The grimace scale: a useful tool for assessing pain in laboratory animals.

Accurately and promptly assessing pain in experimental animals is extremely important to avoid unnecessary suffering of the animals and to enhance the reproducibility of experiments. This is a key concern for veterinarians, animal caretakers, and researchers from the perspectives of veterinary care and animal welfare. Various methods including ethology, immunohistochemistry, electrophysiology, and molecular biology are used for pain assessment. However, the grimace scale, which was developed by taking cues from interpreting pain through facial expressions of non-verbal infants, has become recognized as a very simple and practical method for objectively evaluating pain levels by scoring changes in an animal's expressions. This method, which was first implemented with mice approximately 10 years ago, is now being applied to various experimental animals and is widely used in research settings. This review focuses on the usability of the grimace scale from the "cage-side" perspective, aiming to make it a more user-friendly tool for those involved in animal experiments. Differences in facial expressions in response to pain in various animals, examples of applying the grimace scale, current automated analytical methods, and future prospects are discussed.

Prevalence of staphylococcal enterotoxins in Staphylococcus pseudintermedius isolates from dogs with pyoderma and healthy dogs.

To investigate the role of staphylococcal enterotoxins (SEs) produced by Staphylococcus pseudintermedius in the pathogenesis of pyoderma, isolates from dogs with pyoderma and healthy dogs were analyzed. According to reverse passive latex agglutination, 14/184 isolates (7.6%) from dogs with pyoderma and 9/87 (10.3%) from healthy dogs produced SEs (SEA, SEC or SED). According to multiplex PCR, 99 isolates (53.7%) from dogs with pyoderma and 97 (90.8%) from healthy dogs possessed one or more se genes. There was no significant difference regarding ses between dogs with pyoderma and healthy dogs. Therefore, SEs may not be a direct virulence factor in pyoderma.

Antimicrobial resistance of Staphylococcus pseudintermedius isolates from healthy dogs and dogs affected with pyoderma in Japan.

Staphylococcus pseudintermedius strains were isolated from healthy dogs and dogs with pyoderma in 2000-2002 and 2009. All the isolates from dogs with pyoderma in 1999-2000 and from healthy dogs in 2000-2002 and 2009 were susceptible to cefalexin and/or other cephalosporins and oxacillin. However, 7.1-12.5 and 11.4% of S. pseudintermedius isolates from dogs with pyoderma in 2009 were resistant to cephalosporins and oxacillin, respectively. All S. pseudintermedius isolates from dogs with pyoderma in 1999-2000 and those from healthy dogs in 2000-2002 were susceptible to fluoroquinolones; however, 50% of the S. pseudintermedius strains isolated from dogs with pyoderma in 2009 and 30% of the S. pseudintermedius strains isolated from healthy dogs in 2009 were resistant to fluoroquinolones. Of the 21 oxacillin-resistant S. pseudintermedius (MRSP) isolates, 11 carried SCCmec type V and 10 carried hybrid SCCmec types II-III. Staphylococcus pseudintermedius strains that were resistant to only one of three fluoroquinolones had a mutation in the quinolone resistance determination region of grlA, whereas S. pseudintermedius strains that were resistant to two or more fluoroquinolones had mutations in the quinolone resistance determination regions of both grlA and gyrA.

Development of a high-expression system for staphylococcal exfoliative toxin genes.

We constructed a new expression system for staphylococcal exfoliative toxin (ET). The expression vector, pETA-exp2, was constructed based on Bacillus-Escherichia shuttle vector pHY300PLK. The pETA-exp2 vector includes the regulator of the ETA gene (eta), the promoter and Shine-Dalgarno (SD) sequences of eta, a SalI sequence at the end of the signal sequence of eta, a nucleotide sequence encoding mature ETA, an XhoI site, a 6x His sequence just before the stop codon and the end of the transcription sequence of eta. The nucleotide sequences coding for the mature proteins of ETB, ExhA, ExhB, ExhC, ExhD and SHETB were amplified by polymerase chain reaction (PCR) and inserted into pETA-exp2. These recombinant plasmids were transformed into Bacillus megaterium. The major protein in the culture supernatant of the transformant was recombinant ET (rET). The yields of all rETs were high, and all of them showed exfoliative activity in susceptible animals. The antigenicities of rETs and ETs were not distinguishable from each other.

Detection of Staphylococcus hyicus exfoliative toxin genes by dot blot hybridization and multiplex polymerase chain reaction.

We designed a novel DNA probe and novel PCR primer sets for detecting the genes coding for Staphylococcus hyicus (S. hyicus) exfoliative toxin (ET). In dot blot hybridization, the novel DNA probe hybridized with chromosomal DNA of ExhA-, ExhB-, ExhC-, ExhD-, and SHETA-producing strains. This probe also hybridized with the plasmid DNA of a SHETB-producing strain. In Southern blot hybridization, the probe hybridized with a 1.5 kb HindIII fragment of chromosomal DNA from a SHETA-producing strain. The above fragment was cloned into E. coli and the nucleotide sequence of the SHETA gene determined, this gene proved to have almost the same homology (99.6%) as the ExhB gene. It was therefore thought that SHETA is a subtype of ExhB. In multiplex PCR using five primer sets, each gene gave a band distinguishable from the others. This multiplex PCR system has high specificity among the well-known S. hyicus ET genes. Of the 69 known ET-producing S. hyicus strains, 38, 19, 10, 2 and 1 strains have exhB, exhD exhA, shetb and exhC genes, respectively.