Oral transmission of porcine reproductive and respiratory syndrome virus by muscle of experimentally infected pigs.

The current study was performed to determine if porcine reproductive and respiratory syndrome virus (PRRSV) could be transmitted to pigs by feeding muscle tissue obtained from recently infected pigs. Muscle obtained from pigs infected with either a European strain (EU donor pigs) or American strain (US donor pigs) of PRRSV was fed to PRRSV-free receiver pigs. The donor pigs were slaughtered 11 days post-infection (dpi). PRRSV was detected by conventional virus isolation in muscle at 11 dpi from 7 of 12 EU donor pigs and 5 of 12 US donor pigs. In contrast to conventional virus isolation, all muscle samples from infected pigs were positive for viral nucleic acid by PCR, except for muscle from one animal infected with the American strain of PRRSV. Five hundred grams of raw semimembranosus muscle from each of the donor pigs was fed over a 2 days period (250 g per day) to each of two receiver pigs (48 receiver pigs). The receiver pigs were housed separately in five groups. One of the five groups was fed muscle obtained from US donor pigs that was also spiked with the American strain of PRRSV. Sentinel pigs were placed in-contact with the group of receiver pigs fed spiked muscle. All receiver pigs became viraemic by 6 days post-feeding (dpf). There was evidence of horizontal transmission with sentinel pigs, in-contact with receiver pigs, becoming viraemic. The study demonstrates that PRRSV could be infectious through the oral route via the feeding of meat obtained from recently infected pigs.

Safety and protective efficacy of porcine reproductive and respiratory syndrome recombinant virus vaccines in young pigs.

Three porcine reproductive and respiratory syndrome virus (PRRSV) recombinants, generated by mutagenesis of an infectious cDNA clone of the Lelystad virus (LV) isolate, were tested for their safety and protective efficacy as potential PRRSV vaccines in pigs. Recombinant vABV688 contains two amino acid substitutions in the minor structural protein GP(2) resulting in improved growth on cell line CL2621; in recombinant vABV707 the region encoding the ectodomain of the major unglycosylated membrane protein M has been replaced by that of the murine lactate dehydrogenase-elevating arterivirus; recombinant vABV746 lacks the six C-terminal amino acids of the nucleocapsid protein N. First, we determined the safety of these recombinant viruses by monitoring the stability of the introduced mutations in 8-week-old pigs. We showed that the introduced genomic mutations were maintained throughout the viraemic period. Second, the protective efficacy of immunization with the recombinant viruses against challenge with a homologous and a heterologous PRRSV strain was determined in two pigs and compared with the efficacy of vABV437, a virus derived from the parental LV cDNA. The viraemia in pigs immunized with the recombinant viruses was reduced compared to pigs immunized with vABV437. In addition, the length of viraemia was reduced in the sentinel pigs that were introduced into the groups immunized with vABV746, vABV688, and vABV707, however, all of the sentinel pigs became infected. Pigs immunized with vABV707 and vABV437 were protected against challenge with homologous virus LV-Ter Huurne and transmission of the latter virus. None of the immunized pigs were protected against heterologous challenge with the virulent US isolate SDSU#73, but the vABV707- and vABV746-immunized pigs were protected against transmission of this virus from challenged pigs. In conclusion, the obtained viral recombinants are interesting candidates to be further explored for their use as vaccines against PRRSV.

Virological kinetics and immunological responses to a porcine reproductive and respiratory syndrome virus infection of pigs at different ages.

The objective of this study was to measure the effect of two variables (pig age and virus strain) on selected responses (clinical signs, viraemia, virus excretion and seroconversion) of pigs following exposure to porcine reproductive and respiratory syndrome (PRRS) virus. Therefore, young (6 till 8 weeks old) and old (6 months old) pigs were infected with 3 different PRRSV strains, i.e. LV ter Huurne (LVTH), LV4.2.1 and SDSU#73. Regardless of the strain used for exposure, young pigs were more susceptible to infection as shown by a higher number of viraemic and virus excreting pigs. Strain differences were also evident. LV ter Huurne induced virus excretion in a higher number of pigs and with a higher virus titre, whereas SDSU#73 induced most severe clinical signs. LV4.2.1 induced viraemia and virus excretion in a low number of pigs. The kinetics of the antibody response differed per virus strain. The results presented here are useful in developing a less expensive standardised infection model, consisting of young pigs intranasally infected with a virulent PRRSV strain, to study the efficacy of new vaccine strains.

A quantitative assessment of the effectiveness of PRRSV vaccination in pigs under experimental conditions.

This paper presents a quantitative approach to evaluate effectiveness of vaccination under experimental conditions. We used two consecutive experimental designs to investigate whether PRRSV transmission among vaccinated pigs was reduced compared to control pigs and to estimate the reproduction parameter R. Based upon data analysis and power calculations the series of small-scale vaccination-challenge experiments ended with multiple one-to-one experiments. This new experimental design has considerable power to detect the effect of vaccination on transmission if R is close to but still above one in vaccinated pigs. The last experiment showed that transmission was not significantly reduced and the R for vaccinated pigs was estimated to be larger than 4.9. This is remarkable because duration and level of viremia were significantly reduced by vaccination.

Sequence analysis of the membrane protein gene and nucleocapsid gene of porcine reproductive and respiratory syndrome virus isolated from a swine herd in Hungary.

Porcine reproductive and respiratory syndrome virus (PRRSV) was isolated from blood samples taken at a pig farm in Hungary from pigs showing clinical signs of the disease. The virus (ABV 32) was identified as belonging to the European genotype by using type-specific monoclonal antibodies. This was confirmed by comparing the sequence of the membrane protein gene (ORF 6) and the nucleocapsid gene (ORF 7) with the American VR2332 and the European LV genotype reference strain, respectively. Analysis of the amino acid sequence of the ORF 6 and ORF 7 of ABV 32 revealed five amino acid changes in both ORFs when compared with LV, of which two changes in ORF 7 were only found in the Spanish isolates. Additionally, the ORF 7 sequence was compared with corresponding sequences of a total of 21 other European strains. Phylogenetic analysis using the PHYLIP package confirmed the close relationship between the Hungarian and the Spanish isolates. Of all the isolates analysed, ABV 32 and LV were the least related.

Isolation and characterization of porcine circovirus type 2 from pigs showing signs of post-weaning multisystemic wasting syndrome in The Netherlands.

Pigs with wasting syndrome were examined for macroscopic and histopathological lesions, and for porcine circovirus (PCV). Histopathological lesions were comparable to those previously documented for post-weaning multisystemic wasting syndrome (PMWS). In addition, in seven out of ten examined PMWS-affected pigs focal-to-slight mononuclear meningitis and focal cerebral mononuclear infiltrates (4 out of 10) were observed. A virus was isolated from organs and sera from pigs showing wasting syndrome. An immunoperoxidase monolayer assay and an indirect immunofluorescence assay were performed on the infected PK-15 and Dulac cell cultures, respectively, and both assays indicated the presence of PCV type 2 (PCV2). The nested-polymerase chain reaction (nPCR) technique, based on the use of PCV2 specific oligonucleotides, revealed specific amplified products of 481 bp. Nucleotide sequence analysis of the entire genome of the Dutch PCV isolate 24657 NL showed a homology with known nucleotide sequences of porcine PCV type 1 (PCV1) and PCV2 isolates of 77.1% and >96%, respectively. This is the first report of the isolation and characterization of PCV2 in PMWS-affected pigs in the Netherlands.

Catheter deadspace: a source of error during tonometry.

Tonometry of PCO2 is a promising method for assessing the oxygen supply to demand ratio of the gastrointestinal mucosa in critically ill patients. A balloon-tipped tonometer is introduced into the stomach or sigmoid colon, and saline is instilled into the balloon. After a time to allow partial equilibration with intraluminal PCO2, saline is aspirated and PCO2 is measured. Intermittent instillation and aspiration of saline allows serial PCO2 measurements, provided correction factors are used to calculate the PCO2 value expected at full equilibration from the PCO2 values measured after short dwell times. The technique is not yet widely applied, partly because of methodological controversies. We evaluated the role of the catheter deadspace as a source of error during PCO2 tonometry. The increase in PCO2 in sigmoid-type tonometers with a normal length (normal tonometer (NT)) and in those with a 50% increase in length and thus deadspace (extended tonometer (ET)), in a saline bath at a PCO2 of 4.8 kPa was assessed. Saline dwell times were 10, 20, 30, 45, 60 and 90 min and the time-dependent PCO2 increase was determined at deadspace PCO2 values of approximately 4.0 and 8.0 kPa following contamination of the catheter deadspace after immersion in saline baths at PCO2 values of 4.8 and 9.6 kPa, respectively, before each measurement cycle. In another experiment, the tonometer was rinsed between measurement cycles to remove deadspace saline containing carbon dioxide and to obviate contamination of instilled saline. PCO2 was measured in a blood-gas analyser, taking into account measurement bias in saline. Failure to remove deadspace saline between measurement cycles resulted in an overestimation of 10% and 6% for the NT and 16% and 10% for the ET, at saline dwell times of 10 and 20 min, respectively, at a deadspace PCO2 of approximately 4.0 kPa. At a deadspace PCO2 of approximately 8.0 kPa, PCO2 was overestimated by 17%, 11% and 5% for the NT and 31%, 20% and 11% for the ET, at dwell times of 10, 20 and 30 min, respectively. Rinsing the NT/ET resulted in accurate assessment of PCO2 at all dwell times, but the dwell time-dependent increase in PCO2 was slightly slower in the ET, particularly at 10 min, after a sink effect of the increased deadspace. Hence, a previously unrecognized deadspace effect caused error during PCO2 tonometry, particularly with short dwell times. This potentially large error can be avoided by rinsing the tonometer before each measurement cycle, allowing accurate PCO2 tonometry even at 10-min saline dwell times, provided that correction factors are used that are specific for catheter size. These findings may help to widen the clinical applicability of tonometry.

Characteristics of time-dependent PCO2 tonometry in the normal human stomach.

Factors that affect PCO2 measurement in balloon saline during gastrointestinal tonometry are unclear. They include carbon dioxide diffusion rate, correction factors for calculation of equilibrium PCO2 from measurements at saline dwell times that are shorter than needed for full equilibration, role of blood-gas analyser bias during ex vivo PCO2 measurements in saline, and normal values for intragastric PCO2 (PiCO2) and intramucosal pH (pHi) at equilibrium, and their differences from blood values. In a laboratory study, normal PCO2 changes in a saline-filled tonometer balloon placed in a saline bath at constant PCO2 were described by a non-linear model, with a half-time of mean 4.4 min and 95% equilibration at mean 83 min. In a study in 20 healthy volunteers, PiCO2 build up in a saline-filled tonometer balloon placed in the stomach, measured at dwell times of 10, 20, 30 and 60 min, was slightly (P < 0.05) slower than in vitro, with a half-time of mean 5.8 min and 95% equilibration at mean 110 min. Correction factors to derive equilibrium PiCO2 at short dwell times and independently from blood-gas analyser bias were calculated. The factors differed (P < 0.05) from those currently provided by the manufacturer. Normal threshold values (mean) were: equilibrium PiCO2 < or = 6.6 kPa, pHi > or = 7.33, PiCO2 to blood PCO2 difference < or = 1.1 kPa and pH difference > or = -0.06. PiCO2 did not differ from, and was directly related to, blood PCO2. These values provide a reference base for other studies and show that gastric mucosal PCO2 depends on alveolar ventilation if blood flow is adequate.

Type of solution and PCO2 measurement errors during tonometry.

OBJECTIVE: The choice of solution for gastrointestinal tonometry influences the PCO2 measurement bias, precision and the time required for equilibration. We compared saline with buffered solutions during in vitro tonometry, with respect to systematic and accidental measurement errors and equilibration time. DESIGN: A prospective laboratory study. MEASUREMENTS: Saline, phosphate, phosphate bicarbonate and succinylated gelatin solutions were equilibrated in a specialized blood gas tonometer at PCO2s of 2.7, 3.6, 4.5, 6.2 and 9.0 kPa, using calibration gases. Accidental errors were determined: the within-syringe decline of PCO2 and the effects of handling errors (five up and down movements of the plunger). The PCO2 build up in gastrointestinal tonometers was determined in 5000 ml saline baths with fixed PCO2 levels of 2.7 and 9.0 kPa. RESULTS: The build up of PCO2 in phosphate bicarbonate and gelatin was about 4 and 2 times slower than in saline and phosphate, respectively, both for gas and gastrointestinal tonometers. The bias of the measured PCO2 at equilibrium was -15% for saline, and between -1 and 3% for phosphate, phosphate bicarbonate and gelatin. The precision was comparable among the solutions: 2 +/- 1% for saline, 2 +/- 1% for phosphate, 1 +/- 0% for phosphate bicarbonate and 1 +/- 1% for gelatin. The accidental errors were virtually absent with phosphate bicarbonate, intermediate with gelatin and largest with saline and phosphate. CONCLUSION: Phosphate bicarbonate buffer and succinylated gelatin allow accurate PCO2 measurements, but their equilibration is too slow for clinical application. The advantage of phosphate over saline solution is a smaller bias only. Thus, both saline and phosphate are currently the tonometer solutions of choice, provided that strictly anaerobic conditions are applied and the bias by the blood gas analyzer is known.

Aneurysm of the cranial mesenteric artery in a cow.

An exploratory laparotomy of a four-year-old red Holstein cow with signs of abdominal discomfort revealed a painful pulsating mass in the cranial part of the abdomen. The cow died suddenly. A post mortem examination showed that a ruptured aneurysm of the cranial mesenteric artery had been the cause of death.

Laboratory and clinical evaluation of a chromogenic endotoxin assay for horses with acute intestinal disorders.

In this study the laboratory and clinical performance of a chromogenic endotoxin assay for equine plasma was evaluated. The assay was sensitive (detection limit 3 ng LPS/L plasma), reproducible (within and between-assay CV at 50 ng LPS/L E. coli O111:B4 LPS standard addition was 5% and 7.5%, respectively), and not substantially affected by enhancement or inhibition phenomena (recovery of an in vitro spike was 75-125% in 80% of the samples). LPS added to whole blood was rapidly inactivated upon incubation at 37 degrees C but not at 0 degrees C. A recently developed blood collection tube for LPS testing was found suitable, i.e. LPS-free and providing non-contaminated samples. In 48 horses suffering from acute abdominal diseases requiring surgical treatment, LPS levels were significantly higher in platelet-rich plasma (PRP) than in platelet-poor plasma (PPP), and the proportional difference was related to the PRP platelet count (r = 0.52, p < 0.001, mean difference 48%, range 8-77%). LPS levels were also significantly higher in horses that died or were euthanized than in surviving horses (mean 16.5 and 7.1 ng/L PRP, respectively, p < 0.05). We conclude that LPS can be measured in equine plasma with picogram sensitivity and recommend the use of PRP instead of PPP for clinical LPS testing. For clinical use a decision limit for endotoxaemia of 5 ng LPS/L PRP appeared to be inadequate. Analysis at a higher cut-off level for endotoxaemia and the evaluation of clinical, pathological, and laboratory parameters would be more meaningful.