Genetic characterization of CCL3, CCL4 and CCL5 in leporid genera Oryctolagus, Sylvilagus and Lepus.

The genetic diversity of C-C motif chemokine receptor 5 (CCR5) ligands CCL3, CCL4 and CCL5 in the leporid genera Oryctolagus, Sylvilagus and Lepus was studied. Our results demonstrate that the three CCR5 chemokine ligands are under strong purifying selection as a result of possible functional binding constraints.

The evolution of the immunoglobulin heavy chain variable region (IgVH) in Leporids: an unusual case of transspecies polymorphism.

In domestic rabbit (Oryctolagus cuniculus), three serological types have been distinguished at the variable domain of the antibody H chain, the so-called V(H) a allotypes a1, a2, and a3. They correspond to highly divergent allelic lineages of the V(H) 1 gene, which is the gene rabbit utilizes in more than 80% of VDJ rearrangements. The sharing of serological V(H) a markers between rabbit and snowshoe hare (Lepus americanus) has suggested that the large genetic distances between rabbit V(H) 1 alleles (9-14% nucleotide differences) can be explained by unusually long lineage persistence times (transspecies polymorphism). Because this interpretation of the serological data is uncertain, we have determined the nucleotide sequences of V(H) genes expressed in specimens of Lepus species. Two sequence groups were distinguished, one of which occurred only in hare specimen displaying serological motifs of the rabbit V(H) a-a2 allotype. Sequences of this group are part of a monophyletic cluster containing the V(H) 1 sequences of the rabbit a2 allotype. The fact that this "transspecies a2 cluster" did not include genes of other rabbit V(H) a allotypes (a1, a3, and a4) is incompatible with the existence of a common V(H) a ancestor gene within the species, and suggests that the divergence of the V(H) a lineages preceded the Lepus vs Oryctolagus split. The sequence data are furthermore compatible with the hypothesis that the V(H)a polymorphism can be two times older than the divergence time between the Lepus and Oryctolagus lineages, which was estimated at 16-24 million years.

Development of the antibody repertoire in rabbit: gut-associated lymphoid tissue, microbes, and selection.

Rabbits generate their antibody repertoire in three stages. First, a neonatal repertoire is generated by B lymphopoiesis in fetal liver and bone marrow and is limited by preferential V(H) gene segment usage. Between 4 and 8 weeks after birth a complex primary antibody repertoire is developed by somatically diversifying the neonatal repertoire through somatic hypermutation and a somatic gene conversion-like mechanism in gut-associated lymphoid tissue (GALT). In rabbits, unlike other species, the development of the primary antibody repertoire through somatic diversification of Ig genes appears to be dependent on intestinal microbial flora. The primary antibody repertoire is subsequently modified during antigen-dependent immune responses in which VDJ genes further diversify both by somatic hypermutation and by a gene conversion-like mechanism (the secondary repertoire). During the various stages of development, the antibody repertoire is modified and shaped by selective processes. In this review, we discuss the roles of GALT, microbes, and B-cell selection in generating antibody diversity in rabbits.

Intestinal microflora and diversification of the rabbit antibody repertoire.

The rabbit establishes its primary Ab repertoire by somatically diversifying an initial repertoire that is limited by restricted VH gene segment usage during VDJ gene rearrangement. Somatic diversification occurs in gut-associated lymphoid tissue (GALT), and by about 1-2 mo of age nearly all Ig VDJ genes are somatically diversified. In other species that are known to establish their primary Ab repertoire by somatic diversification, such as chicken, sheep, and cattle, diversification appears to be developmentally regulated: it begins before birth and occurs independent of exogenous factors. Because somatic diversification in rabbit occurs well after birth in GALT, the diversification process may not be developmentally regulated, but may require interaction with exogenous factors derived from the gut. To test this hypothesis, we examined Ab repertoire diversification in rabbits in which the appendix was ligated shortly after birth to prevent microbial colonization and all other organized GALT was surgically removed. We found that by 12 wk of age nearly 90% of the Ig VDJ genes in PBL were undiversified, indicating that intestinal microflora are required for somatically diversifying the Ab repertoire. We also examined repertoire diversification in sterilely derived remote colony rabbits that were hand raised away from contact with conventional rabbits and thereby acquired a different gut microflora. In these remote colony rabbits, GALT was underdeveloped, and 70% of the Ig VDJ genes in PBL were undiversified. We conclude that specific, currently unidentified intestinal microflora are required for Ab repertoire diversification.

Myofibroblast induction with transforming growth factor-beta1 and -beta3 in cutaneous fetal excisional wounds.

BACKGROUND/PURPOSE: In a noncontractile fetal rabbit model, the authors recently have shown the induction of excisional wound contraction with sustained-release cellulose implants formulated with transforming growth factor (TGF)-beta. The purpose of this study was to test the hypothesis that the excisional wound contraction in this model is associated with the induction of myofibroblasts in the surrounding dermis, demonstrated by the presence of alpha-smooth muscle actin. METHODS: Cellulose discs were formulated with either 1.0 microg of TGF-beta1 (n = 6); 1.0 microg of TGF-beta3 (n = 9); 10 microg of TGF-beta3 (n = 6); or their carrier protein, bovine serum albumin (BSA; n = 9), for sustained-release over 5 days. Each disc was implanted into a subcutaneous pocket on the back of a fetal New Zealand White rabbit in utero on day 24 of gestation (term, 31 days). A full-thickness, 3-mm excisional wound (7.4 mm2) was then made next to the implanted cellulose disc. All fetuses were harvested at 3 days. The amount of alpha-smooth muscle (SM) actin in the dermis around the implants and wounds was determined using immunohistochemical techniques. RESULTS: Excisional wounds exposed to 1.0 microg of TGF-beta1 (5.6+/-2.0 mm2), 1.0 microg of TGF-beta3 (6.9+/-1.0 mm2), and 10 microg of TGF-beta3 (2.7+/-1.0 mm2) were significantly smaller when compared with the BSA control group (12.8+/-1.1 mm2; P<.05). Furthermore, there was a significant increase in staining for alpha-SM actin in the TGF-beta1 (1.8+/-0.5) and 10 microg TGF-beta3 (2.8+/-0.2) groups in comparison with the scant staining in the BSA control group (0.5+/-0.2; P<.05). CONCLUSIONS: TGF-beta1 and -beta3 induce alpha-SM actin and contraction of cutaneous excisional wounds in a fetal noncontractile model. This model of inducible cutaneous excisional wound contraction may be useful in further determining the role of the myofibroblast in wound contraction and the physiology underlying this poorly understood aspect of wound healing.

TGF-beta1 alters the healing of cutaneous fetal excisional wounds.

BACKGROUND/PURPOSE: In a number of species, fetal wound healing differs from the adult in the absence of inflammation, fibrosis, scar formation, and excisional wound contraction. The lack of inflammation also may explain the relative absence of any cytokine levels at the wound site, such as transforming growth factor (TGF)-beta, and therefore the unique characteristics of fetal wound healing. The authors hypothesized that exogenous TGF-beta1 would induce contraction, inflammation, fibrosis, and scar formation in cutaneous excisional wounds in the fetal rabbit. METHODS: Cellulose discs (3 mm in diameter) were formulated with either 1.0 microg TGF-beta1 (n = 6) or bovine serum albumin (BSA; n = 7), as a control, for sustained-release over 3 days. Each disc was implanted into the subcutaneous tissue on the backs of fetal New Zealand White Rabbits in utero on day 24 of gestation (term, 31 days). A full-thickness, 3-mm excisional wound (7.4 mm2) was then made next to the implanted cellulose disc. All wounds were harvested 3 days later. RESULTS: At harvest, the excisional wounds in the TGF-beta1 group had contracted (5.6 +/- 2.0 mm2), whereas those in the control group had expanded (13.5 +/- 1.2 mm2, P< .01). The surrounding dermis in the TGF-beta1 group had 16.3 inflammatory cells per grid block compared with 12.4 cells in the control group (not significant). In addition, a greater amount of fibrosis was induced by the TGF-beta1 implant (1.7 +/- 0.3) than the control implant (0.4 +/- 0.2) on a scale of 0 to 3, P < .01. In situ hybridization analysis showed an increase in procollagen type 1alpha1 gene expression in the surrounding dermis of the TGF-beta1 group (36.7 +/- 3.6 grains per grid block) compared with the control group (7.1 +/- 0.9 grains per grid block, P < .001). CONCLUSIONS: These results demonstrate that the cytokine TGF-beta1 can induce fetal excisional wounds to contract, stimulate fibrosis, and increase procollagen type 1alpha1 gene expression. These findings further suggest that the absence of TGF-beta1 atthe wound site may be responsible in part for the lack of a postnatal healing response.

The mouse p52 subunit of the transcription/DNA repair factor TFIIH is located in the class III region of the H2 complex: cloning and sequence polymorphism.

Loci controlling susceptibility to a number of diseases, including cortisone-induced cleft palate, experimental allergic orchitis, and chemically-induced transplacental lung tumors have been mapped to a 27 kilobase (kb) region within the class III region of the mouse major histocompatibility complex (H2). This region, contains three genes G7e, which resembles a viral envelope gene, Bat6 (G7a), which encodes a valyl-tRNA synthetase, and G7c, which has no known function. We cloned a set of overlapping cosmid clones containing 115 kb of DNA surrounding Bat6. Exon trapping has identified a new gene located telomeric of Bat6. Northern blot analysis detected a transcript of 1.7 kb with highest expression in the testis. DNA sequence analysis identified this gene as the mouse homologue of the human gene encoding the p52 subunit of the TFIIH transcription/DNA repair factor. Nucleotide sequence identity was 91% between mouse and human, and the protein sequence was 98% identical. Sequence analysis of p52 cDNA from congenic mouse strains detected an amino acid polymorphism at position 209, which results in the substitution of a threonine in the H2b haplotype to a methionine in the H2a,d haplotypes.

Somatic hypermutation: mutations 3' of rabbit VDJ H-chain genes.

VDJ genes of rabbit B cells are diversified by a somatic gene conversion-like mechanism in which V(H) gene segments 5' of the VDJ gene serve as donors. To assess whether somatic mutation also contributes to Ab diversification in the rabbit, we searched for mutations 3' of VDJ genes because mutations in this region presumably result from somatic mutation rather than gene conversion. We PCR-amplified and cloned the region extending 534 bp 3' of VDJ genes from splenic DNA of young and adult rabbits. Results of nucleotide sequence analysis of the clones revealed a high mutation frequency (2.4%) within the first 120 bp downstream of VDJ genes. This frequency decreased rapidly with increasing distance 3' of the VDJ genes. The mutations demonstrated both strand bias and dinucleotide preferences characteristic of somatic hypermutation. We conclude that Ab diversity in rabbit is generated not only by somatic gene conversion but also by somatic hypermutation.

Bacteriological and serological investigation of persistent Salmonella enteritidis infection in an integrated poultry organisation.

Bacteriological monitoring of broiler breeder farms, the hatchery, rendering plant and animal feed mill during 1991 identified a number of potential cross-contamination hazards, such as the use of processed poultry proteins in the company feed mill and contamination of egg trolleys and trays, which may have led to widespread dissemination of Salmonella enteritidis within an integrated poultry organisation. Serological monitoring of the flocks suggested that, in most cases, substantial exposure to S. enteritidis infection occurred during the mid-rearing stage whereas routine bacteriological monitoring of poultry house litter and dust samples, and meconium samples taken in the hatchery identified infection only after the onset of the laying period. At least 10 phage types and six plasmid profile types of S. enteritidis were identified in historic submissions from the organisation including one apparently specific plasmid profile type that was distributed throughout the various parts of the company. During sampling for this investigation, most of these strains were not identified, and the number of plasmid profile types was reduced to a single common UK type.

The colonization of broiler chickens with Campylobacter jejuni: some epidemiological investigations.

Between June 1990 and July 1991, broiler chickens from 49 flocks from 23 farms were examined for the carriage of Campylobacter jejuni at slaughter. Thirty-seven flocks (76%) were campylobacter-positive. Prevalence of campylobacter-colonization was not associated with any of a variety of factors such as water source and broiler house floor structure. There was also no apparent seasonal variation in carriage. Investigations on one farm indicated that dipping boots in disinfectant before workers entered broiler houses either delayed or prevented colonization with C. jejuni.

Metabolic engineering of Candida tropicalis for the production of long-chain dicarboxylic acids.

We have engineered an industrial strain of the yeast, Candida tropicalis, for the efficient production of long-chain dicarboxylic acids, which are important raw materials for the chemical industry. By sequential disruption of the four genes encoding both isozymes of the acyl-CoA oxidase which catalyzes the first reaction in the beta-oxidation pathway, alkane and fatty acid substrates have been successfully redirected to the omega-oxidation pathway. Consequently, the conversion efficiency and chemical selectivity of their terminal oxidation to the corresponding dicarboxylic acids has been improved to 100 percent. The specific productivity of the bioconversion has been increased further by amplification of the cytochrome P450 monooxygenase and NADPH-cytochrome reductase genes encoding the rate-limiting omega-hydroxylase in the omega-oxidation pathway. The amplified strains demonstrated increased omega-hydroxylase activity and a 30% increase in productivity compared to the beta-oxidation-blocked strain in fermentations. The bioconversion is effective for the selective terminal oxidation of both saturated and unsaturated linear aliphatic substrates with chain-lengths ranging from 12 carbons to 22 carbons and also avoids the undesirable chain modifications associated with passage through the beta-oxidation pathway, such as unsaturation, hydroxylation, or chain shortening. It is now possible to efficiently produce a wide range of previously unavailable saturated and unsaturated dicarboxylic acids with a high degree of purity.

PFGE mapping and RFLP analysis of the S/D region of the mouse H-2 complex.

We have constructed a long range restriction map of the S/D segment of the mouse H-2 complex by pulsed field gel electrophoresis and hybridization with mouse cDNA probes to Bf and Tnfa genes and human cDNA probes to BAT2, BAT3, BAT4, BAT5, and BAT6 genes which have recently been mapped to the human HLA complex between C2 and HLA-B. The distance between the mouse C2 and Tnfa genes was found to be approximately 350 kilobases. The position of the mouse Bat genes in this map were found to be comparable to the position of the BAT genes in the human HLA complex. A panel of recombinant mouse strains was also examined by restriction fragment analysis with probes detecting the Hsp70, Bat5, and Tnfa genes. The results indicate that recombination in this segment is not random. No recombinants were found with crossovers between the C2 and Hsp70 genes and only one recombinant was found with a crossover between Tnfa and H-2D. In contrast, the crossover sites of 16 recombinants were mapped between the Hsp70 and Tnfa genes. Seven of these recombinants were found to have crossovers between Hsp70 and Bat5 and three recombinants were found to have crossover sites between Bat5 and Tnfa.

The vertical transmission of salmonellas and formic acid treatment of chicken feed. A possible strategy for control.

The treatment of feed given to laying hens with 0.5% formic acid reduced significantly the isolation rate of salmonellas and was associated with a reduction in the incidence of infection in newly hatched chicks. These improvements were not sustained until slaughter, however, as growing birds acquired salmonellas, probably from feed which was not acid treated. The data indicate that formic acid treatment of chicken food could have important benefits for the public health.

Salmonella and Campylobacter contamination of broiler chicken carcasses and scald tank water: the influence of water pH.

Scalding at 50 degrees +/- 0.5 degrees C, in water maintained at pH 9.0 +/- 0.2 by the addition of sodium hydroxide, had no effect on the incidence of salmonella or campylobacter contamination of chicken carcasses. There were significant reductions, however, in the numbers of these organisms in the water itself.

The influence of scald water pH on the death rates of Salmonella typhimurium and other bacteria attached to chicken skin.

Factory trials where scald tank water was maintained at pH 9.0 +/- 0.2 showed that compared with the usual system of scalding when the water is at pH 6.0 for much of the working day the bacterial counts on carcases post scalding and plucking were significantly lower. In laboratory experiments, attached Salmonella typhimurium and the naturally occurring skin flora were found to be killed significantly more quickly in water at pH 9.0 +/- 0.2.