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.

Two enzyme-linked immunosorbent assays (ELISAs) for the detecting Salmonella enterica subsp. enterica serovar Dublin antibodies in bulk milk were developed and evaluated for potential use in control programs. The ELISAs were based on either lipopolysacharide (LPS ELISA) or flagellar antigen (GP ELISA). Sensitivity was determined with 79 case herds with a wide range of clinical signs. Specificity was determined with 125 Dutch and 200 Swedish control herds. The relation between antibodies in bulk milk, antibodies in serum, and the level of milk production of individual cows was studied with 61 case herds. The optimal optical density (OD) values of the LPS ELISA and the GP ELISA were determined to be 0.2 and 0.5, respectively. The sensitivities of the LPS ELISA and the GP ELISA were 54 and 63%, respectively, with a specificity of 98% for both ELISAs with samples from the Dutch control herds. The specificities for samples from the Swedish herds were 100% for the LPS ELISA and 95% for the GP ELISA. The sensitivity of the combination of tests was 65% when samples were run in parallel, and the specificity was 100% when samples were run in series, irrespective of whether the samples came from Dutch or Swedish control herds. The variance (R 2 ) in the OD value for bulk milk samples could be explained by the percentage of seropositive lactating cows in a herd with the LPS ELISA for 51% of the samples and with the GP ELISA for 72%. The variance in the OD value was best explained by the combination of the percentage of seropositive lactating cows in the herd and the mean log 10 serum antibody titer for that herd (R 2 ‫؍‬ 62% for the LPS ELISA and R 2 ‫؍‬ 75% for the GP ELISA). Case herds more often tested negative by the ELISA with bulk milk when the percentage of seropositive lactating cows was less than 5%. It is concluded that both ELISAs with bulk milk can be used in control programs to distinguish between infected and noninfected herds. Specificity can be increased by using the two tests in combination. Sensitivity was relatively low for both single tests and both tests combined.

The program for the eradication of SalmoneUa enteritidis from chickens in The Netherlands is based on bacteriological examination of breeding flocks. There is a great need for a specific and sensitive serological screening test. For that purpose, we developed four different enzyme-linked immunosorbent assays (ELISAs), i.e., an indirect ELISA with S. enteritidis flagellin, an indirect ELISA with S. enteritidis lipopolysaccharide, a double-antibody sandwich blocking ELISA that uses monoclonal antibodies against S. enteritidis flagellin (GM-DAS blocking ELISA), and a double-antibody sandwich ELISA that uses monoclonal antibodies against S. enteritidis lipopolysaccharide. In the present study, we compare the results of those ELISAs with sera from experimentally infected 1-day-old chickens and with sera and eggs from experimentally infected laying hens. Experimental infections were induced with strains of S. enteritidis phage types 1 and 2, S. typhimurium, and S. panama. Sera were collected up to days 44 and 39 after infection from 1-day-old chickens and laying hens, respectively. Only the GM-DAS blocking ELISA was able to discriminate between S. enteritidis infections and infections with the other serotypes. This ELISA had both a sensitivity and a specificity of 100%/ for all serum samples from experimentally infected chickens. A field study is in progress to evaluate whether this test can be

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A 18-year-old Dutch Warmblood mare was referred for colic. Upon arrival, lethargy, blindness, head pressing, ataxia, and circling were the main clinical signs. On rectal examination a hard mass and oedema around the cranial mesenteric artery were palpated. Plasma liver enzyme activities and the ammonia level were elevated. Atrial fibrillation with a pulse frequency of 36-52 beats per minute was noticed. On both sides a holosystolic murmer with the maximum intensity on the right side could be auscultated. Postmortem examination revealed eccentric hypertrophy of the right atrium and a pale spotted myocardium, most prominently of the right ventricle, with secondary venous congestion of the azygos and mesenteric veins. The liver changes were indicative of chronic congestion. Despite the normal pulse rate, it appeared that congestive heart failure due to cardiomyopathy, was responsible for the presenting symptoms of this patient.