Temperature-Sensitive Salmonella enterica Serovar Enteritidis PT13a Expressing Essential Proteins of Psychrophilic Bacteria.

Synthetic genes based on deduced amino acid sequences of the NAD-dependent DNA ligase (ligA) and CTP synthetase (pyrG) of psychrophilic bacteria were substituted for their native homologues in the genome of Salmonella enterica serovar Enteritidis phage type 13a (PT13a). The resulting strains were rendered temperature sensitive (TS) and did not revert to temperature resistance at a detectable level. At permissive temperatures, TS strains grew like the parental strain in broth medium and in macrophage-like cells, but their growth was slowed or stopped when they were shifted to a restrictive temperature. When injected into BALB/c mice at the base of the tail, representing a cool site of the body, the strains with restrictive temperatures of 37, 38.5, and 39°C persisted for less than 1 day, 4 to 7 days, and 20 to 28 days, respectively. The wild-type strain persisted at the site of inoculation for at least 28 days. The wild-type strain, but not the TS strains, was also found in spleen-plus-liver homogenates within 1 day of inoculation of the tail and was detectable in these organs for at least 28 days. Intramuscular vaccination of White Leghorn chickens with the PT13a strain carrying the psychrophilic pyrG gene provided some protection against colonization of the reproductive tract and induced an anti-S. enterica antibody response.

Statistical analyses of chicken intestinal lesion scores in battery cage studies of anti-coccidial drugs.

Establishing the efficacy of an anti-coccidial drug in poultry begins with conducting multiple battery cage studies, where the target animals are challenged with single and mixed Eimeria species inoculum under controlled laboratory conditions. One of the primary outcomes in a battery cage study is the intestinal lesion score defined on a discrete ordinal scale of 0 to 4. So far, the statistical analysis of lesion scores has routinely employed the linear mixed model (LMM). This present work proposes to apply the generalized linear mixed model (GLMM) with the cumulative logit link to statistically analyze coccidial lesion scores collected from battery cage studies. Upon applying this new approach on 9 datasets generated by challenging battery-cage-housed broilers with various mixtures of Eimeria species, it is observed that the GLMM fitted adequately to the data, produced variance component estimates that agreed with the experimental setup, and, at the 0.05 significance level, generated statistical results in complete concordance with the LMM approach. Advantages of the proposed GLMM over the LMM are discussed from several standpoints. Parallel to the regulatory requirement of a ≥1-unit reduction in the mean lesion score for clinical relevant efficacy under the LMM, the clinical relevancy criterion under the GLMM could be set as a ≥10-fold increase in the odds of having low lesion scores. That is, the effect of an anti-coccidial drug product would be deemed clinically relevant in battery-cage studies when the odds of having low lesion scores with the medication is 10 times or more than the odds without the medication.