Examples of proper reporting for evaluation (Stage 2 validation) of diagnostic tests for diseases listed by the World Organisation for Animal Health.

Reporting and design standards are key indicators of the quality of diagnostic accuracy (validation) studies but, with the exception of aquatic animal diseases and paratuberculosis in ruminants, there is limited guidance for designing these studies in animals. There is, therefore, a need for generic guidelines that are based on disease characteristics, such as mode of transmission, latent period and pathogenesis. Comprehensive, clear and transparent reporting of primary test accuracy studies for diseases listed by the World Organisation for Animal Health (OIE) has value for the end users of diagnostic tests and, ultimately, for decision-makers, who require systematic reviews and meta-analysis of multiple tests for specified diseases and testing purposes. The recent publication of reporting standards for Bayesian latent class models, to analyse test-accuracy data from naturally occurring disease events, fills an important gap as these methods are being increasingly used for OIE-listed diseases. Adherence to design and reporting standards, as well as to guidelines, helps to ensure that research funding for test validation studies is used appropriately and that the strengths and limitations of single tests or test combinations are made clear to test users. The authors provide a review of key points that are often overlooked or misinterpreted in test validation studies, as well as two concrete examples of good practice for use as a reference point for future studies.

Les normes de notification et de conception sont des indicateurs essentiels de la qualité des études de validation des tests destinées à déterminer leur exactitude diagnostique ; or, en dehors des maladies des animaux aquatiques et de la paratuberculose chez les ruminants, il n'existe guère de lignes directrices pour concevoir ce type d'études pour les tests utilisés en santé animale. À la connaissance des auteurs, il n'existe pas non plus de normes de conception applicables aux études de validation en santé humaine. Par conséquent, il conviendrait de disposer de lignes directrices génériques fondées sur les caractéristiques des maladies telles que leurs modalités de transmission, leur période de latence et leur pathogénie. Une notification complète, claire et transparente des études d'exactitude des tests primaires pour les maladies listées par l'Organisation mondiale de la santé animale (OIE) serait une aide précieuse pour les utilisateurs finaux des tests de diagnostic, mais aussi pour les responsables de l'élaboration des politiques, dont les décisions reposent sur des examens et des méta-analyses systématiques couvrant un grand nombre de tests pour certaines maladies ou pour certains usages d'un test. La publication récente des normes de notification applicables aux modèles bayésiens à classe latente pour analyser les données de performance d'un test à partir de foyers naturels de maladie comble une lacune importante dans la mesure où ces méthodes sont de plus en plus utilisées pour les maladies listées par l'OIE. L'adhésion à des normes de conception et de notification ainsi qu'à des lignes directrices en la matière permettra de garantir que les fonds alloués aux études de validation des tests sont bien utilisés et que les atouts et les limitations de certains tests individuels ou associations de tests sont clairement perçus par les utilisateurs. Les auteurs passent en revue certains points essentiels qui sont souvent ignorés ou mal interprétés lors des études de validation des tests et proposent deux exemples concrets de bonnes pratiques qui pourront servir de références pour les études à venir.

Las normas de comunicación y diseño son indicadores básicos de la calidad de los estudios encaminados a determinar la exactitud de diagnóstico (validación) pero, con la excepción de las enfermedades de los animales acuáticos y la paratuberculosis en rumiantes, hay escasas directrices que se apliquen al diseño de esos estudios en animales. Además, hasta donde saben los autores, en el ámbito de la salud humana no hay normas de diseño. De ahí la necesidad de directrices genéricas que estén basadas en las características de las enfermedades, como modo de transmisión, período de latencia o patogénesis. La comunicación exhaustiva, clara y transparente de estudios primarios sobre la exactitud de pruebas de diagnóstico de enfermedades incluidas en las listas de la Organización Mundial de Sanidad Animal (OIE) reviste utilidad no solo para los usuarios finales de la prueba, sino también, en última instancia, para los órganos decisorios, que necesitan metaanálisis y estudios sistemáticos de múltiples pruebas que se apliquen a una u otra enfermedad y sirvan para una u otra finalidad. La reciente publicación de normas de comunicación de modelos bayesianos de clases latentes para analizar los datos de exactitud de pruebas a partir de episodios de enfermedad de origen natural viene a colmar una importante laguna, en la medida en que estos métodos se aplican cada vez más al diagnóstico de enfermedades incluidas en las listas de la OIE. El cumplimiento de las normas de diseño y comunicación, y también de las directrices, ayuda a garantizar que los fondos de investigación destinados a estudios de validación de pruebas sean utilizados debidamente y que el usuario final de una prueba reciba información clara sobre los puntos fuertes y las limitaciones de una prueba o combinación de pruebas. Los autores pasan revista a los principales aspectos que se suelen pasar por alto o malinterpretar en los estudios de validación de pruebas y ofrecen dos ejemplos concretos de buenas prácticas que se pueden utilizar como referencia en futuros estudios.

Bayesian latent class analysis when the reference test is imperfect.

Latent class analysis (LCA) has allowed epidemiologists to overcome the practical constraints faced by traditional diagnostic test evaluation methods, which require both a gold standard diagnostic test and ample numbers of appropriate reference samples. Over the past four decades, LCA methods have expanded to allow epidemiologists to evaluate diagnostic tests and estimate true prevalence using imperfect tests over a variety of complex data structures and scenarios, including during the emergence of novel infectious diseases. The objective of this review is to provide an overview of recent developments in LCA methods, as well as a practical guide to applying Bayesian LCA (BLCA) to the evaluation of diagnostic tests. Before conducting a BLCA, the suitability of BLCA for the pathogen of interest, the availability of appropriate samples, the number of diagnostic tests, and the structure of the data should be carefully considered. While formulating the model, the model's structure and specification of informative priors will affect the likelihood that useful inferences can be drawn. With the growing need for advanced analytical methods to evaluate diagnostic tests for newly emerging diseases, LCA is a promising field of research for both the veterinary and medical disciplines.

L'analyse à classes latentes a permis aux épidémiologistes de surmonter les problèmes concrets posés par les méthodes traditionnelles d'évaluation des essais de diagnostic, qui nécessitent à la fois un test de référence absolue (étalon ou gold standard) et un grand nombre d'échantillons de référence aux caractéristiques appropriées. Au cours des quatre dernières décennies, les méthodes d'analyse à classes latentes ont acquis de l'ampleur et permettent aux épidémiologistes d'évaluer les essais diagnostiques et d'estimer les taux de prévalence réelle tout en recourant à des tests supposés imparfaits, grâce à l'utilisation de données et de scénarios divers et complexes, y compris dans les situations d'émergence de nouvelles maladies infectieuses. Les auteurs font un tour d'horizon des dernières évolutions dans ce domaine et donnent des orientations pratiques concernant la manière d'utiliser l'analyse bayésienne à classes latentes pour évaluer les performances d'un test de diagnostic. Avant de conduire une telle analyse, il convient de déterminer avec soin si elle est adaptée à l'agent pathogène considéré et si les échantillons disponibles sont appropriés et en nombre suffisant ; il convient également de prendre en compte le nombre de tests de diagnostic à évaluer et la structure des données utilisées. Lors de la conception du modèle, sa structure et la définition préalable des données informatives vont affecter la probabilité que le modèle génère des inférences utiles. Face à la nécessité croissante de disposer de méthodes analytiques sophistiquées pour évaluer les tests de diagnostic utilisés pour les maladies émergentes nouvelles, les analyses à classes latentes offrent des perspectives prometteuses pour la recherche, aussi bien dans le domaine de la santé vétérinaire que de la médecine humaine.

El análisis de clases latentes ha servido a los epidemiólogos para superar las limitaciones prácticas que imponen los métodos tradicionales de evaluación de pruebas de diagnóstico, que requieren a la vez una prueba de diagnóstico que sirva de patrón de referencia perfecto y un gran número de muestras de referencia adecuadas. En los últimos cuatro decenios, los métodos de análisis de clases latentes se han ido ampliando hasta permitir a los epidemiólogos evaluar pruebas de diagnóstico y calcular la prevalencia real empleando pruebas imperfectas ante muy diversas estructuras de datos y situaciones complejas, incluida la aparición de nuevas enfermedades infecciosas. Los autores, tras presentar a grandes líneas los últimos adelantos en cuanto a métodos de análisis de clases latentes, ofrecen indicaciones prácticas para aplicar el análisis bayesiano de clases latentes a la evaluación de pruebas de diagnóstico. Antes de proceder a un análisis bayesiano de este tipo conviene estudiar con detenimiento la idoneidad del método para el patógeno en cuestión, la disponibilidad de muestras apropiadas, el número de pruebas de diagnóstico y la estructura de los datos. A la hora de formular el modelo, la estructura del propio modelo y la especificación de los elementos informativos previos influirán en la probabilidad de poder extraer conclusiones provechosas. Ante la creciente necesidad de disponer de métodos analíticos avanzados con los que evaluar pruebas de diagnóstico de nuevas enfermedades emergentes, el análisis de clases latentes abre un promisorio campo de investigación para las disciplinas veterinarias y médicas.

Bayesian evaluation of three serological tests for the diagnosis of bovine brucellosis in Bangladesh.

We evaluated the performance of three serological tests - an immunoglobulin G indirect enzyme linked immunosorbent assay (iELISA), a Rose Bengal test and a slow agglutination test (SAT) - for the diagnosis of bovine brucellosis in Bangladesh. Cattle sera (n = 1360) sourced from Mymensingh district (MD) and a Government owned dairy farm (GF) were tested in parallel. We used a Bayesian latent class model that adjusted for the conditional dependence among the three tests and assumed constant diagnostic accuracy of the three tests in both populations. The sensitivity and specificity of the three tests varied from 84.6% to 93.7%, respectively. The true prevalences of bovine brucellosis in MD and the GF were 0.6% and 20.4%, respectively. Parallel interpretation of iELISA and SAT yielded the highest negative predictive values: 99.9% in MD and 99.6% in the GF; whereas serial interpretation of both iELISA and SAT produced the highest positive predictive value (PPV): 99.9% in the GF and also high PPV (98.9%) in MD. We recommend the use of both iELISA and SAT together and serial interpretation for culling and parallel interpretation for import decisions. Removal of brucellosis positive cattle will contribute to the control of brucellosis as a public health risk in Bangladesh.

Spatio-temporal modelling of foot-and-mouth disease outbreaks.

We present and analyse data collected during a severe epidemic of foot-and-mouth disease (FMD) that occurred between July and September 2000 in a region of northeastern Greece with strategic importance since it represents the southeastern border of Europe and Asia. We implement generic Bayesian methodology, which offers flexibility in the ability to fit several realistically complex models that simultaneously capture the presence of 'excess' zeros, the spatio-temporal dependence of the cases, assesses the impact of environmental noise and controls for multicollinearity issues. Our findings suggest that the epidemic was mostly driven by the size and the animal type of each farm as well as the distance between farms while environmental and other endemic factors were not important during this outbreak. Analyses of this kind may prove useful to informing decisions related to optimal control measures for potential future FMD outbreaks as well as other acute epidemics such as FMD.

Bayesian validation of a serum and milk ELISA for antibodies against Mycobacterium avium subspecies paratuberculosis in Greek dairy goats across lactation.

We validated a commercial (Idexx Pourquier, Montpellier, France) serum and milk indirect ELISA that detects antibodies against Mycobacterium avium ssp. paratuberculosis (MAP) in Greek dairy goats. Each goat was sampled 4 times, starting from kidding and covering early, mid, and late lactation. A total of 1,268 paired milk (or colostrum) and serum samples were collected during the 7-mo lactation period. Bayesian latent class models, which allow for the continuous interpretation of test results, were used to derive the distribution of the serum and milk ELISA response for healthy and MAP-infected individuals at each lactation stage. Both serum and milk ELISA, in all lactation stages, had average and similar overall discriminatory ability as measured by the area under the curve (AUC). For each test, the smallest overlap between the distribution of the healthy and MAP-infected does was in late lactation. At this stage, the AUC was 0.89 (95% credible interval: 0.70; 0.98) and 0.92 (0.74; 0.99) for the milk and serum ELISA, respectively. Both tests had comparable sensitivities and specificities at the recommended cutoffs across lactation. Lowering the cutoffs led to an increase in sensitivity without serious loss in specificity. In conclusion, the milk ELISA was as accurate as the serum ELISA. Therefore, it could serve as the diagnostic tool of choice, especially during the implementation of MAP control programs that require frequent testing, because milk sampling is a noninvasive, rapid, and easy process.

Epidemiological insights into paratuberculosis in camels in Saudi Arabia: Bayesian estimation of true prevalence and identification of risk factors.

Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a significant concern in the camel population of Saudi Arabia. This study aimed to provide epidemiological insights into the disease by estimating the true prevalence in camels in the Eastern Province and Riyadh, using a Bayesian estimation framework, and exploring the associated risk factors through a frequentist approach. A total of 1200 camel blood samples were collected and analyzed using an indirect ELISA method. The true herd-level prevalence was estimated at 0.7 (95% probability interval: 0.57 to 0.81), and the mean expected true animal-level prevalence was 0.17 (0.14 to 0.20). Risk factors associated with Map seropositivity were identified, including sex, breed, raising system, and production type. Females, single breed camels, and nomadic raising systems were found to have lower odds of seropositivity, while camels used for racing and show had significantly higher odds. The study's Bayesian approach, adjusting for the imperfect accuracy of MAP tests, provides a nuanced understanding of the disease's prevalence in the region. The integration of true prevalence estimates with risk factor analysis offers a comprehensive framework that can guide future policies and strategies in the fight against paratuberculosis in Saudi Arabia. The findings emphasize the importance of targeted control measures, underscoring the urgent need for interventions in Saudi Arabia's camel population. By understanding the true disease prevalence and its associated risk factors, we can enhance disease management strategies, offering valuable insights for future control and eradication efforts in the region.

Sample size estimation to substantiate freedom from disease for clustered binary data with a specific risk profile.

Disease cases are often clustered within herds or generally groups that share common characteristics. Sample size formulae must adjust for the within-cluster correlation of the primary sampling units. Traditionally, the intra-cluster correlation coefficient (ICC), which is an average measure of the data heterogeneity, has been used to modify formulae for individual sample size estimation. However, subgroups of animals sharing common characteristics, may exhibit excessively less or more heterogeneity. Hence, sample size estimates based on the ICC may not achieve the desired precision and power when applied to these groups. We propose the use of the variance partition coefficient (VPC), which measures the clustering of infection/disease for individuals with a common risk profile. Sample size estimates are obtained separately for those groups that exhibit markedly different heterogeneity, thus, optimizing resource allocation. A VPC-based predictive simulation method for sample size estimation to substantiate freedom from disease is presented. To illustrate the benefits of the proposed approach we give two examples with the analysis of data from a risk factor study on Mycobacterium avium subsp. paratuberculosis infection, in Danish dairy cattle and a study on critical control points for Salmonella cross-contamination of pork, in Greek slaughterhouses.

A comparison of cytologic quality in fine-needle specimens obtained with and without aspiration from superficial lymph nodes in the dog.

OBJECTIVES: To assess the impact of aspiration on cytologic quality of fine-needle specimens obtained from lymph nodes of dogs and to compare the level of agreement of the cytologic diagnosis between the two sampling techniques. MATERIALS AND METHODS: Fifty-three client-owned dogs were prospectively enrolled. Client-owned dogs were prospectively enrolled in the study if cytologic examination of lymph nodes was indicated in the course of their clinical diagnostic work-up. In each dog, two superficial, palpably accessible lymph nodes were sampled: one by fine-needle aspiration and the other by fine-needle non-aspiration, using a 21-Gauge needle with or without a 5-mL syringe. Cytologic quality was assessed in duplicate smears assessed by two observers who rated cellularity, blood contamination, thickness, cell preservation and cytoplasmic fragmentation using a predefined scoring system. RESULTS: Fifty-three client-owned dogs were included in the study. No significant difference was found in rating scores between the two sampling techniques for any of the cytologic quality parameters assessed. Cohen's kappa coefficient was 0.84 (95% confidence interval 0.68-1.00), indicating diagnostic agreement between the sampling techniques. CLINICAL SIGNIFICANCE: In this study, both fine-needle aspiration and fine-needle non-aspiration techniques yielded lymph node specimens of comparable cytologic quality and with acceptable agreement in cytologic diagnosis.

Bayesian estimation of the true prevalence of paratuberculosis in Hungarian dairy cattle herds.

Paratuberculosis is a chronic incurable disease caused by Mycobacterium avium subsp. paratuberculosis (MAP), which leads to extensive economic losses on dairy farms, and may also pose serious public health risk to the consumers. The aim of our study was to estimate the true prevalence of paratuberculosis in commercial dairy cattle herds participating in a voluntary MAP testing programme that started in February 2018 in Hungary. Milk samples collected during official milk recording were used for MAP ELISA testing. A Bayesian two-stage hierarchical (herd and animal level) model was fitted to the data. Altogether, 26,437 cows from 51 herds were sampled, which represents 14.4 % of the Hungarian dairy cow population. The median herd size was 477 cows (interquartile range: 331-709). Each studied farm had at least one ELISA positive cow, resulting in a herd-level apparent prevalence of 100 %. The overall within herd apparent prevalence was 5.5 %. Herd-level true prevalence was estimated at 89.1 % [95 % credible interval (CrI): 80.3-95.6%]. Within the infected herds, the median animal-level true prevalence was 4.4 % (3.2-5.8%) for primiparous and 10.3 % (7.9-12.9%) for multiparous cows, respectively. The probability of having an animal-level true prevalence of at least 5% among primiparous cows, within infected herds, was 17.8 %. Similarly, the probability of having an animal-level true prevalence of at least 5% or 10 % among multiparous cows was 100 % and 56 %, respectively. Simulations assuming herd-level true prevalence varying from 50 to 100 % revealed high accuracy of our Bayesian model. Our study showed that a large percentage of the studied Hungarian dairy cattle herds was infected with MAP.

Accuracy of paratuberculosis diagnostic tests in small ruminants: protocol for a systematic review and meta-analysis.

Paratuberculosis is a worldwide infectious disease caused by Mycobacterium avium ssp. paratuberculosis (MAP). Various ruminant species can be affected by the disease, and the diagnosis of the disease is challenging in the absence of a gold standard test. The aim of this systematic review protocol is to determine the accuracy of the direct and indirect diagnostic tests for MAP infection with a special focus on sheep and goats.

Brucellosis underreporting in Greece: assessment based on aggregated laborato-ry data of culture-confirmed cases from public hospitals.

BACKGROUND: Brucella spp. isolation is one of the mainstays of brucellosis diagnosis. Simultaneously, the true brucellosis disease rate may be underrepresented in notification systems. This study aims at assessing the nosocomial capacity for Brucella spp. isolation and the underreporting rate of brucellosis cases in Greece. METHODS: Data for Brucella spp. culture capacity and the number of isolations were collected annually from public hospitals nationwide, during 2015-2018. The number of unreported cases was estimated after subtracting the National Mandatory Notification System cases from the survey-captured isolations, matched by hospital and year. RESULTS: Feedback was provided by 112 public hospitals (response rate: 97.4 %). Brucella spp. isolation capacity was completely absent in 27.7 % of hospitals; during the four years of the study, 11.3 %, 13.9 %, 20.0 %, and 25.2 % of the hospitals had isolation competence for one, two, three, or four years, respectively. Underreporting assessment was possible in hospitals that declared at least one Brucella spp. isolation (n =35) and unreported cases were identified in 19 (54 %). Α mean underreporting of 28.9 % of total cases was estimated for the whole period of the study ranging annually from 24.1 % to 35.0 %. The number of unreported cases per hospital ranged from one to 12 per year (median: 2, IQR: 5). CONCLUSIONS: Interventions for improving diagnosis and reporting of the disease are recommended. Assessment of brucellosis underreporting by comparing raw numerical data of survey-captured isolations and officially notified cases lacks the case by case specificity, however, keeping required data to a minimum achieves high feedback rate from hospitals and provides a tentative estimation of the notification deficit. HIPPOKRATIA 2019, 23(3): 106-110.

A retrospective study of 61 cases of spontaneous canine epistaxis (1998 to 2001).

OBJECTIVES: To determine the prevalence and identify possible clinicopathologic indicators of the diseases associated with canine epistaxis. METHODS: The medical records of 61 dogs with epistaxis were reviewed. RESULTS: Systemic diseases, diagnosed in fifty-six dogs, included canine leishmaniasis in twenty-three dogs, canine monocytic ehrlichiosis in twenty-two, concurrent canine leishmaniasis and canine monocytic ehrlichiosis in six, rodenticide toxicity in two and primary immune-mediated thrombocytopenia, suspected oestrogen toxicity and systemic arterial hypertension in one dog each. Intranasal diseases were documented in the remaining five dogs, including transmissible venereal tumour in three dogs, and nasal adenocarcinoma and nasal aspergillosis in one dog each. Mucosal pallor and a generalised bleeding tendency were significantly more common among dogs with canine monocytic ehrlichiosis compared with those with canine leishmaniasis, whereas the opposite was true for peripheral lymphadenomegaly. Also, dogs with canine monocytic ehrlichiosis presented with pancytopenia more frequently compared with those with canine leishmaniasis; in the latter dogs, the median values of haematocrit, leucocyte and platelet counts and serum total protein concentrations were higher. CLINICAL SIGNIFICANCE: Canine leishmaniasis and canine monocytic ehrlichiosis are the leading causes of canine epistaxis in Greece. Mucosal pallor, bleeding tendency and pancytopenia are more likely to be indicative of canine monocytic ehrlichiosis, as opposed to peripheral lymphadenomegaly and hyperproteinaemia in canine leishmaniasis.

Application of a semi-dependent latent model in the Bayesian estimation of the sensitivity and specificity of two faecal culture methods for diagnosis of paratuberculosis in sub-clinically infected Greek dairy sheep and goats.

In this study, we compared the frequency of isolation of Mycobacterium avium subsp. paratuberculosis (MAP) from faecal samples grown on Herrold's egg-yolk medium (HEYM) or on Lowenstein-Jensen (LJ) medium and estimated the sensitivity (Se) and specificity (Sp) of the methods separately in sub-clinically infected Greek dairy sheep and goats, using latent-class models and Bayesian estimation procedures. Faecal and blood samples were collected from 400 animals > or =1 year old in April-May 2002. The HEYM supported growth of MAP better than the LJ method and their agreement was very poor (weighted kappa=0.062 (95% CI: -0.098, 0.222)). There was no evidence of dependence between the Ses whereas the Sps were positively correlated. Thus, a semi-dependent model that assumed independence of Ses and accounted for the dependence of Sps was adopted. Under this model, the parallel interpretation of the results of the two methods gave median estimates and 95% credible intervals (CrIs) for Se(par), Sp(par) of 15% (CrIs: 3, 45%), 96% (92, 98%) in sheep and 16% (6, 36%) and 97% (94, 99%) in goats.

Bayesian estimation of sensitivity and specificity of serum ELISA and faecal culture for diagnosis of paratuberculosis in Greek dairy sheep and goats.

Latent class models were used to estimate the sensitivity (Se) and the specificity (Sp) of a serum ELISA and a faecal culture (FC) method for the diagnosis of paratuberculosis separately, in sheep and goats. The estimates were obtained by a Bayesian method. Possible dependence of diagnostic errors was investigated by comparing models where independence was assumed to models allowing for conditional dependence given the true disease status. ROC analysis for the serum ELISA was also performed and optimized cut-off values based on the misclassification cost term were determined. No evidence of conditional dependence was found. Assuming independence, posterior medians and 95% credible intervals for the Se(ELISA), Sp(ELISA), Se(FC) and Sp(FC), were 63% (42, 93%), 95% (90, 98%), 8% (2, 17%) and 98% (95, 100%) in goats and 37% (10, 80%), 97% (93, 99%), 16% (2, 48%) and 97% (95, 99%) in sheep. AUC was calculated 0.702 for sheep and 0.847 for goats. For the serum ELISA, there is need of species- and purpose-specific cut-off selection. For instance, with 20% prevalence situation and assuming equal and five-fold cost of a false negative to a false positive test result, the optimal cut-off is 0.3 and 0.05 in sheep, respectively, while it is 0.6 and 0.1 in goats, respectively. Serum ELISA performed better in goats than in sheep. Lowering the cut-off, in relation to the one recommended by the manufacturer, improved Se(ELISA) without seriously compromising Sp(ELISA), in either species.

The association of sub-clinical paratuberculosis with the fertility of Greek dairy ewes and goats varies with parity.

Our cross-sectional study investigated the association of sub-clinical Mycobacterium avium subsp. paratuberculosis (MAP) infection with failing to produce a live offspring the season of lambing/kidding (November 2001 to January 2002) before testing (in April-May 2002), in four dairy-sheep and/or goat flocks in Greece (369 animals >or=1.5-year-old). From each selected animal 10 ml of blood and 10 g of feces from the rectum were obtained. The harvested sera were tested for antibodies to MAP with a commercial ELISA test kit; the feces were cultured on Herrold's egg-yolk medium supplemented with mycobactin J and antibiotics. An animal was considered sub-clinically infected when found either seropositive or culture positive. The true prevalence of sub-clinically infected animals, adjusted for the sensitivity and specificity of the parallel testing, was 14% (0.1-28%) and 35.9% (9.2-62.7%) in sheep and goats, respectively. The association of fertility of sheep and goats with sub-clinical paratuberculosis was investigated in random-effects logistic models. Sub-clinically infected animals (compared to uninfected) had OR for live offspring the previous year of 5.4 for parity <4, OR=0.05 for parity >6, and a non-significant OR for the middle parity category.

Prevalence of BVDV infection in Greek dairy herds.

Thirty-nine Greek dairy herds, totalling 6333 cattle, enrolled in a voluntary bovine viral diarrhoea virus (BVDV) eradication programme based on the identification and removal of persistently infected (PI) animals. The aim of this study was to estimate the prevalences of BVD antigen-positive and PI animals, and investigate the significance of the associations between the prevalence estimates and herd size. Initially, all animals were bled and examined for BVDV, using an antigen ELISA. A second sample was collected from the positive animals, after a period of at least three weeks. Animals retested positive were classified as PI. Antigen positive and PI animals were detected in all herds. The respective mean prevalences, adjusted for the test's accuracy and the herd-clustering effect, were 14% (95%CI: 11-18%) and 1.3% (0.8-1.8%), respectively. Herd size was not associated with the prevalence of antigen-positive or PI animals.

Effect of challenge dose and age in experimental infection of pigs with encephalomyocarditis virus.

Two experiments were performed to compare the severity of encephalomyocarditis virus (EMCV) infection in pigs. The pigs were challenged with the Greek myocardial strain, at different ages and with different doses. In the first experiment, nineteen susceptible pigs, 40 days old, were divided into three groups and were experimentally infected with 10(6) TCID(50), 10(4) TCID(50) or 10(2) TCID(50) of the Greek EMCV strain. In the second experiment, 10 susceptible pigs, of either 20 or 105 days, were divided into two groups according to age and were experimentally infected with 10(6) TCID(50) of the Greek EMCV strain. In addition, five piglets, each one the same age as its experimental group, were used as uninfected controls. No clinical signs were observed after infection, except a transient temperature rise in some pigs. Another important observation was the difference in mortality between groups. The survival rate of the 40-day-old pigs was inversely related to the viral dose. In these pigs, a positive association between the viral dose and the severity of macroscopical and histopathological lesions of the heart was also evident. Viral isolations from various organs of the challenged 40-day-old pigs increased with the increasing dose level. When challenged with 10(6) TCID(50) of EMCV, there was no difference in the fatality rate of the 20- and 40-day-old pigs, but none of the 105-day-old pigs died. The severity of the macroscopical and the histopathological heart lesions was inversely related to the age of the pigs. Furthermore, viral isolations from the various organs were higher in 20- and 40-day-old pigs than in the older ones. In 40-day-old pigs, neutralizing antibodies linearly increased as the dose increased. These antibodies were consistently lower in 20-day-old pigs. Viraemia, and nasal and faecal excretions were detected in all groups and lasted 1-3 days, except for the 105-day-old pigs whose symptoms lasted for an additional day.

Flock-level factors associated with the risk of Mycobacterium avium subsp. paratuberculosis (MAP) infection in Greek dairy goat flocks.

In this cross-sectional study we identified flock-level risk factors for Mycobacterium avium subsp. paratuberculosis (MAP) infection, in Greek dairy goat flocks. We collected 1599 milk samples from does that were at the last stage of lactation in 58 randomly selected dairy goat flocks, during May to September 2012. The collected samples were tested with a commercial milk ELISA (IdexxPourquier, Montpellier, France) and the results were interpreted at a cut-off that optimized the accuracy of the diagnostic process. For the analysis of the data we used Bayesian models that adjusted for the imperfect Se and Sp of the milk-ELISA. Flock was included as a random effect. Does in flocks that used common water troughs and communal grazing grounds had 4.6 [95% credible interval (CI): 1.5; 17.4] times higher odds of being MAP-infected compared to does in flocks that had no contact with other flocks. Does of flocks supplied with surface water from either streams or shallow wells had 3.7 (1.4; 10.4) times higher odds of being infected compared to those in flocks watered by underground and piped water sources. When kids were spending equal to or more than 10h per day with their dams they had 2.6 (1.1; 6.4) times higher odds of being MAP infected compared to kids that were separated from their dams for less than 10h per day. Finally, does in flocks that continuously used the same anti-parasitic compound had 2.2 (1.0; 4.6) times higher odds of MAP infection compared to those in flocks alternating anti-parasitic compounds. These results should be considered in the development of a nationwide future control program fοr caprine paratuberculosis in Greece.

Strain-specific sensitivity estimates of Mycobacterium avium subsp. paratuberculosis culture in Greek sheep and goats.

The requirements for the isolation of Mycobacterium avium subsp. paratuberculosis (Map) may be related to the strain-type [sheep (S)- or cattle (C)-type] and not to the host. The objective of this paper was to estimate and compare strain- and biological sample (faeces or pooled-tissue)--specific sensitivities (Ses) of two solid culture media, Herrold's egg yolk medium (HEYM) and Lowenstein-Jensen (LJ) medium, for the isolation of Map from Greek dairy sheep and goats. From 400 faecal samples collected from sub-clinically infected sheep and goats of four flocks and from 214 pooled-tissue samples (142 from sheep and 72 from goats) collected, at the abattoir, from >1-year-old routinely slaughtered animals, with gross pathology suggestive of paratuberculosis, we isolated 34 Map strains. Of those, by the IS1311 PCR, 18 were categorized into the C-type and nine into the S-type; seven were not typed. We used a Bayesian approach to estimate the strain-specific Ses. SeHEYM-C-faecal=17% (95% credible interval: 7, 40) was higher than SeHEYM-S-faecal=2% (0.3, 11). Also, SeHEYM-C-faecal was higher than SeLJ-C-faecal=4% (1, 12). In pooled-tissue samples, the strain-specific Ses did not differ between the two media.

Isolation of Mycobacterium avium subspecies paratuberculosis from non-ruminant wildlife living in the sheds and on the pastures of Greek sheep and goats.

This study aimed to: (1) investigate whether non-ruminant wildlife interfacing with dairy sheep and goats of four Greek flocks endemically infected with Mycobacterium avium subspecies paratuberculosis (MAP) harboured MAP and (2) genetically compare the strains isolated from the wildlife to those isolated from the small ruminants of these flocks. We cultured and screened, by polymerase chain reaction (PCR), pooled-tissue samples from 327 wild animals of 11 species for the MAP-specific IS900 insertion sequence. We also cultured faecal samples from 100 sheep or goats from each of the four flocks. MAP was detected in samples from 11 sheep, 12 goats, two mice, two rats, a hare and a fox. Only one rat had histopathological findings. Genetic typing categorized 21 isolates as cattle-type strains and two, from a house mouse and a goat respectively, as sheep-type strains; this is the first report of a rodent harbouring a sheep-type strain. The MAP types that were most frequently isolated amongst the sheep and goats of each flock were also the ones isolated from sympatric rodents; those isolated from the fox and hare also belonged to the predominant ruminant strains.