) is a contagious viral disease of small ruminants in Africa and Asia. In 1999, probably the largest survey on PPR ever conducted in Africa was initiated in Ethiopia where 13 651 serum samples from 7 out of the 11 regions were collected and analyzed by competitive enzyme-linked immunosorbent assay (cELISA). The objective of this paper is to present the results of this survey and discuss their practical implications for PPR-endemic regions.
Methods
We explored the spatial distribution of PPR in Ethiopia and we investigated risk factors for positive serological status. Intracluster correlation coefficients (ρ), were calculated for 43 wereda (administrative units).
Results
Seroprevalence was very heterogeneous across regions and even more across wereda, with prevalence estimates ranging from 0% to 52.5%. Two groups of weredas could be distinguished on the basis of the estimated ρ: a group with very low ρ (ρ < 0.12) and a group with very high ρ (ρ > 0.37).
Conclusion
The results indicate that PPRV circulation has been very heterogeneous, the values for the ρ may reflect the endemic or epidemic presence of the virus or the various degrees of mixing of animals in the different areas and production systems. Age appears as a risk factor for seropositive status, the linear effect seeming to confirm in the field that PPRV is highly immunogenic. Our estimates of intracluster correlation may prove useful in the design of serosurveys in other countries where PPR is of importance.
doi:10.1186/1746-6148-4-34
PMCID: PMC2561016  PMID: 18786275
21.  A cross-sectional study to determine the seroprevalence of bluetongue virus serotype 8 in sheep and goats in 2006 and 2007 in the Netherlands 
Background
In August 2006 a major epidemic of bluetongue virus serotype 8 (BTV8) started off in North-West Europe. In the course of 2007 it became evident that BTV8 had survived the winter in North-West Europe, re-emerged and spread exponentially. Recently, the European Union decided to start vaccination against BTV8. In order to improve the understanding of the epidemiological situation, it was necessary to execute a cross-sectional serological study at the end of the BT vector season. Cattle were the target species for cross-sectional serological studies in Europe at the end of 2006 and 2007. However, there was no information on the BTV8-seroprevalence in sheep and goats.
Results
On the basis of our cross-sectional study, the estimated seroprevalence of BTV8-exposed locations in the Netherlands in 2006 was 0% for goats (95% confidence interval: 0 – 5.6%) and 7.0% for sheep (95% confidence interval: 3.5 – 12.9%). The estimated seroprevalence of BTV-8 exposed locations in 2007 was 47% for goats (95% confidence interval: 36 – 58%) and 70% for sheep (95% confidence interval: 63 – 76%). There was a wide range in within-location seroprevalence in locations with goats and sheep (1 – 100%). A gradient in seroprevalence was seen, with the highest level of seroprevalence in the southern Netherlands, the area where the epidemic started in 2006, and a decreasing seroprevalence when going in a northern direction.
Conclusion
There is a much higher estimated seroprevalence of locations with goats exposed to BTV8 than can be inferred from the rather low number of reported clinical outbreaks in goats. This is probably due to the fact that clinical signs in infected goats are far less obvious than in sheep. The wide range in within-location seroprevalence observed means that the proportion of animals protected in 2008 by a natural infection in 2006 and/or 2007 can differ highly between flocks. This should be taken into account when vaccinating animals.
doi:10.1186/1746-6148-4-33
PMCID: PMC2531099  PMID: 18752681
22.  Evaluation of prognostic markers for canine mast cell tumors treated with vinblastine and prednisone 
Background
Canine cutaneous mast cell tumor (MCT) is a common neoplastic disease associated with a variable biologic behavior. Surgery remains the primary treatment for canine MCT; however, radiation therapy (RT) and chemotherapy are commonly used to treat aggressive MCT. The goals of this study were to evaluate the prognostic utility of histologic grade, c-KIT mutations, KIT staining patterns, and the proliferation markers Ki67 and AgNORs in dogs postoperatively treated with vinblastine and prednisone +/- RT, and to compare the outcome of dogs treated with post-operative chemotherapy +/- RT to that of a prognostically matched group treated with surgery alone. Associations between prognostic markers and survival were evaluated. Disease-free intervals (DFI) and overall survival times (OS) of dogs with similar pretreatment prognostic indices postoperatively treated with chemotherapy were compared to dogs treated with surgery alone.
Results
Histologic grade 3 MCTs, MCTs with c-KIT mutations, MCTs with increased cytoplasmic KIT, and MCTs with increased Ki67 and AgNOR values were associated with decreased DFI and OS. Dogs with histologic grade 3 MCT had significantly increased DFI and OS when treated with chemotherapy vs. surgery alone. Although not statistically significant due to small sample sizes, MCTs with c-KIT mutations had increased DFI and OS when treated with chemotherapy vs. surgery alone.
Conclusion and clinical importance
This study confirms the prognostic value of histologic grade, c-KIT mutations, KIT staining patterns, and proliferation analyses for canine MCT. Additionally, the results of this study further define the benefit of postoperative vinblastine and prednisone for histologic grade 3 MCTs.
doi:10.1186/1746-6148-4-32
PMCID: PMC2529280  PMID: 18700956
23.  Loop-mediated isothermal amplification as an emerging technology for detection of Yersinia ruckeri the causative agent of enteric red mouth disease in fish 
Background
Enteric Redmouth (ERM) disease also known as Yersiniosis is a contagious disease affecting salmonids, mainly rainbow trout. The causative agent is the gram-negative bacterium Yersinia ruckeri. The disease can be diagnosed by isolation and identification of the causative agent, or detection of the Pathogen using fluorescent antibody tests, ELISA and PCR assays. These diagnostic methods are laborious, time consuming and need well trained personnel.
Results
A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for detection of Y. ruckeri the etiological agent of enteric red mouth (ERM) disease in salmonids. The assay was optimised to amplify the yruI/yruR gene, which encodes Y. ruckeri quorum sensing system, in the presence of a specific primer set and Bst DNA polymerase at an isothermal temperature of 63°C for one hour. Amplification products were detected by visual inspection, agarose gel electrophoresis and by real-time monitoring of turbidity resulted by formation of LAMP amplicons. Digestion with HphI restriction enzyme demonstrated that the amplified product was unique. The specificity of the assay was verified by the absence of amplification products when tested against related bacteria. The assay had 10-fold higher sensitivity compared with conventional PCR and successfully detected Y. ruckeri not only in pure bacterial culture but also in tissue homogenates of infected fish.
Conclusion
The ERM-LAMP assay represents a practical alternative to the microbiological approach for rapid, sensitive and specific detection of Y. ruckeri in fish farms. The assay is carried out in one hour and needs only a heating block or water bath as laboratory furniture. The advantages of the ERM-LAMP assay make it a promising tool for molecular detection of enteric red mouth disease in fish farms.
doi:10.1186/1746-6148-4-31
PMCID: PMC2531098  PMID: 18700011
24.  Biomarkers for ragwort poisoning in horses: identification of protein targets 
Background
Ingestion of the poisonous weed ragwort (Senecio jacobea) by horses leads to irreversible liver damage. The principal toxins of ragwort are the pyrrolizidine alkaloids that are rapidly metabolised to highly reactive and cytotoxic pyrroles, which can escape into the circulation and bind to proteins. In this study a non-invasive in vitro model system has been developed to investigate whether pyrrole toxins induce specific modifications of equine blood proteins that are detectable by proteomic methods.
Results
One dimensional gel electrophoresis revealed a significant alteration in the equine plasma protein profile following pyrrole exposure and the formation of a high molecular weight protein aggregate. Using mass spectrometry and confirmation by western blotting the major components of this aggregate were identified as fibrinogen, serum albumin and transferrin.
Conclusion
These findings demonstrate that pyrrolic metabolites can modify equine plasma proteins. The high molecular weight aggregate may result from extensive inter- and intra-molecular cross-linking of fibrinogen with the pyrrole. This model has the potential to form the basis of a novel proteomic strategy aimed at identifying surrogate protein biomarkers of ragwort exposure in horses and other livestock.
doi:10.1186/1746-6148-4-30
PMCID: PMC2527303  PMID: 18691403
25.  Gene expression profiles in liver of pigs with extreme high and low levels of androstenone 
Background
Boar taint is the unpleasant odour and flavour of the meat of uncastrated male pigs that is primarily caused by high levels of androstenone and skatole in adipose tissue. Androstenone is a steroid and its levels are mainly genetically determined. Studies on androstenone metabolism have, however, focused on a limited number of genes. Identification of additional genes influencing levels of androstenone may facilitate implementation of marker assisted breeding practices. In this study, microarrays were used to identify differentially expressed genes and pathways related to androstenone metabolism in the liver from boars with extreme levels of androstenone in adipose tissue.
Results
Liver tissue samples from 58 boars of the two breeds Duroc and Norwegian Landrace, 29 with extreme high and 29 with extreme low levels of androstenone, were selected from more than 2500 individuals. The samples were hybridised to porcine cDNA microarrays and the 1% most significant differentially expressed genes were considered significant. Among the differentially expressed genes were metabolic phase I related genes belonging to the cytochrome P450 family and the flavin-containing monooxygenase FMO1. Additionally, phase II conjugation genes including UDP-glucuronosyltransferases UGT1A5, UGT2A1 and UGT2B15, sulfotransferase STE, N-acetyltransferase NAT12 and glutathione S-transferase were identified. Phase I and phase II metabolic reactions increase the water solubility of steroids and play a key role in their elimination. Differential expression was also found for genes encoding 17beta-hydroxysteroid dehydrogenases (HSD17B2, HSD17B4, HSD17B11 and HSD17B13) and plasma proteins alpha-1-acid glycoprotein (AGP) and orosomucoid (ORM1). 17beta-hydroxysteroid dehydrogenases and plasma proteins regulate the availability of steroids by controlling the amount of active steroids accessible to receptors and available for metabolism. Differences in the expression of FMO1, NAT12, HSD17B2 and HSD17B13 were verified by quantitative real competitive PCR.
Conclusion
A number of genes and pathways related to metabolism of androstenone in liver were identified, including new candidate genes involved in phase I oxidation metabolism, phase II conjugation metabolism, and regulation of steroid availability. The study is a first step towards a deeper understanding of enzymes and regulators involved in pathways of androstenone metabolism and may ultimately lead to the discovery of markers to reduce boar taint.
doi:10.1186/1746-6148-4-29
PMCID: PMC2535776  PMID: 18684314

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