Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy

We previously demonstrated that oral application of the recombinant single-domain antibody fragment (VHH) clone K609, directed against Escherichia coli F4 fimbriae, reduced E. coli-induced diarrhoea in piglets, but only at high VHH doses. We have now shown that a large portion of the orally applied K609 VHH is proteolytically degraded in the stomach. Stringent selection for proteolytic stability identified seven VHHs with 7- to 138-fold increased stability after in vitro incubation in gastric fluid. By DNA shuffling we obtained four clones with a further 1.5- to 3-fold increased in vitro stability. These VHHs differed by at most ten amino acid residues from each other and K609 that were scattered over the VHH sequence and did not overlap with predicted protease cleavage sites. The most stable clone, K922, retained 41% activity after incubation in gastric fluid and 90% in jejunal fluid. Oral application of K922 to piglets confirmed its improved proteolytic stability. In addition, K922 bound to F4 fimbriae with higher affinity and inhibited fimbrial adhesion at lower VHH concentrations. K922 is thus a promising candidate for prevention of piglet diarrhoea. Furthermore, our findings could guide selection and improvement by genetic engineering of other recombinant antibody fragments for oral use

Prevalence of <i>Mycobacterium avium</i> in Slaughter Pigs Based on Serological Monitoring Results and Bacteriological Validation

Mycobacterium avium (MA) is a potential food safety hazard in pigs. Blood samples of slaughtered pigs in the Netherlands and Germany were tested for the presence of MA antibodies to estimate the serological prevalence in the tested population. In the Dutch and German population 1.0% and 1.7% samples were positive, and 0.5% and 17.4% of the herds were at risk for having a MA infection respectively. The validity of the applied MA-ELISA was evaluated under field conditions. The specificity of the MA-ELISA was high (>98.4%). The average herd sensitivity was 18%. In the affected herds on average 50% of the animals were tested bacteriological positive for MA. It can be concluded that serological screening for the presence of MA antibodies is capable of identifying pig populations that are at risk for a MA infection

Isolation of mycobacteria other than<i> Mycobacterium avium</i> from porcine lymph nodes

Mycobacterium avium causes lymphadenitis in pigs. This presents an economical burden, as these pigs meat is considered inappropriate for consumption. In humans, lymphadenitis due to nontuberculous mycobacteria (NTM) primarily affects children and is caused by a variety of NTM, though M. avium predominates. Mycobacterial culture was undertaken on lymph nodes of 107 slaughter pigs from a single pig farm. A high number of pigs with mycobacterial lymphadenitis were identified by culture. A commercial line probe assay and 16S rDNA gene sequencing were used to assess the frequency of disease due to mycobacteria other than M. avium. Forty-five pigs had mandibular lymph node samples yielding mycobacteria in culture. The majority yielded M. avium (39; 87%) only. One yielded M. avium and Mycobacterium palustre, five yielded only NTM other than M. avium (2 yielded Mycobacterium malmoense, 1 Mycobacterium bohemicum, 1 Mycobacterium heckeshornense and a possibly novel species related to Mycobacterium scrofulaceum, and 1 grew a possibly novel species related to M. palustre).Several NTM species other than M. avium were cultured from porcine lymph nodes. The species distribution shows interesting parallels with human NTM lymphadenitis. Molecular typing and environmental sampling studies are required to identify the sources of these infections

Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy

We previously demonstrated that oral application of the recombinant single-domain antibody fragment (VHH) clone K609, directed against Escherichia coli F4 fimbriae, reduced E. coli-induced diarrhoea in piglets, but only at high VHH doses. We have now shown that a large portion of the orally applied K609 VHH is proteolytically degraded in the stomach. Stringent selection for proteolytic stability identified seven VHHs with 7- to 138-fold increased stability after in vitro incubation in gastric fluid. By DNA shuffling we obtained four clones with a further 1.5- to 3-fold increased in vitro stability. These VHHs differed by at most ten amino acid residues from each other and K609 that were scattered over the VHH sequence and did not overlap with predicted protease cleavage sites. The most stable clone, K922, retained 41% activity after incubation in gastric fluid and 90% in jejunal fluid. Oral application of K922 to piglets confirmed its improved proteolytic stability. In addition, K922 bound to F4 fimbriae with higher affinity and inhibited fimbrial adhesion at lower VHH concentrations. K922 is thus a promising candidate for prevention of piglet diarrhoea. Furthermore, our findings could guide selection and improvement by genetic engineering of other recombinant antibody fragments for oral use