No abstract

From January 1998 to July 2000, 2,456 clinical samples, including lung, tonsil, lymph node, and serum, from 760 cases submitted to the Animal Health Laboratory, Ontario, Canada, were tested for porcine reproductive and respiratory syndrome viruses (PRRSV) using reverse transcriptase polymerase chain reaction (RT-PCR) and RT-PCR product restriction fragment length polymorphism (RFLP) analysis. A total of 516 samples from 284 cases were RT-PCR positive for the PRRSV open reading frame (ORF) 7 sequence. The RT-PCR RFLP typing assay was performed using 2 different sets of primers, amplifying 716 or 933 base pairs of ORF 4, 5, and 6 of PRRSV. Samples from 254 cases were typeable, yielding 34 different RFLP types. Of these, 164 cases had 32 different RFLP types of field or intermediate strains, 86 had a pattern similar to a commercial PRRSV vaccine or VR 2332 strain of the virus, 4 had a RFLP type shared by another commercial vaccine and a field strain. In 4 cases, 2 different RFLP types were identified from tissues from different pigs that were submitted at the same time from the same farm. Of the 195 farms that submitted PRRSV PCR-positive samples, 48 submitted samples on more than 1 occasion during the specified time frame. In 23 of those 48 farms, RFLP patterns of PRRSV differed between submissions, whereas in the other 25 farms, the RFLP pattern remained unchanged. There were 34 different PRRSV patterns identified from 236 cases using the primer set amplifying 716 base pairs of PRRSV. There were 18 cases, consisting of 9 different patterns, typeable only by using the primers amplifying a 933-base pair fragment of the virus.

The polymerase chain reaction (PCR) was standardized and assessed as a potential diagnostic test for infectious laryngotracheitis using conjunctival swabs collected from experimentally infected chickens. Polymerase chain reaction primers based on the sequence of a 1.1-kb BamHI restriction enzyme fragment of the Ontario 1598 (Ont 1598) strain of infectious laryngotracheitis virus were selected and 300 fg of purified viral DNA were detected by ethidium bromide staining of agarose gels or 30 fg were detected by DNA hybridization. At least five different strains (Ont 1598, ATCC N-71851, LT-IVAX, Laryngo-Vac, and a local strain 322) were amplified whereas other avian pathogens and uninfected cell cultures tested negative. Swabs collected from experimentally infected chickens were analyzed by both PCR and virus isolation on various days postinfection. A comparison of virus isolation to PCR indicated that, in the mid-postinfection phase, PCR and virus isolation appeared to be comparable with a kappa value of greater than 0.8. The polymerase chain reaction was shown to be the better test later in infection, when clinical recovery had occurred.

SUMMARY: A number of different organisms were subjected to violent shaking with minute round glass particles. Vegetative bacteria, spores, and acid-fast species were killed by this treatment, though a t varying rates.Curran & Evans (1942) described the destruction of bacterial spores by violent agitation yvith small inert particles. Several workers (e.g. Gale, 1947) have applied this technique to the extraction of enzymes from vegetative cells. Unlike other mechanical methods (e.g. sonic disintegration or the use of bacterial mills) it requires no elaborate equipment, and the risk of damage to proteins or other labile cell constituents is less than with methods involving autolysis or the use of chemical agents. Cuiran & Evans (1942) obtained large decreases in the viable count of spore suspensions by this method and claimed (though without giving experimental data) that suspensions of Bacterium coli may be completely sterilized. We have investigated a wider range of both sporing and non-sporing bacteria. In confirmation of Curran & Evans's findings we obtained a rapid exponential fall in the number of viable organisms: but a small proportion of the cells appeared to be much more resistant to this treatment than the others present in the same bacterial suspension. METHODSBacterial suspensions. The organisms tested were mainly from our laboratory stock. They were grown for 24 hr. (48 hr. for Mycobacterium megmatis) a t 37' on the surface of nutrient agar, with addition of 0.1 yo glucose and 10 % horse blood for the pneumococcus, washed off with physiological saline, washed twice with saline, and resuspended in the fluid (usually saline) in which they were to be shaken. The cultures of Bacillus subtilis, B. mesentericus and B. anthracis were allowed to stand a t room temperature for a few days before preparing the suspensions, which consequently consisted mainly if not entirely of spores. Suspensions of yeast were prepared from commercial brewers' yeast by repeated washing with saline.All suspensions were adjusted to an opacity equal to Brown's tube no. 3, which corresponds to about los organisms/ml. in most cases. Exact adjustment of the initial count was not practicable since, for reasons stated later, the 'initial' was taken after a short period of shaking during which a variable degree of killing took place. All precautions to avoid extraneous contamination were taken throughout.Particles. Curran & Evans (1942) employed several types of particles and found small smooth glass beads to be the most suitable. We obtained these from Messrs Chance Bros. Ltd., Smethwick, England, under the trade name ' ballotini '. Three grades were supplied whose mean diameters, measured

Background As a pragmatic randomised timing-of-birth trial, WILL adapted its trial procedures in response to the COVID-19 pandemic. These are reviewed here to inform post-pandemic trial methodology. Methods The trial (internal pilot) paused in March 2020, re-opened in July 2020, and is currently recruiting in 37 UK NHS consultant-led maternity units. We evaluated pandemic adaptations made to WILL processes and surveyed sites for their views of these changes (20 sites, videoconference). Results Despite 88% of sites favouring an electronic investigator site file (ISF), information technology requirements and clinical trial unit (CTU) operating procedures mandated the ongoing use of paper ISFs; site start-up delays resulted from restricted access to the CTU. Site initiation visits (SIVs) were conducted remotely; 50% of sites preferred remote SIVs and 44% felt that it was trial-dependent, while few preferred SIVs in-person as standard procedure. The Central team felt remote SIVs provided scheduling and attendance flexibility (for sites and trial staff), the option of recording discussions for missing or future staff, improved efficiency by having multiple sites attend, and time and cost savings; the negative impact on rapport-building and interaction was partially mitigated over time with more familiarity with technology and new ways-of-working. Two methods of remote consent were developed and used by 30/37 sites and for 54/156 recruits. Most (86%) sites using remote consenting felt it improved recruitment. For remote data monitoring (5 sites), advantages were primarily for the monitor (e.g. flexibility, no time constraints, reduced cost), and disadvantages primarily for the sites (e.g. document and access preparation, attendance at a follow-up meeting), but 81% of sites desired having the option of remote monitoring post-pandemic. Conclusions COVID adaptations to WILL trial processes improved the flexibility of trial delivery, for Central and site staff, and participants. Flexibility to use these strategies should be retained post-pandemic. Trial registration ISRCTN77258279. Registered on 05 December 2018.