How well does physician selection of microbiologic tests identify Clostridium difficile and other pathogens in paediatric diarrhoea? Insights using multiplex PCR-based detection.

The objective of this study was to compare the aetiologic yield of standard-of-care microbiologic testing ordered by physicians with that of a multiplex PCR platform. Stool specimens obtained from children and young adults with gastrointestinal illness were evaluated by standard laboratory methods and a developmental version of the FilmArray Gastrointestinal (GI) Diagnostic System (FilmArray GI Panel), a rapid multiplex PCR platform that detects 23 bacterial, viral and protozoal agents. Results were classified according to the microbiologic tests requested by the treating physician. A median of three (range 1-10) microbiologic tests were performed by the clinical laboratory during 378 unique diarrhoeal episodes. A potential aetiologic agent was identified in 46% of stool specimens by standard laboratory methods and in 65% of specimens tested using the FilmArray GI Panel (p < 0.001). For those patients who only had Clostridium difficile testing requested, an alternative pathogen was identified in 29% of cases with the FilmArray GI Panel. Notably, 11 (12%) cases of norovirus were identified among children who only had testing for Clostridium difficile ordered. Among those who had C. difficile testing ordered in combination with other tests, an additional pathogen was identified in 57% of stool specimens with the FilmArray GI Panel. For patients who had no C. difficile testing performed, the FilmArray GI Panel identified a pathogen in 63% of cases, including C. difficile in 8%. Physician-specified laboratory testing may miss important diarrhoeal pathogens. Additionally, standard laboratory testing is likely to underestimate co-infections with multiple infectious diarrhoeagenic agents.

DNA-based animal models of human disease: from genotype to phenotype.

Biomedical research utilizes animal models to elucidate human disease processes at the cellular and molecular level and for the development of new therapies. Traditionally, mammalian models have been limited to the mouse, primarily because of well characterized genetic lines and the ability to manipulate the genome to directly test hypotheses regarding causal mutations and disease phenotypes. The emerging availability of genome sequences of other mammals (bovine, canine, equine, feline, and porcine) now permits utilization of the mammal in which the phenotype best approximates the human condition. Equally important is the use of somatic cell nuclear cloning (SCNT) coupled with targeted germline manipulation to create animals to resolve the molecular mechanisms of the disease state. Our efforts have focused on the pig, which has emerged as an important biomedical mammalian model due to its closer physiology to humans. The utility of porcine genetically-defined tumour, cardiovascular and neurological disease models is described.

Robertsonian metacentrics of the house musk shrew (Suncus murinus, Insectivora, Soricidae) lose the telomeric sequences in the centromeric area.

The house musk shrew, Suncus murinus, is polymorphic for five Robertsonian translocations (Rb8.17, 9.13, 10.12, 11.16, 14.15). Fluorescence in situ hybridisation with a biotin-labelled oligonucleotide, (TTAGGG)7, was performed to localise the telomeric DNA sequences at Rb chromosomes of heterozygous shrews. Hybridisation signals were observed at both ends of all chromosomes, but not at the pericentromeric areas of any of the Robertsonian metacentrics. Our results indicate a complete loss of the telomeric sequences at the fusion points of the Rb metacentrics in S. murinus.

Chromosomal segregation and fertility in Robertsonian chromosomal heterozygotes of the house musk shrew (Suncus murinus, Insectivora, Soricidae).

Crucial to our understanding of chromosomal variation and evolution in mammals are detailed studies of chromosomal heterozygotes, with analyses of chromosomal segregation and chromosome-derived infertility. We studied segregation and fertility in hybrids between karyotypic races of the house musk shrew Suncus murinus. These individuals were heterozygous for up to five Robertsonian fusions (Rbs) and an insertion of heterochromatin in an autosome. All variant chromosomes showed Mendelian segregation and all Rbs segregated independently of each other in the progeny of double heterozygotes. Litter size in single and even multiple Rb heterozygotes was no smaller than that in the less fertile parental strain. The effects of genetic background were more important in determining litter size than Rb heterozygosity for the shrews that we examined.

An XXY sex chromosome constitution in a house musk shrew (Suncus murinus L.) with testicular hypoplasia.

An adult male house muck shrew with an XXY sex chromosome constitution was found in a laboratory-bred colony. Maternal origin of the additional X chromosome was demonstrated. The external appearance of the animal was normal, but the testes were small and displayed a high density of interstitial cells. The seminiferous tubules were narrow and contained only Sertoli cells.

Inheritance of litter size at birth in the house musk shrew (Suncus murinus, Insectivora, Soricidae).

In this research we estimated the contribution of a major-gene effect to the control of litter size in hybrids between two local populations of the house musk shrew (Suncus murinus). Segregation analysis was performed on the basis of a mixed polygene and major-gene model. The model presumes that two parental populations may differ from each other in gene frequencies and in the values of polygenic effects but not in the major-gene contribution of the trait. Moreover, the peculiarity of the trait--litter size--is taken into account. This trait is not an individual attribute. It characterizes the parental couple and may depend on the genotypes of both parents. Results of segregation analysis of a large hybrid pedigree of Suncus murinus indicate that the parental populations differ in the allele frequency of the major gene (one population is homozygous, while the other contains the two alleles in approximately equal proportions) and in the values of average polygenic effects. Both major-gene and polygenic components are necessary for the correct description of litter size inheritance in interracial hybrids of S murinus, inasmuch as the exclusion of either of them leads to a significant drop in likelihood. The Elston-Stewart criterion also confirms the Mendelian inheritance of the major gene.

Chromosome pairing in inter-racial hybrids of the house musk shrew (Suncus murinus, Insectivora, Soricidae).

Two chromosome races of the house shrew Suncus murinus that differ from each other for five Robertsonian translocations (8.17, 9.13, 10.12, 11.16, and 14.15), heterochromatic insertions in chromosomes 7 and X, and multiple rearrangements in the Y chromosome were crossed and then intercrossed in captivity to produce a hybrid stock. Electron-microscopic analysis of synaptonemal complexes in fertile and sterile hybrid males was carried out. Meiosis in sterile males did not progress beyond pachytene and was severely disrupted. Meiotic arrest was not determined by structural heterozygosity: heterozygotes for all variant chromosomes distinguishing two parental races were found in both sterile and fertile male hybrids. Fertile hybrids demonstrated an orderly pairing of all chromosomes. In heterozygotes for Robertsonian fusions, completely paired trivalents were formed between the Robertsonian metacentrics and homologous acrocentrics. In heterozygotes for chromosome 7, bivalents with a small buckle were observed in a small fraction of pachytene cells. No differences were found in the morphology and pairing pattern of sex bivalents, composed of the X and Y chromosomes derived from the same or different parental races. Univalents, multivalents, and associations between X and Y chromosomes and autosomal trivalents, as well as associations of autosomal trivalents with each other, were observed in a small fraction of the pachytene cells of fertile males. Our results indicate that the system controlling male sterility in interracial hybrids of S. murinus is of genic rather than of chromosomal type.

Inheritance of male hybrid sterility in the house musk shrew (Suncus murinus, Insectivora, Soricidae).

Two geographic races of the house musk shrew (Suncus murinus) were crossed and intercrossed in the laboratory. Many cases of male sterility were detected among the hybrids. Segregation analysis of the pedigree data showed that the inheritance of male sterility in interracial hybrids of S. murinus can be described within the framework of monogene polyallele model with sterility of a single allele combination. This model is similar if not identical to that proposed by Dobzhansky and Muller.

Localization of the genes for major ribosomal RNA on chromosomes of the house musk shrew, Suncus murinus, at meiotic and mitotic cells by fluorescence in situ hybridization and silver staining.

The genes for major ribosomal RNA were localized on chromosomes 5pter-p15, 9q64-qter, and 13q38-qter of the house musk shrew, Suncus murinus (Insectivora, Soricidae) by silver staining of mitotic metaphase and meiotic pachytene spreads and fluorescence in situ hybridization using the human 28S-RNA genes as a probe to mitotic metaphase spreads. The data presented indicate a correlation between sites of in situ hybridization and silver staining. The finding of nuclear materials in mitosis was in a good agreement with observation in meiosis: same chromosomes carried active NORs in both meiotic and mitotic cells.

Robertsonian chromosomal variation in the house musk shrew (Suncus murinus, Insectivora:Soricidae) and the colonization history of the species.

A high-resolution G-banding technique was used to identify five metacentrics that characterize Suncus murinus from Sri Lanka. These metacentrics were shown to be the product of Robertsonian fusion of acrocentric chromosomes identical to those in the standard karyotype defined by M.B. Rogatcheva et al. Two of the metacentrics in the Sri Lankan shrews (Rb(10.12) and Rb(14.15)) were the same as those reported by C.H. Sam et al. in Malayan populations of S. murinus. This finding provides strong support for the suggestion of T.H. Yosida that metacentric-carrying shrews colonized Malaya from Sri Lanka and hybridized with individuals of standard karyotype, generating the Robertsonian polymorphism now observed. In addition to the Robertsonian variation in S. murinus, we have used our high resolution technique (G- and C-banding) to characterize variants on chromosome 7, the X chromosome, and the Y chromosome.

Segregation analysis of animal pedigree data from inter-population crosses.

The general method of segregation analysis of pedigree data has been developed and widely used in human genetics. We modified this method to examine pedigree data coming from inter-population crosses. These kinds of pedigrees are common in laboratory and farm animal breeding. This paper describes a rationale for the method and illustrates its application to the study of inheritance of litter size and of male sterility in hybrid stock of the house musk shrew (Suncus murinus) derived from crosses of two geographically isolated populations.

Pattern of X-Y chromosome pairing in the Japanese field vole, Microtus montebelli.

Pairing of X and Y chromosomes at meiotic prophase in males of Microtus montebelli was analyzed. The sex chromosomes form a synaptonemal complex at pachytene and end-to-end association at diakinesis-metaphase I in two species of the genus Microtus (M. montebelli and M. oeconomus) only, while they do not pair at all in the other species of this genus that have been studied so far. These data confirm that M. montebelli and M. oeconomus are very closely related in their origin. It is suggested that the sex chromosomes of M. montebelli and M. oeconomus display the ancestral type of X-Y pairing. The lack of X-Y pairing in most species of Microtus appeared after the split in lineage that led to M. oeconomus and M. montebelli on the one hand and the remaining species on the other.