Prion Protein Gene Variability in Spanish Goats. Inference through Susceptibility to Classical Scrapie Strains and Pathogenic Distribution of Peripheral PrPsc

Classical scrapie is a neurological disorder of the central nervous system (CNS) characterized by the accumulation of an abnormal, partially protease resistant prion protein (PrP^sc) in the CNS and in some peripheral tissues in domestic small ruminants. Whereas the pathological changes and genetic susceptibility of ovine scrapie are well known, caprine scrapie has been less well studied. We report here a pathological study of 13 scrapie-affected goats diagnosed in Spain during the last 9 years. We used immunohistochemical and biochemical techniques to discriminate between classical and atypical scrapie and bovine spongiform encephalopathy (BSE). All the animals displayed PrP^sc distribution patterns and western blot characteristics compatible with classical scrapie. In addition, we determined the complete open reading frame sequence of the PRNP in these scrapie-affected animals. The polymorphisms observed were compared with those of the herd mates (n = 665) and with the frequencies of healthy herds (n = 581) of native Spanish goats (Retinta, Pirenaica and Moncaina) and other worldwide breeds reared in Spain (Saanen, Alpine and crossbreed). In total, sixteen polymorphic sites were identified, including the known amino acid substitutions at codons G37V, G127S, M137I, I142M, H143R, R151H, R154H, R211Q, Q222K, G232W, and P240S, and new polymorphisms at codons G74D, M112T, R139S, L141F and Q215R. In addition, the known 42, 138 and 179 silent mutations were detected, and one new one is reported at codon 122. The genetic differences observed in the population studied have been attributed to breed and most of the novel polymorphic codons show frequencies lower than 5%. This work provides the first basis of polymorphic distribution of PRNP in native and worldwide goat breeds reared in Spain.     

Clinical Examination Protocol to Detect Atypical and Classical Scrapie in Sheep

The diagnosis of scrapie, a transmissible spongiform encephalopathy (TSEs) of sheep and goats, is currently based on the detection of disease-associated prion protein by post mortem tests. Unless a random sample of the sheep or goat population is actively monitored for scrapie, identification of scrapie cases relies on the reporting of clinical suspects, which is dependent on the individual''s familiarization with the disease and ability to recognize clinical signs associated with scrapie. Scrapie may not be considered in the differential diagnosis of neurological diseases in small ruminants, particularly in countries with low scrapie prevalence, or not recognized if it presents as nonpruritic form like atypical scrapie. To aid in the identification of clinical suspects, a short examination protocol is presented to assess the display of specific clinical signs associated with pruritic and nonpruritic forms of TSEs in sheep, which could also be applied to goats. This includes assessment of behavior, vision (by testing of the menace response), pruritus (by testing the response to scratching), and movement (with and without blindfolding). This may lead to a more detailed neurologic examination of reporting animals as scrapie suspects. It could also be used in experimental TSE studies of sheep or goats to evaluate disease progression or to identify clinical end-point.     

Monitoring of clinical signs in goats with transmissible spongiform encephalopathies

Background  

As there is limited information about the clinical signs of BSE and scrapie in goats, studies were conducted to describe the clinical progression of scrapie and BSE in goats and to evaluate a short clinical protocol for its use in detecting scrapie-affected goats in two herds with previously confirmed scrapie cases. Clinical assessments were carried out in five goats intracerebrally infected with the BSE agent as well as five reported scrapie suspects and 346 goats subject to cull from the two herds, 24 of which were retained for further monitoring. The brain and selected lymphoid tissue were examined by postmortem tests for disease confirmation.     

Diversity in neuroanatomical distribution of abnormal prion protein in atypical scrapie

Scrapie is a transmissible spongiform encephalopathy (TSE) in sheep and goats. In recent years, atypical scrapie cases were identified that differed from classical scrapie in the molecular characteristics of the disease-associated pathological prion protein (PrP(sc)). In this study, we analyze the molecular and neuropathological phenotype of nine Swiss TSE cases in sheep and goats. One sheep was identified as classical scrapie, whereas six sheep, as well as two goats, were classified as atypical scrapie. The latter revealed a uniform electrophoretic mobility pattern of the proteinase K-resistant core fragment of PrP(sc) distinct from classical scrapie regardless of the genotype, the species, and the neuroanatomical structure. Remarkably different types of neuroanatomical PrP(sc) distribution were observed in atypical scrapie cases by both western immunoblotting and immunohistochemistry. Our findings indicate that the biodiversity in atypical scrapie is larger than expected and thus impacts on current sampling and testing strategies in small ruminant TSE surveillance.     

Extended scrapie incubation time in goats singly heterozygous for PRNP S146 or K222

Scrapie is the transmissible spongiform encephalopathy (TSE) of sheep and goats, and scrapie eradication in sheep is based in part on strong genetic resistance to classical scrapie. Goats may serve as a scrapie reservoir, and to date there has been no experimental inoculation confirming strong genetic resistance in goats. Two prion protein variants (amino acid substitutions S146 and K222) in goats have been significantly underrepresented in scrapie cases though present in scrapie-exposed flocks, and have demonstrated low cell-free protein conversion efficiency to the disease form (PrP(D)). To test degree of genetic resistance conferred in live animals with consistent exposure, we performed the first oral scrapie challenge of goats singly heterozygous for either PRNP S146 or K222. All N146-Q222 homozygotes became clinically scrapie positive by an average of 24months, but all S146 and K222 heterozygotes remain scrapie negative by both rectal biopsy and clinical signs at significantly longer incubation times (P<0.0001 for both comparisons). Recent reports indicate small numbers of S146 and K222 heterozygous goats have become naturally infected with scrapie, suggesting these alleles do not confer complete resistance in the heterozygous state but rather extend incubation. The oral challenge results presented here confirm extended incubation observed in a recent intracerebral challenge of K222 heterozygotes, and to our knowledge provide the first demonstration of extended incubation in S146 heterozygotes. These results suggest longer relevant trace-back histories in scrapie-eradication programs for animals bearing these alleles and strengthen the case for additional challenge experiments in both homozygotes to assess potential scrapie resistance.     

Transmissibility of caprine scrapie in ovine transgenic mice

ABSTRACT: BACKGROUND: The United States control program for classical ovine scrapie is based in part on the finding that infection is typically spread through exposure to shed placentas from infected ewes. Transmission from goats to sheep is less well described. A suitable rodent model for examining the effect of caprine scrapie isolates in the ovine host will be useful in the ovine scrapie eradication effort. In this study, we describe the incubation time, brain lesion profile, glycoform pattern and PrPSc distribution patterns in a well characterized transgenic mouse line (Tg338) expressing the ovine VRQ prion allele, following inoculation with brain from scrapie infected goats. RESULTS: First passage incubation times of caprine tissue in Tg338 ovinized mice varied widely but second passage intervals were shorter and consistent. Vacuolation profiles, glycoform patterns and paraffin-embedded tissue blots from terminally ill second passage mice derived from sheep or goat inocula were similar. Proteinase K digestion products of murine tissue were slightly smaller than the original ruminant inocula, a finding consistent with passage of several ovine strains in previous reports. CONCLUSIONS: These findings demonstrate that Tg338 mice propagate prions of caprine origin and provide a suitable baseline for examination of samples identified in the expanded US caprine scrapie surveillance program.     

The prevalence of atypical scrapie in sheep from positive flocks is not higher than in the general sheep population in 11 European countries

Background

During the last decade, active surveillance for transmissible spongiform encephalopathies in small ruminants has been intensive in Europe. In many countries this has led to the detection of cases of atypical scrapie which, unlike classical scrapie, might not be contagious. EU legislation requires, that following detection of a scrapie case, control measures including further testing take place in affected flocks, including the culling of genotype susceptible to classical scrapie. This might result in the detection of additional cases. The aim of this study was to investigate the occurrence of additional cases in flocks affected by atypical scrapie using surveillance data collected in Europe in order to ascertain whether atypical scrapie, is contagious.

Results

Questionnaires were used to collect, at national level, the results of active surveillance and testing associated with flock outbreaks in 12 European countries. The mean prevalence of atypical scrapie was 5.5 (5.0-6.0) cases per ten thousand in abattoir surveillance and 8.1 (7.3-9.0) cases per ten thousand in fallen stock. By using meta-analysis, on 11 out of the 12 countries, we found that the probability of detecting additional cases of atypical scrapie in positive flocks was similar to the probability observed in animals slaughtered for human consumption (odds ratio, OR = 1.07, CI_95%: 0.70-1.63) or among fallen stock (OR = 0.78, CI_95%: 0.51-1.2). In contrast, when comparing the two scrapie types, the probability of detecting additional cases in classical scrapie positive flocks was significantly higher than the probability of detecting additional cases in atypical scrapie positive flocks (OR = 32.4, CI_95%: 20.7-50.7).

Conclusions

These results suggest that atypical scrapie is not contagious or has a very low transmissibility under natural conditions compared with classical scrapie. Furthermore this study stressed the importance of standardised data collection to make good use of the analyses undertaken by European countries in their efforts to control atypical and classical scrapie.     

A transfectant RK13 cell line permissive to classical caprine scrapie prion propagation

To assess scrapie infectivity associated with caprine-origin tissues, bioassay can be performed using kids, lambs or transgenic mice expressing caprine or ovine prion (PRNP) alleles, but the incubation periods are fairly long. Although several classical ovine scrapie prion permissive cell lines with the ability to detect brain-derived scrapie prion have been available, no classical caprine scrapie permissive cell line is currently available. Therefore, the aims of this study were to generate a rabbit kidney epithelial cell line (RK13) stably expressing caprine wild-type PRNP (cpRK13) and then to assess permissiveness of cpRK13 cells to classical caprine scrapie prion propagation. The cpRK13 and plasmid control RK13 (pcRK13) cells were incubated with brain-derived classical caprine scrapie inocula prepared from goats or ovinized transgenic mice (Tg338, express ovine VRQ allele) infected with caprine scrapie. Significant PrP^Sc accumulation, which is indicative of scrapie prion propagation, was detected by TSE ELISA and immunohistochemistry in cpRK13 cells inoculated with classical caprine scrapie inocula. Western blot analysis revealed the typical proteinase K-resistant 3 PrP^res isoforms in the caprine scrapie prion inoculated cpRK13 cell lysate. Importantly, PrP^Sc accumulation was not detected in similarly inoculated pcRK13 cells, whether by TSE ELISA, immunohistochemistry, or western blot. These findings suggest that caprine scrapie prions can be propagated in cpRK13 cells, thus this cell line may be a useful tool for the assessment of classical caprine prions in the brain tissues of goats.     

Prospects for applying breeding for resistance to control scrapie in goats: The current situation in Italy

Genetic selection has been used to control transmissible spongiform encephalopathies (TSEs) in sheep populations based on the association between PRNP polymorphisms and resistance to scrapie. In goats even if a protective role has been suggested for several polymorphisms (I142M, H143R, N146D/S, R154H, R211Q, Q222K) by different European studies, a similar, univocal association has not been proved so far.The aim of this paper was to identify target polymorphisms in goats and their potential applicability in a selection plan in Italy. For this purpose, the existing genetic data on modulating resistance to classical and atypical scrapie in goats in this country will be used as starting point.Two Italian case–control studies concluded that the polymorphism at codon 222 (from glutamine to lysine Q/K) is important in influencing the susceptibility of goats to classical scrapie. Moreover, goats are susceptible to an unusual form of scrapie, named Nor98, and the H154 mutation was shown to be statistically associated with this disease in goats in an Italian case–control study. Currently, a strategy based on killing goats carrying the H154 mutation is being applied to manage atypical scrapie outbreaks in Italy.The current situation in Italy bodes well for the applicability of breeding plans based on the K222 mutation; data from independent studies on the role of K222 as a protective or even a resistance factor and its frequency in several Italian breeds are available. In the near future, as new data on K222 will be reported, testing the application of selective culling in classical scrapie goat outbreaks may become feasible.     

Proteinase K-resistant material in ARR/VRQ sheep brain affected with classical scrapie is composed mainly of VRQ prion protein

Classical scrapie is a prion disease in sheep and goats. In sheep, susceptibility to disease is genetically influenced by single amino acid substitutions. Genetic breeding programs aimed at enrichment of arginine-171 (171R) prion protein (PrP), the so-called ARR allele, in the sheep population have been demonstrated to be effective in reducing the occurrence of classical scrapie in the field. Understanding the molecular basis for this reduced prevalence would serve the assessment of ARR adaptation. The prion formation mechanism and conversion of PrP from the normal form (PrP(C)) to the scrapie-associated form (PrP(Sc)) could play a key role in this process. Therefore, we investigated whether the ARR allele substantially contributes to scrapie prion formation in naturally infected heterozygous 171Q/R animals. Two methods were applied to brain tissue of 171Q/R heterozygous sheep with natural scrapie to determine the relative amount of the 171R PrP fraction in PrP(res), the proteinase K-resistant PrP(Sc) core. An antibody test differentiating between 171Q and 171R PrP fragments showed that PrP(res) was mostly composed of the 171Q allelotype. Furthermore, using a novel tool for prion research, endoproteinase Lys-C-digested PrP(res) yielded substantial amounts of a nonglycosylated and a monoglycosylated PrP fragment comprising codons 114 to 188. Following two-dimensional gel electrophoresis, only marginal amounts (<9%) of 171R PrP(res) were detected. Enhanced 171R(res) proteolytic susceptibility could be excluded. Thus, these data support a nearly zero contribution of 171R PrP in PrP(res) of 171R/Q field scrapie-infected animals. This is suggestive of a poor adaptation of classical scrapie to this resistance allele under these natural conditions.     

Design and validation of a high-throughput assay to detect codon 146 polymorphisms in the caprine prion protein gene

In sheep, scrapie susceptibility is so strongly associated with single nucleotide polymorphisms (SNPs) in the gene encoding the prion protein (PrP) that this linkage constitutes the basis for selective breeding strategies directed toward controlling the disease. For goats, in contrast, the association between scrapie susceptibility/resistance and variations in the PrP gene is far weaker, with only a few identified SNPs showing an influence on scrapie susceptibility. A recent survey of PrP genotypes in Cypriot goats, however, revealed the existence of a robust association between polymorphisms at codon 146 of the caprine PrP gene and resistance/susceptibility to natural scrapie. Here we describe here a high-throughput assay, based on homogeneous MassExtend technology coupled with mass spectrometry, for genotyping codon 146 of the caprine PrP gene. Our results demonstrate that this assay exhibits high accuracy, reproducibility, and repeatability, thereby making it suitable for large-scale SNP genotyping, as required for scrapie surveillance programs.     

Estimation of the relative sensitivity of the comparative tuberculin skin test in tuberculous cattle herds subjected to depopulation

Bovine tuberculosis (bTB) is one of the most serious economic animal health problems affecting the cattle industry in Great Britain (GB), with incidence in cattle herds increasing since the mid-1980s. The single intradermal comparative cervical tuberculin (SICCT) test is the primary screening test in the bTB surveillance and control programme in GB and Ireland. The sensitivity (ability to detect infected cattle) of this test is central to the efficacy of the current testing regime, but most previous studies that have estimated test sensitivity (relative to the number of slaughtered cattle with visible lesions [VL] and/or positive culture results) lacked post-mortem data for SICCT test-negative cattle. The slaughter of entire herds ("whole herd slaughters" or "depopulations") that are infected by bTB are occasionally conducted in GB as a last-resort control measure to resolve intractable bTB herd breakdowns. These provide additional post-mortem data for SICCT test-negative cattle, allowing a rare opportunity to calculate the animal-level sensitivity of the test relative to the total number of SICCT test-positive and negative VL animals identified post-mortem (rSe). In this study, data were analysed from 16 whole herd slaughters (748 SICCT test-positive and 1031 SICCT test-negative cattle) conducted in GB between 1988 and 2010, using a Bayesian hierarchical model. The overall rSe estimate of the SICCT test at the severe interpretation was 85% (95% credible interval [CI]: 78-91%), and at standard interpretation was 81% (95% CI: 70-89%). These estimates are more robust than those previously reported in GB due to inclusion of post-mortem data from SICCT test-negative cattle.     

Prion Remains Infectious after Passage through Digestive System of American Crows (Corvus brachyrhynchos)

Avian scavengers, such as American crows (Corvus brachyrhynchos), have potential to translocate infectious agents (prions) of transmissible spongiform encephalopathy (TSE) diseases including chronic wasting disease, scrapie, and bovine spongiform encephalopathy. We inoculated mice with fecal extracts obtained from 20 American crows that were force-fed material infected with RML-strain scrapie prions. These mice all evinced severe neurological dysfunction 196–231 d postinoculation ( = 198; 95% CI: 210–216) and tested positive for prion disease. Our results suggest a large proportion of crows that consume prion-positive tissue are capable of passing infectious prions in their feces ( = 1.0; 95% CI: 0.8–1.0). Therefore, this common, migratory North American scavenger could play a role in the geographic spread of TSE diseases.     

Potential Benefits of Cattle Vaccination as a Supplementary Control for Bovine Tuberculosis

Vaccination for the control of bovine tuberculosis (bTB) in cattle is not currently used within any international control program, and is illegal within the EU. Candidate vaccines, based upon Mycobacterium bovis bacillus Calmette-Guérin (BCG) all interfere with the action of the tuberculin skin test, which is used to determine if animals, herds and countries are officially bTB-free. New diagnostic tests that Differentiate Infected from Vaccinated Animals (DIVA) offer the potential to introduce vaccination within existing eradication programs. We use within-herd transmission models estimated from historical data from Great Britain (GB) to explore the feasibility of such supplemental use of vaccination. The economic impact of bovine Tuberculosis for farmers is dominated by the costs associated with testing, and associated restrictions on animal movements. Farmers’ willingness to adopt vaccination will require vaccination to not only reduce the burden of infection, but also the risk of restrictions being imposed. We find that, under the intensive sequence of testing in GB, it is the specificity of the DIVA test, rather than the sensitivity, that is the greatest barrier to see a herd level benefit of vaccination. The potential negative effects of vaccination could be mitigated through relaxation of testing. However, this could potentially increase the hidden burden of infection within Officially TB Free herds. Using our models, we explore the range of the DIVA test characteristics necessary to see a protective herd level benefit of vaccination. We estimate that a DIVA specificity of at least 99.85% and sensitivity of >40% is required to see a protective benefit of vaccination with no increase in the risk of missed infection. Data from experimentally infected animals suggest that this target specificity could be achieved in vaccinates using a cocktail of three DIVA antigens while maintaining a sensitivity of 73.3% (95%CI: 61.9, 82.9%) relative to post-mortem detection.     

Combined eradication strategy for CAE in a dairy goat farm in Japan

We conducted an eradication program from 2002 to 2006 against caprine arthritis-encephalitis (CAE) virus (CAEV) in an important farm that maintained goat breeds and had a high prevalence of CAEV infection. The program did not involve the slaughter and replacement of entire flocks, but rather the prevention of both vertical and horizontal transmission. The program consisted of (1) removal of kids immediately after birth, (2) segregation of each generation, and (3) culling of positive goats in periodical tests. All goats born before 2002 were regarded as infected and grouped into herd A. Kids born during the program were divided into several herds on the basis of CAEV infection risk, raised with calf milk replacer, and periodically tested by polymerase chain reaction (PCR) and the agar gel immunodiffusion (AGID) test. A total of 205 kids were produced from 137 parents in herd A, 92 of which were distinctly infected. Only 11 of the 205 kids were infected with CAEV and were culled. The remaining 194 kids and all other kids born from other herds were negative by PCR and AGID testing throughout the program. The milk yield of primiparous does was significantly increased after the eradication program. These findings indicate that the combine use of isolated and milk-deprived rearing and periodical detection testing are effective in establishing a CAEV-free flock from an infected flock.     

Global Burden of HIV among Men Who Engage in Transactional Sex: A Systematic Review and Meta-Analysis

Background

Men who engage in transactional sex, the exchange of sex for money, goods, or other items of value, are thought to be at increased risk of HIV, but there have been no systematic attempts to characterize HIV burden in this population. We undertook a systematic review and meta-analysis to quantify the burden in this population compared with that of men in the general population to better inform future HIV prevention efforts.

Methods

We searched seven electronic databases, national surveillance reports, and conference abstracts for studies of men who engage in transactional sex published between 2004–2013. Random effects meta-analysis was used to determine pooled HIV prevalence and prevalence ratios (PR) for the difference in HIV prevalence among men who engage in transactional sex as compared to general population men.

Findings

Of 66 studies included representing 31,924 men who had engaged in transactional sex in 28 countries, pooled biological assay-confirmed HIV prevalence was 10.5% (95% CI = 9.4 to 11.5%). The highest pooled HIV prevalence was in Sub-Saharan Africa (31.5%, 95% CI = 21.6 to 41.5%), followed by Latin America (19.3%, 95% CI = 15.5 to 23.1%), North America (16.6%, 95% CI = 3.7 to 29.5%), and Europe (12.2%, 95% CI = 6.0 to 17.2%). Men who engaged in transactional sex had an elevated burden of HIV compared to the general male population (PR = 20.7, 95% CI = 16.8 to 25.5).

Conclusions

The global burden of HIV is disproportionately high among men who engage in transactional sex compared with the general male population. There is an urgent need to include this population in systematic surveillance as well as to scale-up access to quality HIV prevention programs.     

Caprine PrP variants harboring Asp-146, His-154 and Gln-211 alleles display reduced convertibility upon interaction with pathogenic murine prion protein in scrapie infected cells

Scrapie, the prion disease of sheep and goats, is a devastating malady of small ruminants. Due to its infectious nature, epidemic outbreaks may occur in flocks/herds consisting of highly susceptible animals. Field studies identified scrapie-protective caprine PrP variants, harboring specific single amino acid changes (Met-142, Arg-143, Asp-146, Ser-146, His-154, Gln-211 and Lys-222). Their effects are under further evaluation, and aim to determine the most protective allele. We assessed some of these variants (Asp-146, His-154, Gln-211 and Lys-222), after their exogenous expression as murine-caprine chimeras in a scrapie- infected murine cell line. We report that exogenously expressed PrPs undergo conformational conversion upon interaction with the endogenous pathological murine prion protein (PrP^SC), which results in the detection of goat-specific and partially PK-resistant moieties. These moieties display a PK-resistance pattern distinct from the one detected in natural goat scrapie cases. Within this cellular model, distinct conformational conversion potentials were assigned to the tested variants. Molecules carrying the Asp-146, His-154 and Gln-211 alleles showed significantly lower conversion levels compared to wild type, confirming their protective effects against scrapie. Although we utilized a heterologous conversion system, this is to our knowledge, the first study of caprine PrP variants in a cellular context of scrapie, that confirms the protective effects of some of the studied alleles.     

Differential immunoreactivity of goat derived scrapie following in vitro misfolding versus mouse bioassay

The protein misfolding cyclic amplification (PMCA) assay allows for detection of prion protein misfolding activity in tissues and fluids from sheep with scrapie where it was previously undetected by conventional western blot and immunohistochemistry assays. Studies of goats with scrapie have yet to take advantage of PMCA, which could aid in discerning the risk of transmission between goats and goats to sheep. The aim of the current study was to adapt PMCA for evaluation of scrapie derived from goats. Diluted brain homogenate from scrapie-infected goats (i.e., the scrapie seed, PrP(Sc)) was subjected to PMCA using normal brain homogenate from ovinized transgenic mice (tg338) as the source of normal cellular prion protein (the substrate, PrP(C)). The assay end-point was detection of the proteinase K-resistant misfolded prion protein core (PrP(res)) by western blot. Protein misfolding activity was consistently observed in caprine brain homogenate diluted 10,000-fold after 5 PMCA rounds. Epitope mapping by western blot analyses demonstrated that PrP(res) post-PMCA was readily detected with an N-terminus anti-PrP monoclonal antibody (P4), similar to scrapie inoculum from goats. This was in contrast to limited detection of PrP(res) with P4 following mouse bioassay. The inverse was observed with a monoclonal antibody to the C-terminus (F99/97.6.1). Thus, brain homogenate prepared from uninoculated tg338 served as an appropriate substrate for serial PMCA of PrP(Sc) derived from goats. These observations suggest that concurrent PMCA and bioassay with tg338 could improve characterization of goat derived scrapie.     

Simultaneous Automated Screening and Confirmatory Testing for Vasculitis-Specific ANCA

Anti-neutrophil cytoplasmic antibodies (ANCA) are the serological hallmark of small vessel vasculitis, so called ANCA-associated vasculitis. The international consensus requires testing by indirect immunofluorescence (IIF) on human ethanol-fixed neutrophils (ethN) as screening followed by confirmation with enzyme-linked immunosorbent assays (ELISAs). This study evaluates the combination of cell- and microbead-based digital IIF analysis of ANCA in one reaction environment by the novel multiplexing CytoBead technology for simultaneous screening and confirmatory ANCA testing. Sera of 592 individuals including 118 patients with ANCA-associated vasculitis, 133 with rheumatoid arthritis, 49 with infectious diseases, 77 with inflammatory bowel syndrome, 20 with autoimmune liver diseases, 70 with primary sclerosing cholangitis and 125 blood donors were tested for cytoplasmic ANCA (C-ANCA) and perinuclear ANCA (P-ANCA) by classical IIF and ANCA to proteinase 3 (PR3) and myeloperoxidase (MPO) by ELISA. These findings were compared to respective ANCA results determined by automated multiplex CytoBead technology using ethN and antigen-coated microbeads for microbead immunoassays. There was a good agreement for PR3- and MPO-ANCA and a very good one for P-ANCA and C-ANCA by classical and multiplex analysis (Cohen''s kappa [κ] = 0.775, 0.720, 0.876, 0.820, respectively). The differences between classical testing and CytoBead analysis were not significant for PR3-ANCA, P-ANCA, and C-ANCA (p<0.05, respectively). The prevalence of confirmed positive ANCA findings by classical testing (IIF and ELISA) compared with multiplex CytoBead analysis (IIF and microbead immunoassay positive) resulted in a very good agreement (κ = 0.831) with no significant difference of both methods (p = 0.735). Automated endpoint-ANCA titer detection in one dilution demonstrated a very good agreement with classical analysis requiring dilution of samples (κ = 0.985). Multiplexing by CytoBead technology can be employed for simultaneous screening and quantitative confirmation of ANCA. This novel technique provides fast and cost-effective ANCA analysis by automated digital IIF for the first time.     

Serological Evidence for Non-Lethal Exposures of Mongolian Wild Birds to Highly Pathogenic Avian Influenza H5N1 Virus

Surveillance for highly pathogenic avian influenza viruses (HPAIV) in wild birds is logistically demanding due to the very low rates of virus detection. Serological approaches may be more cost effective as they require smaller sample sizes to identify exposed populations. We hypothesized that antigenic differences between classical Eurasian H5 subtype viruses (which have low pathogenicity in chickens) and H5N1 viruses of the Goose/Guangdong/96 H5 lineage (which are HPAIV) may be used to differentiate populations where HPAIVs have been circulating, from those where they have not. To test this we performed hemagglutination inhibition assays to compare the reactivity of serum samples from wild birds in Mongolia (where HPAIV has been circulating, n = 1,832) and Europe (where HPAIV has been rare or absent, n = 497) to a panel of reference viruses including classical Eurasian H5 (of low pathogenicity), and five HPAIV H5N1 antigens of the Asian lineage A/Goose/Guangdong/1/96. Antibody titres were detected against at least one of the test antigens for 182 Mongolian serum samples (total seroprevalence of 0.10, n = 1,832, 95% adjusted Wald confidence limits of 0.09–0.11) and 25 of the European sera tested (total seroprevalence of 0.05, n = 497, 95% adjusted Wald confidence limits of 0.03–0.07). A bias in antibody titres to HPAIV antigens was found in the Mongolian sample set (22/182) that was absent in the European sera (0/25). Although the interpretation of serological data from wild birds is complicated by the possibility of exposure to multiple strains, and variability in the timing of exposure, these findings suggest that a proportion of the Mongolian population had survived exposure to HPAIV, and that serological assays may enhance the targeting of traditional HPAIV surveillance toward populations where isolation of HPAIV is more likely.