Evaluation of a Commercial ELISA for the Detection of Antibodies to Sarcoptes scabiei in Wild Boar (Sus scrofa) Open Access

Sarcoptic mange is a parasitic skin disease affecting numerous mammal species worldwide and causing high mortality in free-ranging wildlife (Alasaad et al. 2011). However, in wild boar, mange is apparently associated with low mortality despite high morbidity (Haas et al. 2015) and so far the disease has been poorly investigated in this species.

In recent years, sarcoptic mange has been newly detected in the free-ranging wild boar population in Switzerland and the question was raised as whether the disease has been endemic but previously undetected or has truly emerged in the population (Haas et al. 2015). A serosurvey on new and archived samples may contribute to assessing the current and former spread of the infection in the population. We evaluated the sensitivity and specificity of a commercial enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Sarcoptes scabiei in domestic pigs when applied to wild boar sera.

As negative controls, we used blood samples from 96 wild boars from populations without mange history. They included four captive animals from a healthy zoo collection (Basel Zoo, Basel, Switzerland) and 92 free-ranging boars from the region of Geneva (46°6′00″ to 46°18′0″N; 5°54′00″ to 6°18′00″E), Switzerland, where ecological studies including photo-trapping and animal captures have been performed (Hebeisen et al. 2008) and where sarcoptic mange has not been reported in wild boar. Samples from zoo animals were collected at slaughter on 14 February 2014 and immediately centrifuged; serum was aliquoted before shipping to the laboratory. Samples from free-ranging wild boars were collected from hunted animals between December 2012 and April 2013 by professional game wardens and consisted of blood or serosanguinous fluid retrieved from the heart or thoracic cavity. They were sent to the laboratory immediately after collection and centrifuged at 2,000 × G for 20 min. Zoo animals were juvenile females 6–8 mo old. Age of the free-ranging animals was determined in the field based on body weight and coat color (Hebeisen et al. 2008). There were 55 females and 37 males, including 39 juveniles (6–12 mo), 25 subadults (12–24 mo), and 28 adults (≥2 yr).

We also tested blood samples from 141 free-ranging wild boars with mange-like lesions (MLLs). Three animals were shot by hunters in the region of Solothurn (47°12′00″ to 47°24′00″N;7°24′00″ to 7°30′00″E), Switzerland, and submitted to necropsy; S. scabiei was detected in skin samples of all three cases (Haas et al. 2015). Additionally, 138 samples were collected from 50 live wild boars with MLLs that were captured, marked, and recaptured several times in the Petite Pierre National Reserve (48°30′0″N, 7°0′0″E), Bas-Rhin region, France, where mange is common in wild boar (S.R. pers. obs.). Captures were carried out weekly from 10 May to 23 August 2006 for another study (Rossi et al. 2011). Each animal was marked with ear-tags to allow identification and blood was taken from the jugular vein. Animals were venipunctured one to eight times each, but at a maximum of once a week for welfare consideration. Age was determined based on tooth wear of the inferior jaw (Matschke 1967). There were piglets (2–6 mo; 20 males and 23 females), four subadult males, and three adult females. Blood was immediately centrifuged at 1,500 × G for 15 min. Mange-like lesions were classified in four categories: 1) mild (erythema, alopecia without skin thickening), 2) localized and moderate (erythema, alopecia with local skin thickening and crusts), 3) generalized and severe (erythema, alopecia with skin thickening and crusts on more than two-thirds of the body surface), and 4) healing (thickened skin, hair regrowth, absence of erythema; only animals >12 mo). A skin scraping was made on 21 animals. Mites were detected by light microscopy in 17 boars and identified as S. scabiei based on morphologic characteristics (Sloss and Kemp 1978).

Serum samples were stored at −20 C until analysis. The commercial indirect ELISA SARCOPTES-ELISA 2001^® Pig (AFOSA GmbH, Blankenfelde-Mahlow, Germany) was used according to the manufacturer's instructions to detect anti-Sarcoptes antibodies. Product specification indicates a sensitivity of 94% and a specificity of 97% in domestic pigs. It uses Sarcoptes mites from pigs as antigen and was the most sensitive (88.6%) of four commercially available indirect ELISAs for the detection of S. scabiei antigens applied on pig samples (Löwenstein et al. 2004). Twenty samples with doubtful results were tested a second time.

As in former studies of red fox (Vulpes vulpes; Nimmervoll et al. 2013) we defined sensitivity of the test as the proportion of ELISA-positive samples from animals with macroscopically visible MLLs and demonstrated mite infestation at the time of sample collection (truly infected, n = 20), and specificity as the proportion of samples from animals without MLLs (truly noninfected, n = 96) that were found negative.

Considering the 116 samples from animals with either absence of MLLs or confirmed mite infestation, we obtained a sensitivity of 75% and a specificity of 80% (Table 1). Of the 50 wild boars with MLLs that were sampled several times (over 5–90 d), 41 (82%) were antibody-positive at least once. Of the remaining animals, one had a doubtful result and eight (sampled one to five times) were seronegative. Seven of these eight wild boars presented MLLs in stages 1–2, including a piglet with a positive skin scraping, and there was one animal (of the 8) with healing skin lesions. Considering all analyzed samples, the percentage of antibody-positive samples increased with the chronicity of lesions (Table 2).

Overall, 23 wild boars were antibody-positive at the first sampling, and seroconversion was observed in 19 of 27 (70%) individuals with an initially antibody-negative or doubtful result. In these animals, the first antibody-positive result was detected on average 27.5 d (range: 6–63 d) after the first sampling (capture intervals: 5–35 d). False negative results were obtained in two animals with repeated positive results.

The sensitivity (75%) and specificity (80%) of the SARCOPTES-ELISA 2001 Pig for diagnosis of sarcoptic mange in wild boar were lower than indicated by the manufacturer for domestic pigs (sensitivity, 94%; specificity, 97%). They were also lower than data obtained for pigs (sensitivity, 88.6%) with the same test (Löwenstein et al. 2004) and data obtained with the SARCOPTES-ELISA 2001 Dog applied on red fox sera (sensitivity, 98.2%; specificity, 91.9%; Nimmervoll et al. 2013), but they were comparable to results obtained with an in-house ELISA applied on sera of free-ranging Iberian wolf (Canis lupus; sensitivity, 75%; specificity, 87.5%; Oleaga et al. 2011).

Positive results for 12 “truly noninfected” free-ranging animals may be due to cross-reactions with other mites (Dockmann 2004), subclinical S. scabiei infestations (Ippen et al. 1995), or a lack of detection of mild skin lesions by game wardens. Hemolysis is not expected to have affected our results (Casaubon et al. 2013).

Negative results obtained for “truly positive” animals may be due to differences between the antigen used in the ELISA and the mites infesting the host. Changes in antigen conformation may lead to variable epitope exposure to antibodies and lower test sensitivity (Casais et al. 2013). Wild boar can be repeatedly infested by mites from different subpopulations (Rasero et al. 2010), and each mite strain produces some unique antigen (Arlian et al. 1996), which may involve differences in the circulating antibodies. Furthermore, repeated freeze-thaw cycles (as occurred for the used samples from captured wild boar) may cause a decrease in optical density values (Boadella and Gortázar 2011), a factor possibly contributing to the doubtful or negative results in a few wild boars expected to be antibody positive. However, when results are considered at an individual scale including repeated sampling over time, sensitivity increases (82%), indicating a role of the dynamics of the immune response in the success of antibody detection. The date of infection of the wild boars was unknown but, in some cases, the clinical picture evolved and seroconversion was documented 6–63 d after the first sampling. This is in agreement with the observations of Dockmann (2004) in domestic pigs (seroconversion 5–7 wk postinfection or 3–4 wk after appearance of the first clinical signs) and of Arlian et al. (1994) in domestic rabbits (highest antibody titer 64 d postinfection). Thus, samples from piglets in an initial stage of the disease (this study) are more likely to be antibody-negative than severely diseased animals such as foxes with advanced mange lesions (Nimmervoll et al. 2013), indicating an influence of sample composition when evaluating the performance of a serologic test.

The evaluated commercial ELISA for the detection of Sarcoptes scabiei var. suis in domestic pigs was successfully applied to wild boar sera. However, given the delay of the antibody response after infection and potential cross-reactions, it is not reliable for early detection in individual animals but it may represent a useful tool for serosurveys.

We acknowledge the wildlife managers and collaborators of the Centre for Fish and Wildlife Health and the Office National de la Chasse et de la Faune Sauvage who contributed to sample collection, Christian Wenker for providing samples from zoo animals, Mariana Boadella for critical reading of the manuscript, and Hans-Frieder Matthes from AFOSA-Animal Welfare and Food Safety GmbH, Blankenfelde-Mahlow, Germany, for generously offering the ELISA kits. This study is a contribution to “Harmonised Approaches in Monitoring Wildlife Population Health, And Ecology and Abundance,” a project of the Coordination of European Research on Emerging and Major Infectious Diseases of Livestock–European Research Area Net.