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2.  Intracerebral administration of Interleukin-12 (IL-12) and IL-18 modifies the course of mouse scrapie 
Background
Prion diseases are characterised by a neurodegenerative pattern in which the function of immune system remains still elusive. In the present study, we evaluate if an exogenous treatment with Interleukin-12 (IL-12) and IL-18, able to activate microglia, is able to affect scrapie pathogenesis.
Results
Cytokines injected intracranially, induced a strong inflammatory response characterised by TNF-α production and microglia activation. Two groups of mice were injected intracerebrally with high dose of ME7 strain of scrapie containing IL-12 and IL-18 or sterile saline. Cytokines-treated mice showed a more pronounced accumulation of PrPSc in brain tissues at 90 days post-inoculation and a shorter mean survival times than untreated mice.
Conclusion
We can conclude that intracerebral administration of IL-12 and IL-18 can modulate scrapie pathogenesis possibly through a microglia-mediated pattern.
doi:10.1186/1746-6148-2-37
PMCID: PMC1769363  PMID: 17192191
3.  A proteomics study of the response of North Ronaldsay sheep to copper challenge 
Background
The objective of this proteomics study was to identify proteins that changed expression as a result of copper challenge in the uniquely copper sensitive North Ronaldsay sheep and further, to compare those changes in expression with the more copper tolerant Cambridge breed. Such data gives us a proteome-centered perspective of the pathogenesis of copper-induced oxidative stress in this breed.
Results
Many proteins respond to copper challenge, but this study focuses on those exhibiting a differential response between the two breeds, related to liver copper content. As copper accumulated in the tissue, the pattern of expression of several proteins was markedly different, in North Ronaldsay sheep as compared to the Cambridge breed.
Conclusion
The pattern of changes was consistent with the greatly enhanced susceptibility of North Ronaldsay sheep to copper-induced oxidative stress, focused on mitochondrial disturbance with consequent activation of hepatic stellate cells. The expression profiles were sufficiently complex that the response could not simply be explained as a hypersensitivity to copper in North Ronaldsay sheep.
doi:10.1186/1746-6148-2-36
PMCID: PMC1766353  PMID: 17192175
4.  The molecular epidemiology of Foot-and-Mouth Disease virus serotypes A and O from 1998 to 2004 in Turkey 
Background
Foot-and-Mouth Disease (FMD) causes significant economic losses in Turkish livestock.
We have analysed the genetic diversity of the 1D sequences, encoding the hypervariable surface protein VP1, of Turkish isolates of serotype A and O collected from 1998 to 2004 in order to obtain epidemiological and immunological information.
Results
The 1D coding region of 33 serotype O and 20 serotype A isolates, obtained from outbreaks of FMD between 1998 and 2004, was sequenced.
For serotype A, we confirmed the occurrence of the two subtypes IRN99 and IRN96. These subtypes are most divergent within the region encoding the immuno-dominant GH-loop. Also a close relationship to Foot-and-Mouth Disease virus (FMDV) serotype A isolates obtained from outbreaks in Iraq and Iran were detected and a clustering of isolates collected during the same period of time were found.
The analysis of the deduced amino-acid sequences of these subtypes revealed evidence of positive selection in one site and one deletion, both within the GH-loop region. By inferring the ancestral history of the positively selected codon, two potential precursors were found. Furthermore, the structural alignment of IRN99 and IRN96 revealed differences between the tertiary structures of these subtypes.
The similarity plot of the serotype O isolates suggested a more homogeneous group than the serotype A isolates. However, phylogenetic analysis revealed two major groups, each further divided in subgroups, of which some only consisted of Turkish isolates.
Positively selected sites and structural differences of the Turkish isolates analysed, were not found.
Conclusion
The sequence and structural analysis of the IRN99 strains is indicative of positive selection suggesting an immunological advantage compared to IRN96. However, results of antigenic comparison reported elsewhere do not substantiate such a conclusion. There is evidence that IRN99 was introduced to Turkey, in all probability from Iran.
Since, a member of the IRN96 lineage was included as a component of the FMDV vaccine produced since 2000, the outbreaks caused by IRN96 strains in 2004 could be due to incomplete vaccine coverage.
The Turkish type O strains, all with a VP1 structure similar to the O1/Manisa/69 vaccine, appear in several sublineages. Whether these sublineages reflect multiple samplings from a limited number of outbreaks, or if they reflect cross-boundary introductions is not clear.
doi:10.1186/1746-6148-2-35
PMCID: PMC1698480  PMID: 17144917
5.  Characterization of ovine hepatic gene expression profiles in response to Escherichia coli lipopolysaccharide using a bovine cDNA microarray 
Background
During systemic gram-negative bacterial infections, lipopolysaccharide (LPS) ligation to the hepatic Toll-like receptor-4 complex induces the production of hepatic acute phase proteins that are involved in the host response to infection and limit the associated inflammatory process. Identifying the genes that regulate this hepatic response to LPS in ruminants may provide insight into the pathogenesis of bacterial diseases and eventually facilitate breeding of more disease resistant animals. The objective of this research was to profile the expression of ovine hepatic genes in response to Escherichia coli LPS challenge (0, 200, 400 ng/kg) using a bovine cDNA microarray and quantitative real-time PCR (qRT-PCR).
Results
Twelve yearling ewes were challenged iv with E. coli LPS (0, 200, 400 ng/kg) and liver biopsies were collected 4–5 hours post-challenge to assess hepatic gene expression profiles by bovine cDNA microarray and qRT-PCR analyses. The expression of CD14, C3, IL12R, NRAMP1, SOD and IGFBP3 genes was down regulated, whereas the expression of ACTHR, IFNαR, CD1, MCP-1 and GH was increased during LPS challenge. With the exception of C3, qRT-PCR analysis of 7 of these genes confirmed the microarray results and demonstrated that GAPDH is not a suitable housekeeping gene in LPS challenged sheep.
Conclusion
We have identified several potentially important genes by bovine cDNA microarray and qRT-PCR analyses that are differentially expressed during the ovine hepatic response to systemic LPS challenge. Their potential role in regulating the inflammatory response to LPS warrants further investigation.
doi:10.1186/1746-6148-2-34
PMCID: PMC1684251  PMID: 17134499
6.  Evaluation of tandem repeats for MLVA typing of Streptococcus uberis isolated from bovine mastitis 
Background
Streptococcus uberis is a common cause of bovine mastitis and recommended control measures, based on improved milking practice, teat dipping and antibiotic treatment at drying-off, are poorly efficient against this environmental pathogen. A simple and efficient typing method would be helpful in identifying S.uberis sources, virulent strains and cow to cow transmission. The potential of MLVA (Multiple Loci VNTR Analysis; VNTR, Variable Number of Tandem Repeats) for S. uberis mastitis isolates genotyping was investigated.
Results
The genomic sequence of Streptococcus uberis (strain 0104J) was analyzed for potential variable number tandem repeats (VNTRs). Twenty-five tandem repeats were identified and amplified by PCR with DNA samples from 24 S. uberis strains. A set of seven TRs were found to be polymorphic and used for MLVA typing of 88 S. uberis isolates. A total of 82 MLVA types were obtained with 22 types among 26 strains isolated from the milk of mastitic cows belonging to our experimental herd, and 61 types for 62 epidemiologically unrelated strains, i.e. collected in different herds and areas.
Conclusion
The MLVA method can be applied to S. uberis genotyping and constitutes an interesting complement to existing typing methods. This method, which is easy to perform, low cost and can be used in routine, could facilitate investigations of the epidemiology of S. uberis mastitis in dairy cows.
doi:10.1186/1746-6148-2-33
PMCID: PMC1660536  PMID: 17112377
7.  DGAT1 and ABCG2 polymorphism in Indian cattle (Bos indicus) and buffalo (Bubalus bubalis) breeds 
Background
Indian cattle (Bos indicus) and riverine buffalo (Bubalus bubalis) give a poor yield of milk but it has a high fat and protein percentage compared to taurine cattle. The identification of QTLs (Quantitative Trait Loci) on BTA14 and BTA6 and its subsequent fine mapping has led to identification of two non conservative mutations affecting milk production and composition. Our objective was to estimate the frequency of K232A (DGAT1 – diacylglycerol – acyltransferase 1) and Y581S (ABCG2 – ATP binding cassette sub family G member 2) polymorphisms in diverse cattle and buffalo breeds of India having large variation in terms of milk production.
Results
We screened the reported missense mutations in six cattle and five buffalo breeds. The DGAT1K and ABCG2Y alleles were found to be fixed in Indian cattle and buffalo breeds studied.
Conclusion
This study provides an indirect evidence that all the Indian cattle and buffalo breeds have fixed alleles with respect to DGAT1 and ABCG2 genes reported to be responsible for higher milk fat yield, higher fat and protein percent.
doi:10.1186/1746-6148-2-32
PMCID: PMC1636029  PMID: 17087837
8.  Different prion disease phenotypes result from inoculation of cattle with two temporally separated sources of sheep scrapie from Great Britain 
Background
Given the theoretical proposal that bovine spongiform encephalopathy (BSE) could have originated from sheep scrapie, this study investigated the pathogenicity for cattle, by intracerebral (i.c.) inoculation, of two pools of scrapie agents sourced in Great Britain before and during the BSE epidemic.
Two groups of ten cattle were each inoculated with pools of brain material from sheep scrapie cases collected prior to 1975 and after 1990. Control groups comprised five cattle inoculated with sheep brain free from scrapie, five cattle inoculated with saline, and for comparison with BSE, naturally infected cattle and cattle i.c. inoculated with BSE brainstem homogenate from a parallel study. Phenotypic characterisation of the disease forms transmitted to cattle was conducted by morphological, immunohistochemical, biochemical and biological methods.
Results
Disease occurred in 16 cattle, nine inoculated with the pre-1975 inoculum and seven inoculated with the post-1990 inoculum, with four cattle still alive at 83 months post challenge (as at June 2006). The different inocula produced predominantly two different disease phenotypes as determined by histopathological, immunohistochemical and Western immunoblotting methods and biological characterisation on transmission to mice, neither of which was identical to BSE. Whilst the disease presentation was uniform in all scrapie-affected cattle of the pre-1975 group, the post-1990 inoculum produced a more variable disease, with two animals sharing immunohistochemical and molecular profile characteristics with animals in the pre-1975 group.
Conclusion
The study has demonstrated that cattle inoculated with different pooled scrapie sources can develop different prion disease phenotypes, which were not consistent with the phenotype of BSE of cattle and whose isolates did not have the strain typing characteristics of the BSE agent on transmission to mice.
doi:10.1186/1746-6148-2-31
PMCID: PMC1636635  PMID: 17044917
9.  Evaluation of three 3ABC ELISAs for foot-and-mouth disease non-structural antibodies using latent class analysis 
Background
Foot-and-mouth disease (FMD) is a highly contagious viral disease of even-toed ungulates. Serological diagnosis/surveillance of FMD presents several problems as there are seven serotypes worldwide and in the event of vaccination it may be necessary to be able to identify FMD infected/exposed animals irrespective of their vaccination status. The recent development of non-structural 3ABC protein (NSP) ELISA tests has greatly advanced sero-diagnosis/surveillance as these tests detect exposure to live virus for any of the seven serotypes of FMD, even in vaccinated populations. This paper analyses the performance of three NSP tests using a Bayesian formulation of the Hui-Walter latent class model to estimate test sensitivity and specificity in the absence of a "gold-standard" test, using sera from a well described cattle population in Cameroon with endemic FMD.
Results
The analysis found a high sensitivity and specificity for both the Danish C-ELISA and the World Organisation for Animal Health (O.I.E.) recommended South American I-ELISA. However, the commercial CHEKIT kit, though having high specificity, has very low sensitivity. The results of the study suggests that for NSP ELISAs, latent class models are a useful alternative to the traditional approach of evaluating diagnostic tests against a known "gold-standard" test as imperfections in the "gold-standard" may give biased test characteristics.
Conclusion
This study demonstrates that when applied to naturally infected zebu cattle managed under extensive rangeland conditions, the FMD ELISAs may not give the same parameter estimates as those generated from experimental studies. The Bayesian approach allows for full posterior probabilities and capture of the uncertainty in the estimates. The implications of an imperfect specificity are important for the design and interpretation of sero-surveillance data and may result in excessive numbers of false positives in low prevalence situations unless a follow-up confirmatory test such as the enzyme linked immunoelectrotransfer blot (EITB) is used.
doi:10.1186/1746-6148-2-30
PMCID: PMC1629010  PMID: 17042948
10.  Canine faecal contamination and parasitic risk in the city of Naples (southern Italy) 
Background
Dogs are associated with more than 60 zoonotic diseases among which, parasitosis and, in particular, helminthosis, can pose serious public-health concerns worldwide. Many canine gastrointestinal parasites eliminate their dispersion elements (eggs, larvae, oocysts) by the faecal route. The quantity of canine faeces deposited on public and private property in cities worldwide is both a perennial nuisance and an important health issue. Public sites such as playgrounds, parks, gardens, public squares and sandpits may be an important source of human infection. The aim of this study was to evaluate the extent of both canine faecal contamination in the city of Naples (southern Italy), and presence of canine parasitic elements, with particular regard to those which are potential agents of zoonosis. A regular grid of sub-areas (1 km × 700 m) was overlaid on the city map using a Geographical Information System (GIS). In each sub-area the straightest 1 km transect was drawn and digitalized on-screen in the GIS. Between February and May 2005 canine faeces were counted along the 1 km transects in 143 sub-areas, and 415 canine faecal samples were collected and submitted to coprological examinations. Negative binomial regression models and Gaussian random effects models were used to analyze the association between faeces count and human population density taking into account for extraPoisson variability. Logistic regression model was used to evaluate the association between positivity to parasitic elements and number of canine faeces.
Results
Out of the 143 studied sub-areas, 141 (98.6%) contained canine faeces. There was a strong spatial gradient with 48% of the total variability accounted by between neighbourhood variability; a positive association between the number of faeces and the human population density was found. Seventy (over 415, 16.9%) canine faecal samples were positive for parasitic elements. There was no association between positivity to parasitic elements and the number of canine faeces. Eggs of Toxocara canis, Toxascaris leonina, Ancylostoma caninum and Trichuris vulpis were found, as well as oocysts of Isospora canis.
Conclusion
In conclusion, the results of the present study, conducted using GIS both for planning and sampling and for evaluation and presentation of findings, showed the presence of canine faecal contamination in the city of Naples, and the presence of canine parasitic elements, some of which are potential agents of zoonosis.
doi:10.1186/1746-6148-2-29
PMCID: PMC1590007  PMID: 16995934
11.  Characterization of the aldo-keto reductase 1C gene cluster on pig chromosome 10: possible associations with reproductive traits 
Background
The rate of pubertal development and weaning to estrus interval are correlated and affect reproductive efficiency of swine. Quantitative trait loci (QTL) for age of puberty, nipple number and ovulation rate have been identified in Meishan crosses on pig chromosome 10q (SSC10) near the telomere, which is homologous to human chromosome 10p15 and contains an aldo-keto reductase (AKR) gene cluster with at least six family members. AKRs are tissue-specific hydroxysteroid dehydrogenases that interconvert weak steroid hormones to their more potent counterparts and regulate processes involved in development, homeostasis and reproduction. Because of their location in the swine genome and their implication in reproductive physiology, this gene cluster was characterized and evaluated for effects on reproductive traits in swine.
Results
Screening the porcine CHORI-242 BAC library with a full-length AKR1C4 cDNA identified 7 positive clones and sample sequencing of 5 BAC clones revealed 5 distinct AKR1C genes (AKR1CL2 and AKR1C1 through 4), which mapped to 126–128 cM on SSC10. Using the IMpRH7000rad and IMNpRH212000rad radiation hybrid panels, these 5 genes mapped between microsatellite markers SWR67 and SW2067. Comparison of sequence data with the porcine BAC fingerprint map show that the cluster of genes resides in a 300 kb region. Twelve SNPs were genotyped in gilts observed for age at first estrus and ovulation rate from the F8 and F10 generations of one-quarter Meishan descendants of the USMARC resource population. Age at puberty, nipple number and ovulation rate data were analyzed for association with genotypes by MTDFREML using an animal model. One SNP, a phenylalanine to isoleucine substitution in AKR1C2, was associated with age of puberty (p = 0.07) and possibly ovulation rate (p = 0.102). Two SNP in AKR1C4 were significantly associated with nipple number (p ≤ 0.03) and another possibly associated with age at puberty (p = 0.09).
Conclusion
AKR1C genotypes were associated with nipple number as well as possible effects on age at puberty and ovulation rate. The estimated effects of AKR1C genotypes on these traits suggest that the SNPs are in incomplete linkage disequilibrium with the causal mutations that affect reproductive traits in swine. Further investigations are necessary to identify these mutations and understand how these AKR1C genes affect these important reproductive traits.
The nucleotide sequence data reported have been submitted to GenBank and assigned accession numbers [GenBank:DQ474064–DQ474068, GenBank:DQ494488–DQ494490 and GenBank:DQ487182–DQ487184].
doi:10.1186/1746-6148-2-28
PMCID: PMC1586007  PMID: 16970816
12.  Is equine colic seasonal? Novel application of a model based approach 
Background
Colic is an important cause of mortality and morbidity in domesticated horses yet many questions about this condition remain to be answered. One such question is: does season have an effect on the occurrence of colic? Time-series analysis provides a rigorous statistical approach to this question but until now, to our knowledge, it has not been used in this context. Traditional time-series modelling approaches have limited applicability in the case of relatively rare diseases, such as specific types of equine colic. In this paper we present a modelling approach that respects the discrete nature of the count data and, using a regression model with a correlated latent variable and one with a linear trend, we explored the seasonality of specific types of colic occurring at a UK referral hospital between January 1995–December 2004.
Results
Six- and twelve-month cyclical patterns were identified for all colics, all medical colics, epiploic foramen entrapment (EFE), equine grass sickness (EGS), surgically treated and large colon displacement/torsion colic groups. A twelve-month cyclical pattern only was seen in the large colon impaction colic group. There was no evidence of any cyclical pattern in the pedunculated lipoma group. These results were consistent irrespective of whether we were using a model including latent correlation or trend. Problems were encountered in attempting to include both trend and latent serial dependence in models simultaneously; this is likely to be a consequence of a lack of power to separate these two effects in the presence of small counts, yet in reality the underlying physical effect is likely to be a combination of both.
Conclusion
The use of a regression model with either an autocorrelated latent variable or a linear trend has allowed us to establish formally a seasonal component to certain types of colic presented to a UK referral hospital over a 10 year period. These patterns appeared to coincide with either times of managemental change or periods when horses are more likely to be intensively managed. Further studies are required to identify the determinants of the observed seasonality. Importantly, this type of regression model has applications beyond the study of equine colic and it may be useful in the investigation of seasonal patterns in other, relatively rare, conditions in all species.
doi:10.1186/1746-6148-2-27
PMCID: PMC1570133  PMID: 16930473
13.  Flock-level risk factors for scrapie in Great Britain: analysis of a 2002 anonymous postal survey 
Background
In November 2002, an anonymous postal survey of sheep farmers in Great Britain (GB) was conducted to identify factors associated with the flock-level occurrence of scrapie. This survey was undertaken to update an earlier postal survey in 1998, and was the first occasion in which a large-scale postal survey had been repeated.
Results
The results of the 2002 survey indicated that scrapie was more likely to occur in certain geographic regions; in purebred compared to commercial flocks; in larger flocks; in flocks which lambed in group pens compared to those which lambed in individual pens; in flocks which always lambed in the same location compared to those which did not; and in farms which kept certain breeds of sheep. In addition to these factors, the likelihood of the disease occurring in homebred animals was higher in flocks which bred a greater proportion of replacement animals or which bought-in lambs. Finally, within-flock transmission following exposure was more likely to occur in hill flocks compared to other farm types; in flocks which bred a greater proportion of replacement animals; and in farms which kept a certain crossbreed of ewe.
Conclusion
The risk factors identified from the 1998 and 2002 anonymous postal surveys in Great Britain were similar. However, differences between the surveys were identified in the influence of region and of purchasing behaviour on the risk of scrapie. These differences are most likely a consequence of changes in farmer awareness and the impact of the 2001 foot-and-mouth disease epidemic, respectively.
doi:10.1186/1746-6148-2-25
PMCID: PMC1557843  PMID: 16887027
14.  Molecular characterization of Anaplasma platys strains from dogs in Sicily, Italy 
Background
The genetic diversity of Anaplasma platys (Rickettsiales: Anaplasmataceae) strains is currently poorly defined. The present study was designed to characterize A. patys strains in dogs from Palermo, Sicily, Italy, using a combination of PCR and sequence analysis of the 16S rDNA, heat shock operon groESL and citrate synthase (gltA) genes.
Results
Blood was collected from 344 dogs (111 pet dogs, 122 pound dogs and 111 hunting dogs) during 2003–2005 in the Province of Palermo, Sicily, Italy. The prevalence of A. platys in dogs in Sicily, as demonstrated by PCR and sequence analysis of the 16S rDNA, groESL and gltA genes, was 4%. None of the samples were positive for A. marginale, A. centrale, A. ovis and A. phagocytophilum DNA. Three different gltA genotypes of A. platys were identified in dogs from Sicily. Two of the gltA sequences of Sicilian A. platys strains were different from sequences reported previously. However, one of the gltA, 16S rDNA and groESL sequences were identical to the sequence of A. platys strains from other regions of the world characterized previously.
Conclusion
At least three different strains of A. platys were identified in dogs from Sicily by PCR and sequence analyses of the 16S rDNA, groESL and gltA genes. The results reported herein suggested that genetic diversity of A. platys strains may be similar to A. ovis, but lower than the diversity reported for A. marginale and A. phagocytophilum. This lower genetic diversity may have resulted from restricted movement of infected hosts compared to A. marginale-infected cattle and/or the limited host range of A. ovis and A. platys as compared with A. phagocytophilum. These results expand our knowledge about A. platys and encourage further research for analysis of the genetic variation of A. platys strains worldwide.
doi:10.1186/1746-6148-2-24
PMCID: PMC1550391  PMID: 16872489
15.  Porcine congenital splayleg is characterised by muscle fibre atrophy associated with relative rise in MAFbx and fall in P311 expression 
Background
Porcine congenital splayleg (PCS) is the most important congenital condition of piglets, associated with lameness and immobility, of unknown aetiology and pathogenesis, hence the need to better understand the condition by defining, in the first instance, its histopathology and molecular pathology.
Results
Semitendinosus, longissimus dorsi, and gastrocnemius muscles were removed from 4 sets of 2-day-old splayleg piglets, each with a corresponding normal litter mate. Based on immunohistochemistry and histological image analysis, PCS piglets showed significantly smaller fibre size without any accompanying sign of inflammation. Although there was no dramatic change in fibre type composition in affected muscles, several structural myosin heavy chain genes were significantly down-regulated. MAFbx, a major atrophy marker, was highly up-regulated in nearly all PCS muscles, in comparison with controls from normal litter mates. In contrast, P311, a novel 8 kDa protein, was relatively down-regulated in all the PCS muscles. To investigate a functional role of P311 in skeletal muscle, its full-length cDNA was over-expressed in murine C2C12 muscle cells, which resulted in enhanced cell proliferation with reduced myotube formation. Hence, reduced P311 expression in PCS piglets might contribute to atrophy through reduced muscle cell proliferation. P311, predictably, was down-regulated by the over-expression of calcineurin, a key signalling factor of muscle differentiation.
Conclusion
We demonstrated that PCS is a condition characterised by extensive fibre atrophy and raised fibre density, and propose that the combined differential expression of MAFbx and P311 is of potential in the diagnosis of subclinical PCS.
doi:10.1186/1746-6148-2-23
PMCID: PMC1550227  PMID: 16869957
16.  Protection of rabbits against enteropathogenic Escherichia coli (EPEC) using an intimin null mutant 
Background
Diarrhea and mortality resulting from infections with enteropathogenic Escherichia coli (EPEC) are of major economic importance in the rabbit meat industry. There is a growing need for an effective vaccine to cope with these problems and to reduce the use of antibiotics. EPEC are characterized by an attaching and effacing virulence mechanism. This is partly mediated by the intimate binding between an adhesin, called intimin, and a translocated receptor (Tir) of prokaryote origin. We constructed an intimin deletion mutant of the rabbit EPEC (REPEC) wild-type strain 97/241.6 (bio-/serogroup 3-/O15) and examined its protective capacity.
Results
After verifying its complete loss of virulence, we used the attenuated strain in vaccination-challenge experiments in which complete protection against a homologous, but virulent, strain was observed. The attenuated strain was able to persist in the intestinal lumen, where it elicited an immune response against EPEC-related virulence proteins, as was shown using an EspB-specific ELISA. Despite the priming of an immune response and the generation of specific antibodies, the intimin mutant was not able to fully protect rabbits against challenges with REPEC strains of other bio-/serogroups.
Conclusion
These data indicate that protection against REPEC infections is at least partly bio-/serogroup dependent and a multivalent vaccine may be needed for protection against the full range of REPEC types. Such a combination vaccine may be developed using intimin null mutants, as the latter were clearly shown to be safe and effective against homologous infections.
doi:10.1186/1746-6148-2-22
PMCID: PMC1544329  PMID: 16796739
17.  Scrapie infectivity is quickly cleared in tissues of orally-infected farmed fish 
Background
Scrapie and bovine spongiform encephalopathy (BSE) belongs to the group of animal transmissible spongiform encephalopathy (TSE). BSE epidemic in the UK and elsewhere in Europe has been linked to the use of bovine meat and bone meals (MBM) in the feeding of cattle. There is concern that pigs, poultry and fish bred for human consumption and fed with infected MBM would eventually develop BSE or carry residual infectivity without disease. Although there has been no evidence of infection in these species, experimental data on the susceptibility to the BSE agent of farm animals other than sheep and cow are limited only to pigs and domestic chicken. In the framework of a EU-granted project we have challenged two species of fish largely used in human food consumption, rainbow trout (Oncorhynchus mykiss) and turbot (Scophthalmus maximus), with a mouse-adapted TSE strain (scrapie 139A), to assess the risk related to oral consumption of TSE contaminated food. In trout, we also checked the "in vitro" ability of the pathological isoform of the mouse prion protein (PrPSc) to cross the intestinal epithelium when added to the mucosal side of everted intestine.
Results
Fish challenged with a large amount of scrapie mouse brain homogenate by either oral or parenteral routes, showed the ability to clear the majority of infectivity load. None of the fish tissues taken at different time points after oral or parenteral inoculation was able to provoke scrapie disease after intracerebral inoculation in recipient mice. However, a few recipient mice were positive for PrPSc and spongiform lesions in the brain. We also showed a specific binding of PrPSc to the mucosal side of fish intestine in the absence of an active uptake of the prion protein through the intestinal wall.
Conclusion
These results indicate that scrapie 139A, and possibly BSE, is quickly removed from fish tissues despite evidence of a prion like protein in fish and of a specific binding of PrPSc to the mucosal side of fish intestine.
doi:10.1186/1746-6148-2-21
PMCID: PMC1513558  PMID: 16776828
18.  The time-course of a scrapie outbreak 
Background
Because the incubation period of scrapie has a strong host genetic component and a dose-response relationship, it is possible that changes will occur during an outbreak, especially in the genotypes of cases, age-at-onset of disease and, perhaps, the clinical signs displayed. We investigated these factors for a large outbreak of natural scrapie, which yielded sufficient data to detect temporal trends.
Results
Cases occurred mostly in two genotypes, VRQ/VRQ and VRQ/ARQ, with those early in the outbreak more likely to be of the VRQ/VRQ genotype. As the epidemic progressed, the age-at-onset of disease increased, which reflected changes in the genotypes of cases rather than changes in the age-at-onset within genotypes. Clinical signs of cases changed over the course of the outbreak. As the epidemic progressed VRQ/VRQ and VRQ/ARQ sheep were more likely to be reported with behavioural changes, while VRQ/VRQ sheep only were less likely to be reported with loss of condition.
Conclusion
This study of one of the largest scrapie outbreaks in the UK allowed investigation of the effect of PrP genotype on other epidemiological parameters. Our analysis indicated that, although age-at-onset and clinical signs changed over time, the observed changes were largely, but not exclusively, driven by the time course of the PrP genotypes of cases.
doi:10.1186/1746-6148-2-20
PMCID: PMC1513210  PMID: 16772026
19.  Rapid and discriminatory diagnosis of scrapie and BSE in retro-pharyngeal lymph nodes of sheep 
Background
Diagnosis based on prion detection in lymph nodes of sheep and goats can improve active surveillance for scrapie and, if it were circulating, for bovine spongiform encephalopathy (BSE). With sizes that allow repetitive testing and a location that is easily accessible at slaughter, retropharyngeal lymph nodes (RLN) are considered suitable organs for testing. Western blotting (WB) of brain homogenates is, in principle, a technique well suited to both detect and discriminate between scrapie and BSE. In this report, WB is developed for rapid diagnosis in RLN and to study biochemical characteristics of PrPres.
Results
Optimal PrPres detection in RLN by WB was achieved by proper tissue processing, antibody choice and inclusion of a step for PrPresconcentration. The analyses were performed on three different sheep sources. Firstly, in a study with preclinical scrapie cases, WB of RLN from infected sheep of VRQ/VRQ genotype – VRQ represents, respectively, polymorphic PrP amino acids 136, 154, and 171 – allowed a diagnosis 14 mo earlier compared to WB of brain stem. Secondly, samples collected from sheep with confirmed scrapie in the course of passive and active surveillance programmes in the period 2002–2003 yielded positive results depending on genotype: all sheep with genotypes ARH/VRQ, VRQ/VRQ, and ARQ/VRQ scored positive for PrPres, but ARQ/ARQ and ARR/VRQ were not all positive. Thirdly, in an experimental BSE study, detection of PrPres in all 11 ARQ/ARQ sheep, including 7 preclinical cases, was possible. In all instances, WB and IHC were almost as sensitive. Moreover, BSE infection could be discriminated from scrapie infection by faster electrophoretic migration of the PrPres bands. Using dual antibody staining with selected monoclonal antibodies like 12B2 and L42, these differences in migration could be employed for an unequivocal differentiation between BSE and scrapie. With respect to glycosylation of PrPres, BSE cases exhibited a greater diglycosylated fraction than scrapie cases. Furthermore, a slight time dependent increase of diglycosylated PrPres was noted between individual sheep, which was remarkable in that it occurred in both scrapie and BSE study.
Conclusion
The present data indicate that, used in conjunction with testing in brain, WB of RLN can be a sensitive tool for improving surveillance of scrapie and BSE, allowing early detection of BSE and scrapie and thereby ensuring safer sheep and goat products.
doi:10.1186/1746-6148-2-19
PMCID: PMC1544330  PMID: 16764717
20.  Evaluation of the persistence and gene expression of an anti-Chlamydophila psittaci DNA vaccine in turkey muscle 
Background
DNA vaccination has been shown to elicit specific cellular and humoral immune responses to many different agents in a broad variety of species. However, looking at a commercial use, the duration of the immune response against the vaccine is critical. Therefore the persistence of the DNA vaccine, as well as its expression, should be investigated. We conducted these investigations on a DNA vaccine against Chlamydophila psittaci, a Gram-negative intracellular bacterium which causes respiratory disease in turkeys and humans. Previous studies showed that the DNA vaccine confers partial protection against C. psittaci infection in turkeys. Turkeys were injected intramuscularly with the DNA vaccine : a eukaryotic expression vector (pcDNA1::MOMP) expressing the major outer membrane protein (MOMP) of an avian C. psittaci serovar D strain. Over a period of 11 weeks, cellular uptake of the DNA vaccine was examined by PCR, transcription of the insert by reverse transcript-PCR (RT-PCR) and mRNA translation by immunofluorescence staining of muscle biopsies.
Results
The results indicate that the DNA vaccine persists in turkey muscle for at least 10 weeks. Moreover, during this period of time MOMP was continuously expressed, as evidenced by the immunofluorescence staining and RT-PCR.
Conclusion
Since C. psittaci infections occur at the age of 3 to 6 and 8 to 12 weeks, a vaccine persistence of 10 weeks seems adequate. Therefore, further research should concentrate on improving the elicited immune response, more specifically the cell-mediated immune response, rather than prolonging the lifespan of the plasmid.
doi:10.1186/1746-6148-2-18
PMCID: PMC1524740  PMID: 16764710
21.  Comparison of paraffin-embedded skin biopsies from different anatomical regions as sampling methods for detection of Leishmania infection in dogs using histological, immunohistochemical and PCR methods 
Background
We compared skin biopsy samples from different anatomical regions for detecting Leishmania in dogs, using histological (HE), immunohistochemical (IHC) and polymerase chain reaction (PCR) techniques.
Results
The sensitivity was 82.8 percent for PCR, 62.1 percent for IHC and 44.8 percent for HE. These methods do not appear to depend on the clinical status of the animal or the anatomical source of the skin sample; there is no "best region" for any method. However, PCR was more effective than IHC and HE for ear and nose skin samples whereas IHC was better than HE for nose samples. There was weak agreement between PCR and HE for all tissue samples; good agreement between PCR and IHC for ear and abdomen samples, and weak agreement for nose; and optimal agreement between IHC and HE for ear and abdomen and good agreement for nose samples.
Conclusion
The PCR on ear skin could be the best procedure for diagnosing canine visceral leishmaniasis. The good agreement between PCR and IHC indicates that IHC can be used as an alternative method. Finally, tissue samples from ears, nose and abdomen, particularly ears and nose, are potentially useful for diagnosing canine visceral leishmaniasis independently of the animal's clinical status.
doi:10.1186/1746-6148-2-17
PMCID: PMC1553440  PMID: 16762067
22.  Molecular diagnosis of Theileria and Babesia species infecting cattle in Northern Spain using reverse line blot macroarrays 
Background
Piroplasmosis in cattle is caused by tick-borne haemoprotozoan parasites of the genera Theileria and Babesia. Molecular detection techniques offer higher sensitivity and specificity than microscopy examination methods and serological tests. A reverse line blot (RLB) macroarray that included generic and species-specific probes for Theileria annulata, Theileria buffeli, Babesia bovis, Babesia bigemina, Babesia divergens and Babesia major was used to study the presence and identity of the piroplasm species infecting 263 bovine blood samples from 79 farms, most of them in Northern Spain. Microscopy examination of blood smears and haematology were also performed whenever possible to identify animals with parasitaemia.
Results
RLB hybridisation identified infection in 54.0% of the samples, whereas only 28.8% were positive by microscopy examination. The most frequently found species was T. buffeli, present in 42.6% of the samples. T. annulata was found in 22 samples (8.4%) from 12 farms, including 9 farms (14 samples) located in Northern Spain where presence of the vector is not very common. Babesia infections were less frequently detected: B. major was found in 3.0% of the samples, B. bigemina in 2.7%, B. bovis in 2.3% and B. divergens in 1.1%. Mixed infections were detected in 14 samples, accounting for six different combinations of species.
Conclusion
This is the first report in which B. major and B. divergens have been detected in Spain using molecular identification techniques and the first time that B. bovis has been detected in Northern Spain. The detection of T. annulata in Northern Spain suggests that the distribution of Mediterranean theileriosis might be changing. Samples with positive RLB hybridisation but negative microscopy had haematology values within the normal ranges suggesting that they corresponded to chronic carriers that may serve as reservoirs of the infection. In this sense, sensitive and specific laboratorial tests like RLB that clearly identify the parasite and can detect subclinical infections are essential to establish good control measures.
doi:10.1186/1746-6148-2-16
PMCID: PMC1482696  PMID: 16684356
23.  Genetic evaluation of Addison's disease in the Portuguese Water Dog 
Background
Addison's disease, also known as hypoadrenocorticism, has been reported in many individual dogs, although some breeds exhibit a greater incidence than the population as a whole. Addison's is presumed to be an autoimmune mediated hereditary defect but the mode of inheritance remains unclear. In particular, the heritability and mode of inheritance have not been defined for the Portuguese Water Dog although Addison's is known to be prevalent in the breed.
Results
The analyses present clear evidence that establishes Addison's disease as an inherited disorder in the Portuguese Water Dog with an estimate of heritability of 0.49 (± 0.16); there were no differences in risk for disease across sexes (p > 0.49). Further, the complex segregation analysis provides suggestive evidence that Addison's disease in the Portuguese Water Dog is inherited under the control of a single, autosomal recessive locus.
Conclusion
The high heritability and mode of inheritance of Addison's disease in the Portuguese Water Dog should enable the detection of segregating markers in a genome-wide scan and the identification of a locus linked to Addison's. Though the confirmation of Addison's disease as an autosomal recessive disorder must wait until the gene is identified, breeders of these dogs may wish to keep the present findings in mind as they plan their breeding programs to select against producing affected dogs.
doi:10.1186/1746-6148-2-15
PMCID: PMC1481556  PMID: 16670022
24.  Molecular characterization and exclusion of porcine GUSB as a candidate gene for congenital hernia inguinalis/scrotalis 
Background
Inguinal hernias are usually caused by a congenital defect, which occurs as a weakness of the inguinal canal. Porcine β-glucuronidase gene (GUSB) was chosen as functional candidate gene because of its involvement in degradation of hyaluronan within gubernacular tissue during descent of testes. Since a genome-wide linkage analysis approach has shown evidence that two regions on porcine chromosome 3 (SSC 3) are involved in the inheritance of hernia inguinalis/scrotalis in German pig breeds, GUSB also attained status as a positional candidate gene by its localization within a hernia-associated chromosomal region.
Results
A contig spanning 17,157 bp, which contains the entire GUSB, was assembled. Comparative sequence analyses were conducted for the GUSB gene locus. Single nucleotide polymorphisms (SNPs) located within the coding region of GUSB were genotyped in 512 animals. Results of transmission disequilibrium test (TDT) for two out of a total of five detected SNPs gave no significant association with the outcome of hernia in pigs.
Conclusion
On the basis of our studies we are able to exclude the two analyzed SNPs within the porcine GUSB gene as causative for the transmission of inguinal hernia.
doi:10.1186/1746-6148-2-14
PMCID: PMC1471780  PMID: 16646965
25.  In vitro comparison of antiviral drugs against feline herpesvirus 1 
Background
Feline herpesvirus 1 (FHV-1) is a common cause of respiratory and ocular disease in cats. Especially in young kittens that have not yet reached the age of vaccination, but already lost maternal immunity, severe disease may occur. Therefore, there is a need for an effective antiviral treatment. In the present study, the efficacy of six antiviral drugs, i.e. acyclovir, ganciclovir, cidofovir, foscarnet, adefovir and 9-(2-phosphonylmethoxyethyl)-2, 6-diaminopurine (PMEDAP), against FHV-1 was compared in Crandell-Rees feline kidney (CRFK) cells using reduction in plaque number and plaque size as parameters.
Results
The capacity to reduce the number of plaques was most pronounced for ganciclovir, PMEDAP and cidofovir. IC50 (NUMBER) values were 3.2 μg/ml (12.5 μM), 4.8 μg/ml (14.3 μM) and 6 μg/ml (21.5 μM), respectively. Adefovir and foscarnet were intermediately efficient with an IC50 (NUMBER) of 20 μg/ml (73.2 μM) and 27 μg/ml (140.6 μM), respectively. Acyclovir was least efficient (IC50 (NUMBER) of 56 μg/ml or 248.7 μM). All antiviral drugs were able to significantly reduce plaque size when compared with the untreated control. As observed for the reduction in plaque number, ganciclovir, PMEDAP and cidofovir were most potent in reducing plaque size. IC50 (SIZE) values were 0.4 μg/ml (1.7 μM), 0.9 μg/ml (2.7 μM) and 0.2 μg/ml (0.7 μM), respectively. Adefovir and foscarnet were intermediately potent, with an IC50 (SIZE) of 4 μg/ml (14.6 μM) and 7 μg/ml (36.4 μM), respectively. Acyclovir was least potent (IC50 (SIZE) of 15 μg/ml or 66.6 μM). The results demonstrate that the IC50 (SIZE) values were notably lower than the IC50 (NUMBER) values. The most remarkable effect was observed for cidofovir and ganciclovir. None of the products were toxic for CRFK cells at antiviral concentrations.
Conclusion
In conclusion, measuring reduction in plaque number and plaque size are two valuable and complementary means of assessing the efficacy of an antiviral drug. By using these parameters for six selected antiviral drugs, we found that ganciclovir, PMEDAP, and cidofovir are the most potent inhibitors of FHV-1 replication in CRFK cells. Therefore, they may be valuable candidates for the treatment of FHV-1 infection in cats.
doi:10.1186/1746-6148-2-13
PMCID: PMC1475582  PMID: 16640781

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