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On the basis of DNA-DNA hybridization and numerical taxonomy analysis, the mycobacterial species Mycobacterium avium is subdivided into three subspecies, M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum, which share extensive sequence identity (8). Nevertheless, these subspecies can be differentiated from each other on the basis of host range, mycobactin dependence, and the presence of specific insertion elements (17). M. avium subsp. paratuberculosis causes Johne's disease, a severe gastroenteritis in ruminants, with a significant impact on the agricultural economy, particularly the dairy industry (17). In the Belgian cattle population, paratuberculosis prevalence was determined by a serological survey, conducted from December 1997 to March 1998. This approach resulted in an estimated true herd prevalence of M. avium subsp. paratuberculosis infection of 6% (6). Dairy cattle usually start fecal shedding at 2 years of age and develop clinical symptoms around 4 years of age. Infection with M. avium subsp. paratuberculosis is commonly acquired early in life via the fecal-oral route through the ingestion of contaminated colostrum, milk, water, or feed (46) and possibly through intrauterine transmission (42). M. avium subsp. paratuberculosis is extremely robust, and bacteria were reported to survive up to 250 days in water and feces and on pastures (27).Cell-mediated immune responses seem to control the initial infection for a sustained period of time, and clinical symptoms only appear in cows after a number of years, often after the first or second calving, possibly because of enhanced intracellular multiplication of M. avium subsp. paratuberculosis organisms caused by alterations in the hormonal milieu (15). Decreased cell-mediated responses are likely related to a loss of antigen-specific CD4 ϩ T cells, which is most prominent in the ileum lesions from symptomatic animals (24). Also, Khalifeh et al. demonstrated that transforming growth factor ␤ and interleukin-10 (IL-10) mRNA levels are higher in cows that have progressed to the clinical stage of the disease, compared to subclinically infected or healthy cows (23). It is not clear for the moment, whether this reflects a shift from a Th1-to a Th2-biased immune response or rather the development of a regulatory T-cell circuit (10). Vaccines consisting of whole killed or attenuated live M. avium subsp. paratuberculosis bacilli can provide partial protection by delaying fecal shedding and reducing the number of clinically affected animals, but they do not protect against infection. In the context of bovine tuberculosis (M. bovis) control and eradication programs, it is worth mentioning that animals immunized with these paratuberculosis vaccines develop positive reactions in the tuberculin skin test (the reference bovine tuberculosis detection method), and therefore paratuberculosis vaccination is subject to approval by

Persistence of H5N1 high pathogenicity avian influenza virus (HPAIV), isolated during the epidemic in wild birds in Poland in 2006, was evaluated in three water samples derived from the sources known to host wild water birds (city pond, Vistula river mouth, and Baltic Sea). The virus was tested at two concentrations (10(4) and 10(6) median tissue culture infective dose per milliliter) and at three temperatures (4 C, 10 C, and 20 C), representing average seasonal temperatures in Poland. All tested water samples were filtered before virus inoculation, and one unfiltered sample (Baltic seawater) was also tested. Infectivity was determined twice a week over a 60-day trial period by microtiter endpoint titration. The persistence of the virus varied considerably depending on its concentration and also on physico-chemical parameters of the water, such as temperature and salinity. Avian influenza virus survival was the highest at 4 C and the lowest at 20 C. Prolonged infectivity of the virus in Baltic seawater (brackish, 7.8 ppt) was also seen. In distilled water, the virus retained its infectivity beyond the 60-day study period. Interestingly, a devastating effect of the unfiltered fraction of seawater was seen as the virus disappeared in this fraction the quickest in all studied combinations; thus, biologic factors may also affect infectivity of HPAIV.

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In this study, shedding and transmission of three H5/H7 low pathogenic avian influenza viruses (LPAIVs) in poultry was characterized and the impact of floor system on transmission was assessed. Transmission experiments were simultaneously conducted with two groups of animals housed on either a grid or a floor covered with litter. Transmission was observed for H5N2 A/Ch/Belgium/150VB/99 LPAIV. This virus was shed almost exclusively via the oropharynx and no impact of floor system was seen. Transmission was also seen for H7N1 A/Ch/Italy/1067/v99 LPAIV, which was shed via both the oropharynx and cloaca. A slight increase in transmission was seen for animals housed on litter. H5N3 A/Anas Platyrhynchos/Belgium/09-884/2008 LPAIV did not spread to susceptible animals, regardless of the floor system. This study shows that environmental factors such as floor systems used in poultry barns may act upon the transmission of LPAIVs. However, the level of influence depends on the virus under consideration and, more specifically, its principal replication sites.

Severe emaciation and mortalities suggestive of mycobacterial infections were recently reported for both adult and young wild red deer (Cervus elaphus) in the southeastern part of Belgium. In deer, tuberculous lesions are not pathognomonic of Mycobacterium bovis infection due to gross and microscopic similarities with lesions caused by Mycobacterium avium subsp. paratuberculosis or M. avium subsp. avium. The aim of this study was to improve molecular methods for the species-specific identification of M. bovis, M. avium subsp. avium, and M. avium subsp. paratuberculosis in mycobacterial infections of deer. DNA banding patterns were assessed prior to and after Hpy188I restriction of f57-upstream (us)-p34 duplex amplicons. The duplex f57-us-p34 PCR differentiated M. bovis from M. avium subsp. paratuberculosis and M. avium subsp. avium infections, whereas the restriction step differentiated single M. avium subsp. paratuberculosis or M. avium subsp. avium infections from mixed M. avium subsp. paratuberculosis/M. avium subsp. avium infections. The endonuclease Hpy188I cleaves DNA between nucleotides N and G in the unique TCNGA sequence. This restriction site was found at position 168 upstream of the us-p34 initiation codon in all M. avium subsp. avium strains tested, regardless of their origin and the results of IS901 PCR. In contrast, the restriction site was abrogated in all M. avium subsp. paratuberculosis strains tested, independent of their origin, Mycobactin J dependency, and IS900 PCR results. Consequently, a two-step strategy, i.e., duplex us-p34-f57 PCR and Hpy188I restriction, allowed us to exclude M. bovis infection and to identify single (M. avium subsp. paratuberculosis or M. avium subsp. avium) or mixed (M. avium subsp. paratuberculosis/M. avium subsp. avium) infections in wild red deer in Belgium. Accordingly, we propose to integrate, in a functional molecular definition of M. avium subsp. paratuberculosis, the absence of the Hpy188I restriction site from the us-p34 amplicon.The most significant mycobacterial diseases of free-living, captive, and farmed red deer (Cervus elaphus) are bovine tuberculosis, caused by Mycobacterium bovis, Johne's disease (paratuberculosis), caused by Mycobacterium avium subsp. paratuberculosis, and avian tuberculosis, caused principally by M. avium subsp. avium (20).Differential diagnoses of infections by M. bovis, M. avium subsp. paratuberculosis, and M. avium subsp. avium are essential because the epidemiology, zoonotic concern, and prevention and control methods are different for each of these mycobacterial infections (20). In deer, tuberculous lesions are not pathognomonic of M. bovis infection and are actually indistinguishable from lesions due to M. avium subsp. paratuberculosis or M. avium subsp. avium infection because of their gross and microscopic similarities. Moreover, the actual differential diagnosis of these mycobacterial infections is difficult due to similarities in the clinical expression and pathology associated with these infections. Antigenic similarity ...

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Due to their probable role in the spread of Asian highly pathogenic avian influenza (HPAI) H5N1 virus, and in order to explore its implication in the low pathogenic avian influenza (LPAI) virus epidemiology, mute swans represent one particular wild bird species specifically targeted in the avian influenza (AI) surveillance elaborated in Belgium. A total of 640 individual mute swans have been sampled during a 4-yr AI surveillance program (2007-2010) to determine the AI seroprevalence and viroprevalence in this species; all were analyzed through age, temporal, and habitat (flowing and stagnant water) factors. Using a nucleoprotein (NP)-based ELISA, a global antibody prevalence of 35% has been found and was characterized by two peaks in the winter and the summer that might be indicative of a greater LPAI virus circulation in the autumn than in the spring. A significantly higher antibody prevalence was detected in adult swans (53.8%) as compared to juveniles (15.5%). In contrast, a low prevalence of infection (2.7%) was found, mainly in juvenile mute swans and only during the autumn migration period. Interestingly, an impact of water habitat was observed based on the comparison of the antibody prevalence and prevalence of infection from swan populations living on stagnant water vs. flowing water, suggesting that stagnant water provides a more-favorable environment for LPAI persistence and transmission.