Accuracy estimation of an indirect ELISA for the detection of West Nile Virus antibodies in wild birds using a latent class model.

West Nile virus (WNV) and Usutu virus (USUV), genus Flavivirus, are members of the Japanese encephalitis virus antigenic complex, and are maintained primarily in an enzootic cycle between mosquitoes and birds. WNV is zoonotic, and poses a threat to public health, especially in relation to blood transfusion. Serosurveillance of wild birds is suitable for early detection of WNV circulation, although concerns remain to be addressed as regards i) the type of test used, whether ELISA, virus neutralization test (VNT), plaque reduction neutralization test (PRNT), ii) the reagents (antigens, revealing antibodies), iii) the different bird species involved, and iv) potential cross-reactions with other Flaviviruses, such as USUV. The authors developed an indirect IgG ELISA with pan-avian specificity using EDIII protein as antigen and a monoclonal antibody (mAb 1A3) with broad reactivity for avian IgG. A total of 140 serum samples were collected from juvenile European magpies (Pica pica) in areas where both WNV and USUV were co-circulating. The samples were then tested using this in-house ELISA and VNT in parallel. Estimation of test accuracy was performed using different Bayesian two latent class models. At a cut-off set at an optical density percentage (OD%) of 15, the ELISA showed a posterior median of diagnostic sensitivity (DSe) of 88% (95%PCI: 73-99%) and a diagnostic specificity (DSp) of 86% (95%PCI: 68-99%). At this cut-off, ELISA and VNT (cut-off 1/10) performances were comparable: DSe=91% (95%PCI: 79-99%), and DSp=77% (95%PCI: 59-98%). With the cut-off increased to 30 OD%, the ELISA DSe dropped to 78% (95%PCI: 52-99%), and the DSp rose to 94% (95%PCI: 83-100%). In field conditions, the cut-off that yields the best accuracy for the ELISA appears to correspond to 15 OD%. In areas where other Flaviviruses are circulating, however, it might be appropriate to raise the cut-off to 30 OD% in order to achieve higher specificity and reduce the detection of seropositive birds infected by other Flaviviruses, such as USUV.

Suitability of a Salmonella control programme based on serology in slaughter heavy pigs.

The key component of most European pig Salmonella control programmes is the classification of herds according to seroprevalence at slaughter. The objectives of this study were to estimate the true Salmonella seroprevalence, and investigate the association between the true status of infection and serology in slaughter heavy pigs. Blood of 3340 pigs was collected and tested with ELISA. From 385 pigs, also lymph nodes and cecal content were collected for bacteriology. Analysis was performed in a Bayesian framework. Results showed that a large proportion of pigs was serologically positive (herd seroprevalence 93% and within-herd seroprevalence higher than 81% in half of herds at cut-off 10 OD%). The association between the true status of infection and serology was not significant, and therefore the classification of heavy pig herds according to seroprevalence at slaughter would not be suitable to reduce the risk of introducing Salmonella into the food chain.

Effect of a homeopathic complex on reproductive performance in a commercial pig farm.

BACKGROUND AND AIM: Alternative therapies based on homeopathy can be effective in improving reproductive performance in intensive pig breeding. In this study, the effect of a homeopathic complex on reproductive performance of sows under intensive farming has been investigated. MATERIAL AND METHODS: Over period of three years, 186 sows were recruited from a farm where a large proportion of animals were suffering from prolonged weaning-to-oestrus intervals (WEI) and weaning-to-service intervals (WSI). Sows were allocated to two groups; once per month, one group was given a homeopathic complex (Borax 10 mK plus Lycopodium 10 mK), while the other group was given a hydro-alcoholic solution (placebo). The follow-up period started one week before the expected date of oestrus, continued for two pregnancies and ended after the weaning of the second farrowing. To evaluate reproductive performance, during the follow-up we collected data on quantitative parameters such as the average number of stillbirths, newborns, and repeat services per farrowing. Time-related data such as WEI, WSI, length of the two pregnancies and weaning periods were also collected to measure the length of the follow-up of each sow. Differences in quantitative parameters between the two groups were evaluated using parametric and non-parametric statistics. Time-related data were used to plot Kaplan-Meier curves and in Cox regression models to evaluate whether treated sows had a higher probability of experiencing a shorter follow-up in comparison to untreated sows. RESULTS: We did not found significant differences in the number of newborns, while the number of stillbirths was higher in the treatment group, even if the difference was slightly significant (p-value = 0.03). The number of repeat services was lower in the treatment group, and this difference was highly significant (p-value < 0.001). Results from the Cox regression models suggest that the end of the follow-up was reached by sows of the treatment group at about twice the rate of sows of the control group (model 2, Hazard Ratiotreatment = 2.27; 95%CI: 1.56-3.24).

Comparison of three in-house ELISAs for the detection of hepatitis E virus infection in pigs under field conditions.

Hepatitis E virus (HEV) is a RNA non-enveloped virus that comprises four genotypes. The genome of HEV is organized into three Open Reading Frames (ORFs), and the ORF2 is responsible for encoding capsid proteins. HEV can infect a wide range of hosts, and pigs are considered the main reservoir. HEV infection is considered a zoonosis and it is responsible for acute hepatitis in humans, especially in developing countries. The development of a blocking ELISA would be of high value for screening purpose, because there is no need of species specific reagents. The present study was conducted to assess three in-house ELISAs for the detection of HEV infection in 779 sera collected from breeding and fattening farms under field conditions. Two assays were indirect ELISAs, while the third was a blocking ELISA. Two different recombinant antigens were generated from specific sequences of the HEV-ORF2, and a Latent Class approach in a Bayesian framework was used to evaluate the diagnostic accuracy of each ELISA. Because the three ELISAs cannot be thought of as independent, all possible dependence structures were modelled starting from the general case of conditional independence to the most complex situation of three mutually dependent assays. Results showed that none of the three ELISAs was significantly superior to the others in terms of sensitivity (posterior median value ranging from 89% to 94%, all 95% posterior credible intervals (95%PCI) overlapped). In terms of specificity, one of the indirect ELISAs was superior to blocking ELISA (posterior median indirect ELISA: 99%, 95%PCI: 98-100%; blocking ELISA: 90%; 95%PCI: 86-94%). However, this difference could be due to the potential wider spectrum of antibodies that blocking ELISA can actually detect.

A surveillance program on canine leishmaniasis in the public kennels of Emilia-Romagna Region, Northern Italy.

Since 2007, a canine leishmaniasis (CanL) surveillance program has been carried out in public kennels of the Emilia-Romagna region with the aim of providing health guarantees for dog adoptions. According to this program, monitoring activities were performed to verify the presence of sandflies and infected dogs, and a specific CanL risk class was assigned to each kennel, resulting in different control approaches (entomological and/or serological monitoring, clinical surveillance, therapeutic treatment of infected dogs, protections against vector bites). From 2007 to 2012, 20,931 dogs, 89.8% of which were identified by microchip and housed in 73 kennels, were examined using an indirect fluorescent antibody test. In all, 528 (2.8%) dogs tested positive, and 43.0% of these were asymptomatic. The authors used monitoring results, in particular serological tests performed on dogs at admittance to the kennel and annual controls of sentinel dogs, to estimate CanL risk in the whole region and to evaluate the efficacy of the preventive measures adopted. CanL seroprevalence in dogs tested at the admittance in kennels increased significantly from 2010 (1.0%; 29/2858) to 2012 (2.4%; 69/2841). In contrast, the number of seroconversions in sentinel dogs was stable in 2010 (1.2%; 11/896) and 2011 (1.6%; 13/825) and decreased in 2012 (0.9%; 8/850), suggesting the efficacy of the preventive measures applied.

Completeness of the dog registry and estimation of the dog population size in a densely populated area of Rome.

In most European countries, registration and identification of dogs is compulsory. In Italy, the national dog registry is composed of regional dog registries. Although dog registries have been established for many years, the issue related to completeness of data has not been addressed so far. The objective of this study was twofold: first to assess the completeness of data of the dog registry through telephone interview of a sample of dog owners drawn from the dog registry, then to estimate the total owned dog population in 4 boroughs of Rome. For the second objective, a capture-recapture method was applied using data from the dog registry and data from a face-to-face questionnaire submitted to people waiting in the sitting room of 5 points of access for booking and payment of primary and specialist care. Different scenarios are proposed to verify the assumptions of the estimation procedure and potential biases are discussed. The completeness of data of the dog registry was 88.9% (95% CI: 85.8-91.9%) and the owned-dog population was estimated at 26,244 dogs (95% CI: 24,110-28,383). The dog registry is an important source of information especially when it is properly updated and completeness of data is known.

A spatial, statistical approach to map the risk of milk contamination by β-hexachlorocyclohexane in dairy farms.

In May 2005, beta-hexachlorocyclohexane (ß-HCH) was found in a sample of bovine bulk milk from a farm in the Sacco River valley (Latium region, central Italy). The primary source of contamination was suspected to be industrial discharge into the environment with the Sacco River as the main mean of dispersion. Since then, a surveillance programme on bulk milk of the local farms was carried out by the veterinary services. In order to estimate the spatial probability of ß- HCH contamination of milk produced in the Sacco River valley and draw probability maps of contamination, probability maps of ß-HCH values in milk were estimated by indicator kriging (IK), a geo-statistical estimator, and traditional logistic regression (LR) combined with a geographical information systems approach. The former technique produces a spatial view of probabilities above a specific threshold at non-sampled locations on the basis of observed values in the area, while LR gives the probabilities in specific locations on the basis of certain environmental predictors, namely the distance from the river, the distance from the pollution site, the elevation above the river level and the intrinsic vulnerability of hydro-geological formations. Based on the ß-HCH data from 2005 in the Sacco River valley, the two techniques resulted in similar maps of high risk of milk contamination. However, unlike the IK method, the LR model was capable of estimating coefficients that could be used in case of future pollution episodes. The approach presented produces probability maps and define high-risk areas already in the early stages of an emergency before sampling operations have been carried out.

[Beta-hexachlorocyclohexane contamination in dairy farms of the Sacco River Valley, Latium, Italy, 2005. A retrospective cohort study]. / Contaminazione da beta-esaclorocicloesano delle aziende bovine da latte della Valle del Sacco (Lazio). Gestione dell'emergenza 2005 e conseguente studio di coorte retrospettivo.

OBJECTIVE: in March 2005, the Italian National Monitoring System on Chemical Residuals in Food of Animal Origin detected levels of the pesticide beta-hexachlorocyclohexane (ß-HCH) that were 20 times higher than the legal limit of 0.003 mg/kg in bulk milk from a dairy farm in the Sacco River valley. ß-HCH, a lindane isomer and possible human carcinogen, was subsequently found in milk from several neighboring farms. A study was therefore undertaken to evaluate the extent and risk factors for contamination. DESIGN: all dairy cattle farms in the valley were enrolled in a retrospective cohort study and their bulk milk analyzed for ß-HCH. A questionnaire was administered to farmers to evaluate possible exposure factors. SETTING AND PARTICIPANTS: cases: dairy farms with at least one result indicating ß-HCH ≥ 0.002 mg/kg in bulk milk during the period april-june 2005; exposure: feeding animals on fodder cultivated in soils watered with and/or flooded by river water; participants: IZSLT, RMG Local Health Unit, FR Local Health Unit. MAIN OUTCOME MEASURES: attack rate, relative risk, attributable proportion among exposed. RESULTS: of 244 farms tested, 34 met the case definition (attack rate 14%). The exposure to fodder cultivated in soils watered with and/or flooded by river water was observed in 33/34 (97%) case-farms and in 23/210 (10.9%) of those with contamination <0.002 mg/kg in bulk milk (RR 110.8; 95%CI 15.5- 792). Attributable proportion among exposed was more than 99%. CONCLUSION: fodder cultivated near a contaminated river was the main risk factor for ß-HCH contaminated milk. On the basis of the epidemiologic evidence and laboratory testing, watering local fields with river water and production of fodder in farms with contaminated soil was banned, and all the animals from positive farms were culled.