Detection of virulent strains of Streptococcus suis type 2 and highly virulent strains of Streptococcus suis type 1 in tonsillar specimens of pigs by PCR.

We developed a PCR assay for the rapid and sensitive detection of virulent Streptococcus suis type 2 and highly virulent S. suis type 1 in tonsillar specimens from pigs. The PCR primers were based on the sequence of the gene encoding the EF-protein of virulent S. suis type 2 strains (MRP+EF+) and highly virulent S. suis type 1 strains (MRP(s)EF+) and of the EF protein of weakly virulent S. suis type 2 strains (MRP+EF). The latter strains give rise to larger PCR products than the virulent strains of S. suis type 1 and 2. A positive control template was included in the assay to identify false negative results. The PCR was evaluated using tonsillar specimens from herds known (or suspected) to be infected and herds without an S. suis history. The results obtained with the PCR assay were compared with the results obtained with a newly developed bacteriological examination. In this bacteriological examination we were able to identify the EF-positive strains directly in the tonsillar specimens. From the 99 tonsils examined, 48 were positive in the PCR and 51 negative. All specimens from which EF-positive S. suis strains were isolated were also positive in the PCR assay. Three samples were positive in the PCR, but negative by bacteriological examination. The results demonstrated that the PCR is a highly specific and sensitive diagnostic tool for the detection of pigs carrying virulent strains of S. suis type 2 and highly virulent strains of type 1. Application of the assay may contribute to the control of S. suis infections.

A specific and sensitive PCR assay suitable for large-scale detection of toxigenic Pasteurella multocida in nasal and tonsillar swabs specimens of pigs.

A polymerase chain reaction (PCR) assay for the detection of toxigenic Pasteurella multocida in nasal and tonsillar swab specimens collected from pigs was developed. Target DNA was isolated with guanidine thiocyanate and diatomite, and 2 primer sets derived from sequences in the gene that encodes the dermonecrotic toxin of P. multocida were used simultaneously. The method was adapted to microtiter plate format allowing large-scale use of the PCR assay. To identify false-negative test results caused by failure of amplification, a positive control template was constructed that was spiked to each DNA sample. The PCR assay was evaluated with clinical samples and compared with 2 routinely used methods for detection of toxigenic P. multocida: isolation from a selective agar and direct detection of the toxin in extracts of primary cultures by an enzyme-linked immunosorbent assay (ELISA). The sensitivity of the PCR assay was tested with 346 nasal and tonsillar swabs specimens collected from pigs of 9 herds known to be infected with toxigenic P. multocida. Toxigenic P. multocida was isolated from 22 specimens, only 28 specimens tested positive in ELISA, but 40 tested positive in the PCR assay; thus the PCR assay is the most sensitive of the 3 methods. The specificity of the PCR assay was tested with 372 swab specimens collected from pigs of 6 herds certificated to be free from toxigenic P. multocida. Toxigenic P. multocida was not isolated from any of these specimens, all tested negative in ELISA, and 370 tested negative in PCR. The 2 positive specimens came from 2 pigs of 1 litter and tested only weakly positive in the PCR assay. From these results, it was concluded that the PCR assay is not only highly sensitive but also highly specific.

Use of Multiscreen plates for the preparation of bacterial DNA suitable for PCR.

A rapid and inexpensive method for isolating bacterial DNA for use in PCR is described. The method is based on the guanidinium thiocyanate (GuSCN)-lysis method of Boom et al. (J. Clin. Microbiol. 28:495-503, 1990) and enables a multiple of 96 samples to be prepared in only one hour. We use Multiscreen plates and a vacuum manifold from Millipore. Clinical samples are lysed and washed in the wells of a Multiscreen plate, and DNA is eluted in a standard microplate. Purified DNA was recovered with high yields (over 25%). The method allows multichannel or robotic pipetting for both the sample preparation as well as for the PCR step. The method has been applied successfully to detect pathogenic Streptococcus suis type 2 in nasal and tonsil swab specimens of pigs.

[Swine vesicular disease: threat or challenge for Dutch pig farming?]. / Vesiculaire varkensziekte: bedreiging of uitdaging voor de Nederlandse varkenshouderij?

Swine vesicular disease (SVD) caused problems in 1993 when it was detected in Dutch pigs in Italy. As a result, the EC took measures against the export of live pigs. In all cases the animals had been retained at an Italian abattoir or farm for three days or more, which is longer than the minimum incubation time. Extensive clinical inspections and serological testing on the farms from where the pigs originated revealed no evidence of SVD infection. Serological testing for SVD of over 1.5 million blood samples collected from herds within the framework of export- and herd certification, and the testing of slaughter sows and slaughter boars (EC directive), was negative as well. In view of these results it has to be assumed that the Dutch pig population is free from SVD and that the pigs were infected in Italy. However, a complaint from Italy in 1994 led to the detection of two SVD virus-contaminated export collection centres. If the existing regulations on the cleansing and disinfection of the transport chains are stringently enforced and implementation of the rules is continuously supervised, then it may be expected that the problems have been overcome.

Repeats in an extracellular protein of weakly pathogenic strains of Streptococcus suis type 2 are absent in pathogenic strains.

Streptococcus suis type 2 strains that are pathogenic for pigs produce a 110-kDa extracellular protein factor (EF). Nonpathogenic and weakly pathogenic strains do not produce EF or produce a protein (EF*) that is immunologically related to EF. To study the pathogenesis of S. suis type 2 in pigs and to develop tools and methods for the control of S. suis type 2 infections, we cloned and characterized the genes encoding EF and various EF* proteins. Analysis of the deduced amino acid sequences showed that the first 833 amino acids at the N terminus of the EF and EF* proteins were nearly identical. The proteins differed, however, at their C termini. Unlike the 110-kDa EF protein, the EF* proteins contained several repeated units of 76 amino acids. The number and arrangement of the repeats in the EF* proteins varied. The data suggest that the gene encoding EF could have evolved from an epf* gene by a specific deletion event. The lack of repeated amino acid units in the EF protein may be related to virulence.

Human gamma-crystallin genes. A gene family on its way to extinction.

During hominoid evolution the gamma-crystallins of the lens have decreased in quantity as well as complexity, a change correlated with an increased water content of the lens. To trace the molecular basis for the decrease in gamma-crystallin gene expression, we have characterized the structure and expression of the human gamma-crystallin gene family. We show that the human gamma-crystallin gene family consists of six complete genes (gamma A, gamma B, gamma C, gamma D, psi gamma E and psi gamma F) and one second exon fragment, the gamma G gene. Model experiments showed that, although the gamma G sequence is bordered by consensus splice sites, it is most likely transcriptionally inactive in the lens. In the human embryonic lens the gamma C and gamma D genes accounted for 81% of the gamma-crystallin transcripts, the gamma A gene contributed 14% and the gamma B gene only 5%. The composition of the gamma-crystallin mRNA pool changed only after birth, with the gamma D transcript as the only detectable transcript at ten years of age. The relative activities of the gamma A, gamma C and gamma D promoters in a transient expression system were in agreement with the ratio of their in vivo RNA levels, suggesting that the difference in accumulation of these transcripts is due to differences in the rate of transcription. The gamma B promoter was much more active than expected and had lost its tissue-specificity. Model experiments showed that the low yield of the gamma B transcript is due to post-transcriptional processes, most likely RNa instability mediated by third exon sequences. Together with previous data, our results show that the decrease in expression of the gamma-crystallin genes in the human lens is the consequence of gene loss (gamma G), inactivation of coding sequences (psi gamma E and psi gamma F), decrease in rate of transcription (gamma A), increase in rate of RNA turn-over (gamma B) and a delay in the onset of transcription during development.