F.G. van Zijderveld

The Q fever epidemic in The Netherlands: history, onset, response and reflection

The 2007-2009 human Q fever epidemic in The Netherlands attracted attention due to its magnitude and duration. The current epidemic and the historical background of Q fever in The Netherlands are reviewed according to national and international publications. Seroprevalence studies suggest that Q fever was endemic in The Netherlands several decades before the disease was diagnosed in dairy goats and dairy sheep. This was in 2005 and the increase in humans started in 2007. Q fever abortions were registered on 30 dairy goat and dairy sheep farms between 2005 and 2009. A total of 3523 human cases were notified between 2007 and 2009. Proximity to aborting small ruminants and high numbers of susceptible humans are probably the main causes of the human Q fever outbreak in The Netherlands. In general good monitoring and surveillance systems are necessary to assess the real magnitude of Q fever.

Early and late pathogenesis of natural scrapie infection in sheep.

The pathogenesis of scrapie infection was studied in sheep carrying the PrPVRQ/PrPVRQ genotype, which is associated with a high susceptibility for natural scrapie. The sheep were killed at sequential time points during a scrapie infection covering both the early and late stages of scrapie pathogenesis. Various lymphoid and neural tissues were collected and immunohistochemically examined for the presence of the scrapie‐associated prion protein PrPSc, a marker for scrapie infectivity. The first stage of scrapie infection consisted of invasion of the palatine tonsil and Peyers patches of the caudal jejunum and ileum, the so‐called gut‐associated lymphoid tissues (GALT). At the same time, PrPSc was detected in the medial retropharyngeal lymph nodes draining the palatine tonsil and the mesenteric lymph nodes draining the jejunal and ileal Peyers patches. From these initial sites of scrapie replication, the scrapie agent disseminated to other non‐GALT‐related lymphoid tissues. Neuroinvasion started in the enteric nervous system followed by retrograde spread of the scrapie agent via efferent parasympathetic and sympathetic nerve fibres innervating the gut, to the dorsal motor nucleus of the vagus in the medulla oblongata and the intermediolateral column of the thoracic spinal cord segments T8–T10, respectively.

Discrimination between Scrapie and Bovine Spongiform Encephalopathy in Sheep by Molecular Size, Immunoreactivity, and Glycoprofile of Prion Protein

ABSTRACT A procedure for discrimination between scrapie and bovine spongiform encephalopathy (BSE) in sheep is of importance for establishing whether BSE has entered the sheep population. Since BSE has not yet been found in sheep at the farm level, such discrimination procedures can be developed only with experimental sheep BSE. Two distinctive molecular features of the prion protein (PrP)—molecular size and glycosylation profile—in proteinase K digests of brain stem tissue from sheep were used here; upon Western blotting, these features led to an unequivocal discrimination among natural scrapie, experimental scrapie, and experimental BSE. The higher electrophoretic mobility of PrP in sheep BSE could be best observed after deglycosylation treatment with N-glycosidase F. A simpler method for confirmation of this size difference involved comparison of the ratios for the binding of two monoclonal antibodies: P4 and 66.94b4. Based on epitope mapping studies with P4 and peptides, it appeared that N-terminal amino acid sequence WGQGGSH was intact only in sheep scrapie digests. Another feature typical for PrP in sheep BSE was the large fraction of diglycosylated PrP (70% or more). These data were obtained for a large group of positive sheep, consisting of 7 sheep with experimental BSE infection (genotypes: six ARQ/ARQ and one AHQ/AHQ), 48 sheep naturally infected with scrapie (six different genotypes), and 3 sheep with primary experimental scrapie infection. Routine tests of slaughter material serve well for the initial detection of both BSE and scrapie. With Western blotting as a rapid follow-up test, a 66.94b4/P4 antibody binding ratio above 1.5 is a practical indicator for serious suspicion of BSE infection in sheep.

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.

A long-term perfusion test to measure net absorption in the small intestine of weaned pigs

To study the effects of bacteria on net absorption of fluid and electrolytes in the small intestine of newly weaned pigs, a more comprehensive and ethical alternative to the ligated loop test was developed. Five paired segments, located at 10, 25, 50, 75 and 95 per cent sites along the small intestine, were cannulated at both ends and solutions perfused continuously through the segments for 10 hours. Net absorption was determined by both a volume method and a method using a non-absorbable marker. Net absorption of fluid, sodium, potassium and chloride was significantly less in segments infected with an enterotoxigenic Escherichia coli than in control segments. This method was superior to the ligated loop test because (i) it was performed entirely under anaesthesia, (ii) the small intestine did not distend during a test, (iii) net absorption was determined per cm2 and along the whole length of the small intestine. Net absorption determined by the nonabsorbable marker was significantly less than that determined by the volume method.

Effects of weaning and enterotoxigenic Escherichia coli on net absorption in the small intestine of pigs

The purpose of this study was to measure the net absorption of fluid, sodium, potassium and chloride in the small intestine of weaned pigs and of their unweaned littermates and to correlate these values with villus height and crypt depth. Five pairs of segments of the small intestine were prepared in each of 80 pigs; the cranial segment of each pair was injected with an enterotoxigenic strain of Escherichia coli and the caudal segment with a control solution. Net absorption was measured on the day of weaning and four, seven, 11 and 14 days after weaning. In unweaned pigs the net absorption of fluid, potassium and chloride did not vary with time. In weaned pigs the net absorption of fluid in the control segments was significantly less on days 4, 7 and 14 after weaning and of sodium and chloride on days 4 and 7 than in unweaned littermates. In infected segments of weaned pigs the net absorption of fluid was significantly less than in unweaned littermates on day 11 and 14, of sodium and potassium on day 11 and of chloride on days 4 and 11 after weaning. Net absorption was negatively correlated with villus height but only in the infected segments of weaned pigs; no other correlations were found. It was concluded that after weaning the net absorption of fluid and electrolytes in the small intestine of pigs is temporarily decreased, a condition that may initiate diarrhoea.

Clinical and microbiological field studies in the Netherlands of diarrhoea in pigs at weaning

A longitudinal study investigated the clinical signs and aspects of the aetiology of diarrhoea in pigs at weaning. Two litters of pigs were randomly selected from each of four Dutch herds with a history of diarrhoea after weaning. The pigs were inspected and faecal samples were collected daily. Before weaning, enterotoxigenic Escherichia coli was isolated from only two pigs but after weaning from all the pigs. Before and after weaning, rotaviruses were detected in almost all the pigs during one to four episodes. Rotavirus or enterotoxigenic E coli were generally detected when the pigs had diarrhoea; however, they were also encountered in normal faeces. In many faecal samples from pigs with diarrhoea no pathogenic agent was detected. In almost all the pigs after weaning, E coli types appeared and predominated transiently before they were superseded by another type. This study confirms the results of others in showing that rotaviruses and E coli are important in the aetiology of diarrhoea in pigs at weaning. However, diarrhoea after weaning is probably not caused by these two agents alone; other factors also probably contribute to the syndrome.

Paratuberculosis recognized as a problem at last: a review.

Summary This article attempts to review briefly current opinions on Johnes disease, or paratuberculosis, in ruminants caused by Mycobacterium avium subsp. paratuberculosis. Paratuberculosis has been known to be prevalent in domestic livestock, such as cattle, goats, and sheep, for more than a century. Despite this knowledge only minor efforts have been made to control the disease and, with the attention being focussed on the eradication of other diseases, the problem of paratuberculosis has been neglected in most countries in the past decades. However, recent epidemiological surveys performed in Europe showed a high prevalence of paratuberculosis in cattle and sheep, indicating that the situation has become quite alarming. In addition, the possible role of M. avium subsp. Paratuberculosis in the aetiology of Crohns disease in humans is still debated, as discussed in this article. Therefore, there is suddenly renewed interest in paratuberculosis, and the disease is recognized as a significant problem. As a consequence, there is a need for reliable diagnostic tools for large‐scale use to allow the introduction of programmes to control and eventually eradicate the disease. The current status and the possibilities for such programmes are discussed.

Proteinase K-resistant material in ARR/VRQ sheep brain affected with classical scrapie is composed mainly of VRQ prion protein.

ABSTRACT Classical scrapie is a prion disease in sheep and goats. In sheep, susceptibility to disease is genetically influenced by single amino acid substitutions. Genetic breeding programs aimed at enrichment of arginine-171 (171R) prion protein (PrP), the so-called ARR allele, in the sheep population have been demonstrated to be effective in reducing the occurrence of classical scrapie in the field. Understanding the molecular basis for this reduced prevalence would serve the assessment of ARR adaptation. The prion formation mechanism and conversion of PrP from the normal form (PrPC) to the scrapie-associated form (PrPSc) could play a key role in this process. Therefore, we investigated whether the ARR allele substantially contributes to scrapie prion formation in naturally infected heterozygous 171Q/R animals. Two methods were applied to brain tissue of 171Q/R heterozygous sheep with natural scrapie to determine the relative amount of the 171R PrP fraction in PrPres, the proteinase K-resistant PrPSc core. An antibody test differentiating between 171Q and 171R PrP fragments showed that PrPres was mostly composed of the 171Q allelotype. Furthermore, using a novel tool for prion research, endoproteinase Lys-C-digested PrPres yielded substantial amounts of a nonglycosylated and a monoglycosylated PrP fragment comprising codons 114 to 188. Following two-dimensional gel electrophoresis, only marginal amounts (<9%) of 171R PrPres were detected. Enhanced 171Rres proteolytic susceptibility could be excluded. Thus, these data support a nearly zero contribution of 171R PrP in PrPres of 171R/Q field scrapie-infected animals. This is suggestive of a poor adaptation of classical scrapie to this resistance allele under these natural conditions.

Evaluation of Two Enzyme-Linked Immunosorbent Assays for Detecting Salmonella enterica subsp.enterica Serovar Dublin Antibodies in Bulk Milk

ABSTRACT Two enzyme-linked immunosorbent assays (ELISAs) for the detectingSalmonella enterica subsp. entericaserovar 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 (R2) 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 log10 serum antibody titer for that herd (R2 = 62% for the LPS ELISA andR2 = 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.