Similar antibody response observed in single-dose-vaccination vs. revaccination against Aujeszky's disease in wild boar.

Wild boar is an important reservoir of Aujeszky's disease virus (ADV). There is concern that transmission of this virus from wild boar to domestic pigs is possible. The aim of this work was to compare the antibody response produced by single dose of a gE-deleted ADV vaccine in wild boar to revaccinated animals, to assess if simple single-dose vaccination plans should be examined as a possible control measure against ADV in wild boar. Twenty-five wild boar (ages ranging between 2.5 to 5 months) were included in this study and distributed in three different groups: a control group (n = 5), a single-dose group (10 animals vaccinated only with one dose (day 0)) and a revaccinated group [10 animals vaccinated (day 0) and revaccinated (day 28)]. Mean antibody titers against ADV were determined in three groups using an ELISA assay at three different time points [day 0 (pre-vaccination), 28 (post 1st dose) and 56 (post 2nd dose)]. At day 28, single-dose and revaccinated groups showed a significant increment of antibody titers whereas antibodies in the control group remained stable. At day 56, revaccinated animals did not show a significant increment and antibody titers were similar to those found in animals vaccinated with one dose. These results indicate that vaccination with one dose produces a similar early antibody response to revaccination and therefore, should be examined as a possible control measure against ADV in wild boar. Keywords: Aujeszky's disease; serology; vaccination; wild boar.

Increasing isolation of rapidly growing mycobacteria in a low-incidence setting of environmental mycobacteria, 1994-2015.

To determine trends in incidence and clinical relevance of rapidly growing mycobacteria (RGM) in a low-prevalence region of non-tuberculous mycobacteria. We retrospectively identified all patients with RGM-positive cultures between January 1994 and December 2015. Trends in incidence, clinical significance, and outcomes were assessed. One hundred and forty patients had RGM-positive cultures (116 respiratory and 24 extra-respiratory sources). The incidence of RGM isolates increased steadily from 2003 (0.34 per 100,000) to 2015 (1.73 per 100,000), with an average annual increase of 8.3%. Thirty-two patients (22.9%) had clinical disease, which trended to cluster in the second half of the study period. A positive acid-fast bacilli smear (odds ratio [OR] 97.7, 95 % CI 13.8-689.4), the presence of extra-respiratory isolates (OR 19.4, 95 % CI 5.2-72.7), and female gender (OR 5.9, 95 % CI 1.9-19.1) were independently associated with clinical disease. Cure rates were 73.3 and 87.5% for pulmonary and extra-pulmonary disease respectively. Although the burden of disease remains low, the presence of RGM isolates is increasing in our geographical setting. Whether this rise will be sustained over time and will coincide with an increase in clinical disease, or whether it is merely a cycle in the poorly understood epidemiological behaviour of environmental mycobacteria, will be seen in the near future.

Oral supplementation with postbiotics modulates the immune response produced by myxomatosis vaccination in wild rabbits.

Rabbits (Oryctolagus cuniculus) are vitally important species in the Iberian Peninsula ecosystem. However, since 1950, there has been a significant population decline, with major repercussions. This situation is mainly due to the presence of infectious diseases, such as myxomatosis, which is expanding and is characterized by severe and fatal clinical manifestations. Current control measures, mainly those based on vaccinations, are ineffective. Therefore, new strategies need to be developed and implemented. This study aimed to evaluate whether supplementation with postbiotic products modulates the immune response in wild rabbits vaccinated against myxomatosis. For this purpose, two groups of rabbits were established: a control group fed with standard feed ad libitum from weaning (28 days) until two months of age, and a treated group, which was fed under the same conditions but supplemented with postbiotics (3 kg/Tm). All the studied rabbits were vaccinated against this disease during weaning. In addition, a blood samples were obtained from all animals immediately before vaccination and 30 days later, which allowed us to evaluate the level of antibodies against myxomatosis virus (ELISA detection) and the relative expression of gene encoding to cytokines related to the immune response (IL6, TNFα and IFNγ), at both times of the experience. Weight and length measurements were also taken at both times to calculate body index and mean daily gain (MDG). No statistically significant differences in growth parameters were observed. There were also no differences in the serological response among groups. However, a relative underexpression of gene codifying to TNFα (p-value = 0.03683) and a higher expression on IFNγ (p-value = 0.045) were observed in the treated group. This modulation in cytokines could lead to less severe lesions in wild rabbit naturally infected with myxomatosis virus.

Impact of updating the MALDI-TOF MS database on the identification of nontuberculous mycobacteria.

Conventional identification of mycobacteria species is slow, laborious and has low discriminatory power. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proved highly effective for identifying conventional bacteria, and it may also be useful for identifying mycobacteria. The aim of this study was to evaluate and compare MALDI-TOF MS with currently recommended molecular methods for the identification of nontuberculous mycobacteria (NTM), applying Mycobacteria Libraries v3.0 (ML3.0) and v2.0 (ML2.0). A total of 240 clinical isolates of 41 NTM species grown on solid media were analysed: 132 isolates of slow-growing mycobacteria and 108 of rapid-growing mycobacteria. MALDI-TOF MS, using ML3.0, identified 192 (80%) NTM isolates with a score ≥1.7, encompassing 35 (85.4%) different species, that is, 17 (7.1%; p = 0.0863) isolates and 15 (36.6%; p = 0.0339) species more than currently recommended molecular techniques (polymerase chain reaction reverse hybridization). All these isolates were correctly identified according to molecular identification methods. The application of ML3.0 also identified 15 (6.2%) NTM isolates more than ML2.0 (p < 0.01). The scores obtained with MALDI-TOF MS using ML3.0 (mean score: 1.960) were higher in 147 (61.2%) isolates than when using ML2.0 (mean score: 1.797; p < 0.01). Three of the species analysed were not included in either database, so they were not recognized by this system. In conclusion, MALDI-TOF MS identified more isolates and species than the recommended polymerase chain reaction reverse hybridization assays. Although the new ML3.0 is not the definitive database, it yielded better results than ML2.0. This shows that the updating of the MALDI-TOF MS database plays an essential role in mycobacterial identification. Copyright © 2017 John Wiley & Sons, Ltd.

Association between vitamin D supplementation and severity of tuberculosis in wild boar and red deer.

Tuberculosis (TB) is a chronic disease affecting humans and other mammal species. Severity of TB caused by Mycobacterium tuberculosis in humans seems to be influenced by nutritional factors like vitamin D3 intake. However, this relationship has been scarcely studied in cattle and other mammals infected with Mycobacterium bovis. The aim of this work was to assess if wildlife reservoirs of M. bovis show different levels of TB severity depending on the level of vitamin D found in serum after supplementation with vitamin D3. Forty hunted wildlife mammals were included in this study: 20 wild boar and 20 red deer. Ten wild boar and ten red deer had been supplemented with a vitamin D3-enriched food, whereas the remaining animals had received no supplementation. TB diagnosis was carried out in each animal based on microbiological isolation of M. bovis. Animals infected with M. bovis were then classified as animals with localized or generalized TB depending on the location and dissemination of the lesions. Furthermore, serum levels of vitamin D2 and D3 were determined in each animal to evaluate differences not only between supplemented and non-supplemented animals but also between those with localized and generalized TB. Levels of vitamin D3 found in both, supplemented wild boar and red deer, were significantly higher than those found in the non-supplemented animals. Interestingly, higher levels of vitamin D3 were observed in animals suffering localized TB when compared to animals with generalized TB suggesting that vitamin D3 concentration correlates negatively with TB severity in these wildlife reservoirs.

Non-tuberculous mycobacteria in wild boar (Sus scrofa) from Southern Spain: epidemiological, clinical and diagnostic concerns.

Non-tuberculous mycobacteria (NTM) are widely distributed in the environment, particularly in wet soil, marshland, rivers or streams, but also are causative agents of a wide variety of infections in animals and humans. Little information is available regarding the NTM prevalence in wildlife and their effects or significance in the bovine tuberculosis (bTB) epidemiology and diagnosis. This research shows the most frequently NTM isolated in lymph nodes of wild boar (Sus scrofa) from southern Spain, relating the NTM presence with the individual characteristics, the management of animals and the possible misdiagnosis of Mycobacterium bovis in concurrent infections. A total of 219 NTM isolates were obtained from 1249 wild boar mandibular lymph nodes sampled between 2007 and 2011. All but 75 isolates were identified by the PCR-restriction analysis-hsp65, and a partial sequencing of the 16S rDNA was carried out to identify the rest of the isolates. Results showed that Mycobacterium chelonae was the most frequently isolated NTM specie (133 isolates, 60.7%), followed by Mycobacterium avium (24 isolates, 11%). No relation was found regarding sex, body condition and management, but M. chelonae was more frequently detected in adults, whereas M. avium was more prevalent in subadults. The high NTM prevalence observed in the studied wild boar populations could make difficult the bTB diagnostic.

High-density dependence but low impact on selected reproduction parameters of Brucella suis biovar 2 in wild boar hunting estates from South-Western Spain.

Porcine brucellosis is a disease caused by Brucella suis, which is characterized by reproductive disorders in pigs. The number of cases of swine brucellosis has risen in many European countries, likely because of the presence of a wild reservoir of B. suis in wild boar. This study aimed at evaluating factors that may influence the probability of infection with Brucella spp. in wild boar and at assessing the impact of a previous contact with Brucella spp. on reproductive parameters of wild boar. Two hundred and four wild boar living in Extremadura (south-western Spain) were studied. The presence of anti-Brucella antibodies was determined using an indirect ELISA, while the presence of living bacteria in genital organs was evaluated through microbiological cultures. Sex, age, density of wild boar in summer and presence of outdoor pigs were selected as possible risk factors for being seropositive for Brucella spp. in wild boar. In addition, reproductive parameters such as breeding status or potential fertility in females and testis weight in males were estimated and related to the presence of anti-Brucella antibodies. A total of 121 animals were seropositive, resulting in a prevalence of 59.3% (95% CI). In addition, seven isolates of B. suis biovar 2 were obtained. Wild boar density in summer, as well as age and sex, was proposed as factors to explain the probability of Brucella seroconversion, although wild boar density in summer was the key factor. Current measures of reproductive parameters were not influenced by a previous contact with Brucella spp. Isolation of B. suis confirms that wild boar could represent a risk to domestic pig health in the study area. Wild boar density seems to have a great influence in the probability of infections with B. suis and suggests that density management could be useful to control Brucella infection in wild boar.

Immunopathology of granulomas produced by Mycobacterium bovis in naturally infected wild boar.

Limited information has been published on the wild boar immune response against bovine tuberculosis (bTB) and the immunopathogenesis of the pathological hallmark (granuloma) in this species. The main objectives of this study were, on the one hand, to characterize the histopathological features (number of acid-fast bacilli (AFB) and multinucleated giant cells (MNGCs) and the immunohistochemical distribution of different cell subsets (CD3+, CD79a+ and MAC387+) and chemical mediators (iNOS and IFN-γ) in the different developmental stages of granulomas produced by the natural infection of Mycobacterium bovis (M. bovis) in wild boar. On the other hand, the study also aimed to analyze the mechanisms underlying the marked differences in the typical lesional patterns observed in M. bovis infections of wild boar (contained, not generalized) and those previously described in fallow deer (poorly contained, generalized). The majority of granulomas analyzed (95.3%) did not show any AFB with the ZN stain and a low number of MNGCs were identified in the different granuloma stages. The immunohistochemical analysis showed that MAC387 was the only immune marker that produced decreasing positivity by granuloma stage, being statistically significantly lower in stages III and IV when compared to stage I and II. Immune markers for lymphocyte cells (CD3 and CD79a) showed a slight rise in the positivity (which was not statistically significant) in the advanced granuloma stages. In keeping with the presence of large numbers of T cells and macrophages, there was a consistently high level of expression of IFN-γ at all stages of granuloma development without a statistical significant decrease in advanced stages. Also related with the higher presence of macrophages in stage I and II, the expression of iNOS was higher in early stages and sustained until stage III, showing a non statistical significant decrease in stage IV. The macrophage and iNOS activity are more intense and sustained along the granuloma development than those described in fallow deer. Immunohistochemical protocols with a panel of markers for wild boar different cells subsets (CD3+, CD79a+ and MAC387+) and chemical mediators (iNOS and IFN-γ), and their use to further investigate the immune response in this species are provided.

Influence of porcine circovirus type 2 infections on bovine tuberculosis in wild boar populations.

The wild boar is an important reservoir of bovine tuberculosis (bTB) in south-western Spain. Some risk factors such as wild boar density or age have been associated with the presence of high prevalences of bTB in wild boar. However, the influence of other risk factors such as co-infections with other pathogens has not yet been studied. This work aims to assess the influence of porcine circovirus type 2 (PCV-2) infection on bTB prevalence and bTB lesional patterns observed in wild boar. The presence of bTB-like lesions was evaluated in 551 hunted wild boar from 11 different game estates in south-western Spain, with a known history of bTB. Tuberculosis prevalences in each estate were calculated based on the percentage of animals found with bTB-like lesions. The percentage of animals with generalized bTB lesional patterns (bTB lesions in more than one organ) was also assessed. The prevalence of PCV-2 was studied in each estate using a specific PCR assay. The relationship between PCV-2 and bTB prevalences and between PCV-2 infections and the presence of generalized lesional patterns in wild boar were analysed. A statistical relationship between the prevalences of bTB and PCV-2 was found, with bTB prevalences being higher in estates where prevalences of PCV-2 were high. On the other hand, animals infected with PCV-2 were more likely to develop a generalized lesional pattern. Porcine circovirus type 2 prevalences seem to be associated with prevalences of bTB in wild boar. PCV-2 infection may aggravate the development and severity of bTB, favouring the presence of generalized lesional patterns and raising the risk of contagion in these estates. The implementation of sanitary measures that focus on the control of PCV-2 infection may be necessary as a preliminary measure in bTB control programmes for wild boar.

Reducing Eurasian wild boar (Sus scrofa) population density as a measure for bovine tuberculosis control: effects in wild boar and a sympatric fallow deer (Dama dama) population in Central Spain.

Research on management of bovine tuberculosis (bTB) in wildlife reservoir hosts is crucial for the implementation of effective disease control measures and the generation of practical bTB management recommendations. Among the management methods carried out on wild species to reduce bTB prevalence, the control of population density has been frequently used, with hunting pressure a practical strategy to reduce bTB prevalence. However, despite the number of articles about population density control in different bTB wildlife reservoirs, there is little information regarding the application of such measures on the Eurasian wild boar (Sus scrofa), which is considered the main bTB wildlife reservoir within Mediterranean ecosystems. This study shows the effects of a management measure leading to a radical decrease in wild boar population density at a large hunting estate in Central Spain, in order to assess the evolution of bTB prevalence in both the wild boar population and the sympatric fallow deer population. The evolution of bTB prevalence was monitored in populations of the two wild ungulate species over a 5-year study period (2007-2012). The results showed that bTB prevalence decreased in fallow deer, corresponding to an important reduction in the wild boar population. However, this decrease was not homogeneous: in the last season of study there was an increase in bTB-infected male animals. Moreover, bTB prevalence remained high in the remnant wild boar population.

Comparative pathology of the natural infections by Mycobacterium bovis and by Mycobacterium caprae in wild boar (Sus scrofa).

The potential role of wild animals in the maintenance and spread of tuberculosis (TB) infection in domestic livestock is of particular importance in countries where eradication programs have substantially reduced the incidence of bovine tuberculosis but sporadic outbreaks still occur. Mycobacterium bovis is the agent mainly isolated in wildlife in Spain, but recently, infections by Mycobacterium caprae have increased substantially. In this study, we have analysed 43 mandibular lymph nodes samples containing TB-like lesions from 43 hunted wild boar from Madrid and Extremadura (central and south-western regions of Spain). After isolation, identification and typing of Mycobacterium tuberculosis complex isolates, we found that 23 mandibular lymph nodes involved M. caprae infections and 20 M. bovis. The lesions were compared for histopathology (different granuloma stage and number of multinucleated giant cells (MNGCs)), and acid-fast bacilli (AFBs) were quantified in the Ziehl-Neelsen-stained slides. Granulomas produced by M. caprae showed more stage IV granulomas, more MNGCs and higher AFBs counts than those induced by M. bovis. In conclusion, lesions caused by M. caprae would be more prone to the excretion of bacilli, and infected animals result as a high-risk source of infection for other animals.

Histological and immunohistochemical characterisation of Mycobacterium bovis induced granulomas in naturally infected fallow deer (Dama dama).

Mycobacterium bovis infections in fallow deer have been reported in different countries and play an important role in the epidemiology of bovine tuberculosis (bTB), together with other deer species. There is little knowledge of the pathogenesis of bTB in fallow deer. The aim of this study was to perform a histopathological characterisation of the granulomas induced by M. bovis in this species and the immunohistochemical distribution of different cell subsets (CD3+, CD79+, macrophages) and chemical mediators (iNOS, TNF-α, IFN-γ) in the different developmental stages of granulomas. Stage I/II granulomas showed a marked presence of macrophages (MAC387+) expressing high iNOS levels while stage III/IV granulomas showed a decrease in the number of these cells forming a rim surrounding the necrotic foci. This was correlated with the presence of IFN-γ expressing cell counts, much higher in stage I/II than in stage III/IV. The number of B cells increased alongside the developmental stage of the granuloma, and interestingly the expression of TNF-α was very low in all the stages. This characterisation of the lesions and the local immune response may be helpful as basic knowledge in the attempts to increase the vaccine efficacy as well as for disease severity evaluation and for the development of improved diagnostic tools. Immunohistochemical methods using several commercial antibodies in fallow deer tissues are described.

Outbreak of swine erysipelas in a semi-intensive wild boar farm in Spain.

Swine erysipelas (SE) is a disease caused by the bacterium Erysipelothrix rhusiopathiae and is one of the best-known and most serious diseases affecting domestic pigs. However, few studies exist concerning the susceptibility of wild boars to this disease and the role of this species as a reservoir. This study investigates and describes an outbreak of SE that occurred on a semi-intensive wild boar breeding farm housing 40 boars in Extremadura (SW Spain) on 11-18 February 2010. Seven animals died, of which four were examined post-mortem. Of these, three (two females and one male) were approximately 3 months old, and one was 1 year old (male). Lesions were consistent with acute septicaemia, consisting of cutaneous erythema/cyanosis and petechial haemorrhages in kidneys, urinary bladder, lungs and meninges. The 1-year-old male also had proliferative polyarthritis. Histopathology confirmed the presence of disseminated intravascular coagulation and vasculitis. Additionally, a bilateral acute panuveitis with concurrent necrotizing vasculitis and diffuse corneal oedema, neither of which have been described before in this disease, were found in the 3-month-old male boar. E. rhusiopathiae was isolated from all four animals in pure cultures from several tissues. Of these four animals, antibodies against E. rhusiopathiae, using an indirect ELISA test, were only detected in the 1-year-old male boar with polyarthritis. Posteriorly, of nine live adults tested for antibodies, four (including an adult male with polyarthritis) were positive.