Amplification of sequence tagged sites in five avian species using heterologous oligonucleotides.

Short of a complete genomic DNA sequence, sequence tagged sites (STSs) have emerged as major genomic reagents for the genetic analysis of little-studied ecologically and agriculturally important organisms. Here, we report STS developed for the turkey (Meleagris gallopavo), guinea fowl (Numidea meleagris), Japanese quail (Coturnix coturnix) and pigeon using primers specific for reference DNA sequences of two chicken (Gallus gallus) genes, aggrecan (agc1) and type X collagen (col10). Additional STSs were also developed for turkey, quail and chicken using primers specific for the human apobec-1 gene. The total length of the STSs developed was 5990, 2522, 4127, 1539 and 6600 bp for the turkey, guinea fowl, Japanese quail, pigeon and chicken, respectively. Based on splice site consensus GT and AG sequences, four of the seven agc1-based chicken STS appear to contain introns. The human gene-based STSs showed no significant sequence identity with the reference GenBank sequences. Maximum likelihood, maximum parsimony and neighbour-joining analysis of an agc1-based STS that was common to all five species showed phylogenetic relationships consistent with those previously defined using mitochondria DNA sequences and nuclear gene restriction maps. Additionally, several putative single nucleotide polymorphisms (SNPs) were detected within the STSs, including eight in the turkey, two in the quail, and two in the chicken when multiple sequences were evaluated from each species. This report describes new STSs that are resources for genetic and physical mapping and genome analysis within and among avian species. These resources should further aid in our understanding of the biology of agriculturally important but little-studied guinea fowl and turkey.

Comparative DNA sequence analysis of genetic variation in the African grey parrot, Psittacus erythacus.

Comparative genome analysis promises to provide an insight into avian species that have been very little studied. To test the feasibility of this approach, we investigated the use of heterologous primers to generate single nucleotide polymorphisms (SNP) in the African grey parrot, Psittacus erythacus, using primers specific for chicken and turkey DNA fragments. Three of the primers were specific for three expressed sequence tagged sites in the turkey and the fourth for a chicken proteoglycan core protein-like DNA sequence. A total of about 2200 bp of the parrot genome was evaluated for DNA sequence variation. Seven SNPs were identified and confirmed by Mendelian segregation. The frequency distribution of the most common nucleotide at each SNP locus in an unrelated group of parrots ranged from 0.84 to 0.97. The percent similarity of each parrot sequence to the reference sequence was inconsistent and ranged from zero to 100%. The primers as well as the nucleotide variants described represent valuable resources for genetic analysis in the parrot.

Supplementation with Lactobacillus reuteri or L. acidophilus reduced intestinal shedding of cryptosporidium parvum oocysts in immunodeficient C57BL/6 mice.

The effect of L. acidophilus supplementation to reduce fecal shedding of Cryptosporidium parvum oocysts was compared to L. reuteri using C57BL/6 female mice immunosuppressed by murine leukemia virus (strain LP-BM5) inoculation. After 12 weeks post LP-BM5 inoculation, 15 immunosuppressed mice each were randomly assinged to one of the following treatment groups: historical control (group A), LP-BM5 control (group B), C. parvum (group C), L. reuteri plus C. parvum (group D) or L. acidophilus plus C. parvum (group E). Mice were pre-fed the L. reuteri or L. acidophilus bacteria strains daily for 13 days, challenged with C. parvum oocysts and thereafter fed the specified Lactobacillus regimens daily during the experimental period. Animals supplemented with L. reuteri shed fewer (p<0.05) oocysts on day-7 post C. parvum challenge compared to controls. Mice supplemented with L. acidophilus also shed fewer (p<0.05) oocysts on days 7 and 14 post-challenge compared to controls. Overall, Lactobacillus supplementation reduced C. parvum shedding in the feces but failed to suppress the production of T-helper type 2 cytokines [interleukin-4 (IL-4), IL-8)] which are associated with immunosuppression. Additionally, Lactobacillus supplementation did not restore T-helper type 1 cytokines (interleukin-2 (IL-2) and gamma interferon (IFN-gamma), which are required for recovery from parasitic infections. Altered T-helper types 1 and 2 cytokine production as a consequence of immunodysfunction permitted the development of persistent cryptosporidiosis while mice with intact immune system were refractory to infection with C. parvum. Reduction in shedding of oocysts observed in the Lactobacillus supplemented mice during deminished IL-2 and IFN-gamma production may be mediated by factors released into the intestinal lumen by the Lactobacillus and possibly other host cellular mechanisms. These observations suggest that L. reuteri or L. acidophilus can reduce C. parvum parasite burdens in the intestinal epithelium during cryptosporidiosis and may serve potential benefits as probiotics for host resistance to intestinal parasitic infections. L. acidophilus was more efficacious in reducing fecal shedding than L. reuteri and therefore may also have implication in the therapy of cryptosporidiosis during immunosuppressive states including human AIDS.

Effect of Lactobacillus reuteri on intestinal resistance to Cryptosporidium parvum infection in a murine model of acquired immunodeficiency syndrome.

Efficacy of Lactobacillus reuteri as a probiotic for the control of Cryptosporidium parvum infection was evaluated in C57BL/6 female mice that were immunosuppressed by intraperitoneal inoculation with the LP-BM5 leukemia virus. Four months after inoculation, mice developed lymphadenopathy, splenomegaly, and susceptibility to C. parvum infection. After daily prefeeding with L. reuteri (10(8) cfu/day) for 10 days, mice were challenged with 6.5 x 10(6) C. parvum oocysts and fed L. reuteri during the entire study. Mice supplemented with L. reuteri and challenged with C. parvum cleared parasite loads from the gut epithelium. However, unsupplemented animals developed persistent cryptosporidiosis and shed high levels of oocysts in the feces. L. reuteri feeding increased its colonization of the intestinal tract, which was inversely related to the fecal shedding of oocysts. These findings suggest that L. reuteri may help prevent C. parvum infection in immunodeficient subjects.

LP-BM5 virus causes changes in lactic dehydrogenase and oxygen radical production in splenocytes of C57BL/6J mice.

The LP-BM5 murine leukemia virus causes acquired immunodeficiency syndrome in C57BL/6J mice (MAIDS), similar to that of AIDS in humans. The objective of this study was to determine the effect of LP-BM5 viral infection on cellular activation and membrane integrity of splenocytes. Oxidative burst in splenocytes in response to exposure to PMA (20 microg/ml) was significantly higher (p<.02) in infected than in control mice at two weeks post-infection using luminol-enhanced chemiluminescence. By 13 weeks post-infection superoxide anion production in infected mice was significantly lower when compared to controls coinciding with decreased proliferative response to mitogens. The extent of cell membrane damage as indicated by lactic dehydrogenase (LDH) activity in serum was significantly higher in infected than in control mice (p<.001). The results from this study suggests that LP-BM5 virus causes an initial stimulation of cellular activity followed by a decreased cell activation characterized by decreased proliferation of splenocytes and decreased oxygen radical production. Decreased cell membrane integrity indicated by increased LDH activity may partly be responsible for these changes.