Disorders of sexual development and associated changes in the pituitary-gonadal axis in dogs.

Normal sexual differentiation depends on completion of chromosomal sex determination, gonadal differentiation, and development of the phenotypic sex. An irregularity in any of these three steps can lead to a disorder in sexual development (DSD). We examined nine dogs with DSD by abdominal ultrasonography, laparotomy, histologic examination of the gonads, and reproductive tract, cytogenetic analysis, and mRNA expression of the SRY gene. We also determined the plasma concentrations of luteinizing hormone (LH), estradiol-17ß, and testosterone before and after administration of gonadotropin-releasing hormone (GnRH) and compared these results with those obtained in anestrous bitches and male control dogs. The gonads of three dogs with DSD contained both testicular and ovarian tissue, while in the other six only testicular tissue was found. Each of the dogs had a uterus. Based on gynecologic examination, cytogenetic analysis, and the histology of the gonads, seven of the nine dogs appeared to be XX sex reversals. Three of these were XX true hermaphrodites and four were XX males; the other two dogs had incomplete XY gonadal dysgenesis. All seven XX sex-reversed dogs were found to be negative for the SRY gene by polymerase chain reaction. The basal plasma luteinizing hormone (LH) concentration was significantly higher in dogs with DSD than in anestrous bitches but not significantly different from that in male dogs. The basal plasma LH concentration increased significantly after GnRH administration in all dogs with DSD. The basal plasma estradiol concentration was significantly higher in dogs with DSD than in anestrous bitches but not significantly different from that in male dogs. The basal plasma testosterone concentration was lower in dogs with DSD than in male dogs. In all dogs with DSD both the basal and GnRH-induced plasma testosterone concentrations were above the upper limit of their respective ranges in the anestrous bitches. In conclusion, the secretion of LH and estradiol in these dogs with DSD, all of which had testicular tissue in their gonads, was similar to that in male control dogs. These results indicate that the basal and/or GnRH-stimulated plasma testosterone concentration might be used to detect the presence of testicular tissue in dogs with DSD.

Identification of genes targeted by FSH and oocytes in porcine granulosa cells.

In the mammalian ovarian follicle maturing oocytes are nurtured and supported by surrounding somatic cells, the mural granulosa cells and the cumulus cells. These cells are regulated by follicle-stimulating hormone (FSH), originating from the pituitary, and paracrine factors derived from the oocyte. To gain insight into the mechanisms involved in the regulation of granulosa cell function, this study aimed to identify genes in mural granulosa cells that are regulated by FSH and oocyte secreted factors using the pig as a model organism. Mural granulosa cells were collected from 3-6 mm follicles from sow ovaries and cultured in serum free medium in the presence or absence of FSH and/or isolated cumulus oocyte complexes (COCs). FSH significantly increased both the metabolic activity and progesterone production of granulosa cells, while the presence of COCs reversed these FSH effects. Expression levels of mRNA in the absence/presence of FSH and COCs were analyzed on porcine specific microarrays representing 11,300 genes. Both previously identified and novel FSH target genes as well as some oocyte affected genes were found. Expression of inhibitor of DNA binding protein 2 and 3, ID2 and ID3, was decreased by FSH but increased by COCs, as validated by quantitative PCR. These proteins function as dominant negative basic helix loop helix (bHLH) transcription factors and since all regulated genes contain the consensus E-box sequence that can bind bHLH factors, our data suggest that FSH and COCs may regulate granulosa cell function by tuning the activity of bHLH factors, through ID2 and ID3.

Persistent Mullerian duct syndrome in a Miniature Schnauzer dog with signs of feminization and a Sertoli cell tumour.

A 5-year-old male Miniature Schnauzer was presented with unilateral cryptorchidism and signs of feminization. Abdominal ultrasonography revealed an enlarged right testis and a large, fluid-filled cavity that appeared to arise from the prostate. Computed tomography revealed the cavity to be consistent with an enlarged uterine body, arising from the prostate, and showed two structures resembling uterine horns that terminated close to the adjacent testes. The dog had a normal male karyotype, 78 XY. Gonadohysterectomy was performed and both the surgical and the histological findings confirmed the presence of a uterus in this male animal, resulting in a diagnosis of persistent Mullerian duct syndrome (PMDS). The enlarged intra-abdominal testis contained a Sertoli cell tumour. Computed tomography proved to be an excellent diagnostic tool for PMDS.

Minimal external masculinization in a SRY-negative XX male Podenco dog.

Normal mammalian sex differentiation takes place in three genetically controlled steps: chromosomal sex determination (XX or XY), gonadal differentiation and development of the phenotypic sex. Animals are considered to be sex reversed if chromosomal sex determination and gonadal development are not in agreement. In this report, sex reversal is described in a 1.5-year-old Podenco dog that was referred because of suspected recurrent growth of a previously removed os clitoridis in the vulva. With that exception the dog was phenotypically female, but had never been in oestrus and exhibited male behaviour. Abdominal ultrasonography showed a small tubular structure dorsal to the bladder, consistent with a uterus. An ovoid structure resembling a gonad was visible between the right kidney and inguinal canal. Plasma testosterone concentrations before and after GnRH administration indicated the presence of functional testicular tissue. Two testes, each with its epididymis and ductus deferens, and a complete bicornuate uterus were removed surgically. Cytogenetic analysis of peripheral blood lymphocytes showed a normal female karyotype (78, XX). These findings are consistent with the diagnosis of an XX male. PCR analysis of genomic DNA revealed that the SRY gene was absent. In summary, this report describes the first SRY-negative XX male Podenco dog with an almost complete female phenotype despite high basal and stimulated plasma testosterone concentrations. It is hypothesized that the clinical observations in this dog may have been caused by reduced and delayed Müllerian-inhibiting substance secretion and the absence of conversion of testosterone to dihydrotestosterone due to 5alpha-reductase deficiency.

Blastocyst morphology, actin cytoskeleton quality and chromosome content are correlated with embryo quality in the pig.

Embryo survival rates obtained after transfer of in vitro produced porcine blastocysts are very poor. This is probably related to poor quality of the embryos. The aim of the present study was to determine markers for good quality blastocysts. Therefore, we tried to link blastocyst morphology to several morphological and cell biological properties, and evaluated the survival of in vitro produced, morphologically classified, blastocysts following non-surgical transfer. In vitro and in vivo produced blastocysts were allocated to two groups (classes A and B) on the basis of morphological characteristics. The quality of their actin cytoskeleton, their total cell number, their ability to re-expand after cytochalasin-B treatment and the occurrence of numerical chromosome aberrations were studied and compared. In vivo produced blastocysts were used as a control. Our results indicate that the ability of blastocysts to re-expand after cytochalasin-B-induced actin depolymerization was positively correlated with the morphology of the blastocyst, and associated with the quality of the actin cytoskeleton. Chromosome analysis revealed that mosaicism is inherent to the in vitro production of porcine embryos, but also that in vivo produced blastocysts contained some non-diploid cells. In non-surgical embryo transfer experiments more recipients receiving class A blastocysts were pregnant on Day 20 than those receiving class B blastocysts. One recipient gave birth to six piglets from class A in vitro produced blastocysts, providing a verification of the enhanced viability of blastocysts that were scored as 'good' on the basis of their morphology.

Intravitreal triamcinolone for the treatment of refractory macular edema in idiopathic intermediate or posterior uveitis.

PURPOSE: Cystoid macular edema (CME) is the most significant cause of visual loss associated with idiopathic uveitis. The authors report on the use of intravitreal triamcinolone acetonide (IVTA) in a group of patients with macular edema due to idiopathic intermediate and posterior uveitis. METHODS: Retrospective, noncomparative, interventional case series. Thirty-three eyes were included with uveitic CME that was refractory to topical steroids, oral prednisone, or a combination thereof. Previous steroid treatment did not result in elevated intraocular pressure (IOP). The eyes received an intravitreal injection with 10 mg triamcinolone acetonide, after best-corrected visual acuity (BCVA) and fluorescein angiography (FA) were assessed. Ophthalmologic examination including FA was regularly performed during a 1-year follow-up period. RESULTS: Within 12 weeks after injection of IVTA, 50% of the eyes responded with an improvement in vision of more than two lines and 30% of the eyes reached an IOP of > or = 21 mmHg (p<0.01). All eyes with an elevated IOP responded well on topical antiglaucoma medication. After 12 months follow-up 40% of the eyes responded with an improvement in vision of more than two lines and 28% of the affected eyes underwent phacoemulsification during the follow-up. No other complications occurred within a year after the treatment. CONCLUSIONS: In macular edema due to idiopathic intermediate or posterior uveitis IVTA improves the visual acuity within the first 3 months. However, thereafter the visual acuity decreases again. Cataract and elevated IOP are common side effects.

Physiological and molecular responses of Lactuca sativa to colonization by Salmonella enterica serovar Dublin.

This paper describes the physiological and molecular interactions between the human-pathogenic organism Salmonella enterica serovar Dublin and the commercially available mini Roman lettuce cv. Tamburo. The association of S. enterica serovar Dublin with lettuce plants was first determined, which indicated the presence of significant populations outside and inside the plants. The latter was evidenced from significant residual concentrations after highly efficient surface disinfection (99.81%) and fluorescence microscopy of S. enterica serovar Dublin in cross sections of lettuce at the root-shoot transition region. The plant biomass was reduced significantly compared to that of noncolonized plants upon colonization with S. enterica serovar Dublin. In addition to the physiological response, transcriptome analysis by cDNA amplified fragment length polymorphism analysis also provided clear differential gene expression profiles between noncolonized and colonized lettuce plants. From these, generally and differentially expressed genes were selected and identified by sequence analysis, followed by reverse transcription-PCR displaying the specific gene expression profiles in time. Functional grouping of the expressed genes indicated a correlation between colonization of the plants and an increase in expressed pathogenicity-related genes. This study indicates that lettuce plants respond to the presence of S. enterica serovar Dublin at physiological and molecular levels, as shown by the reduction in growth and the concurrent expression of pathogenicity-related genes. In addition, it was confirmed that Salmonella spp. can colonize the interior of lettuce plants, thus potentially imposing a human health risk when processed and consumed.

Comparison of methods of extracting Salmonella enterica serovar Enteritidis DNA from environmental substrates and quantification of organisms by using a general internal procedural control.

This paper compares five commercially available DNA extraction methods with respect to DNA extraction efficiency of Salmonella enterica serovar Enteritidis from soil, manure, and compost and uses an Escherichia coli strain harboring a plasmid expressing green fluorescent protein as a general internal procedural control. Inclusion of this general internal procedural control permitted more accurate quantification of extraction and amplification of S. enterica serovar Enteritidis in these samples and reduced the possibility of false negatives. With this protocol it was found that the optimal extraction method differed for soil (Mobio soil DNA extraction kit), manure (Bio101 soil DNA extraction kit), and compost (Mobio fecal DNA extraction kit). With each method, as little as 1.2 x 10(3) to 1.8 x 10(3) CFU of added serovar Enteritidis per 100 mg of substrate could be detected by direct DNA extraction and subsequent S. enterica-specific TaqMan PCR. After bacterial enrichment, as little as 1 CFU/100 mg of original substrate was detected. Finally, the study presents a more accurate molecular analysis for quantification of serovar Enteritidis initially present in soil or manure using DNA extraction and TaqMan PCR.

A Multiplex Real-Time Polymerase Chain Reaction (TaqMan) Assay for the Simultaneous Detection of Meloidogyne chitwoodi and M. fallax.

ABSTRACT This study describes a multiplex real-time polymerase chain reaction (PCR) approach for the simultaneous detection of Meloidogyne chitwoodi and M. fallax in a single assay. The approach uses three fluorogenic minor groove binding (MGB) TaqMan probes: one FAM-labeled to detect M. chitwoodi, one VIC-labeled to detect M. fallax, and one NED-labeled to detect the internal amplification control (IAC) to monitor false negative results. One common primer set is used for the amplification of part of the internal transcribed spacer (ITS) region of M. chitwoodi and M. fallax and one primer set for the amplification of the IAC. The test enabled detection of M. chitwoodi and/or M. fallax in DNA samples extracted from batches of juveniles, from single juveniles, and from infected plant material. Compared with current assays to detect M. chitwoodi and M. fallax, the multiplex real-time PCR offers the following advantages: it is faster because the test can simultaneously detect both quarantine species without the need for post-PCR processing; and it is at least 10 times more sensitive than a comparable regular PCR also targeting the ITS sequence. Inclusion of the IAC facilitates the interpretation of the FAM and VIC cycle threshold (Ct) values and can prevent the scoring of false negative results when FAM, VIC, and NED Ct values are high. The test allows precise quantification when only one of the two species is present in the sample. However, experiments with mixtures of genomic DNA of M. chitwoodi and M. fallax revealed that the ability of the multiplex real-time PCR assay to detect small quantities of DNA of one species is reduced when large quantities of DNA of the other species are present.

The canine sarcoglycan delta gene: BAC clone contig assembly, chromosome assignment and interrogation as a candidate gene for dilated cardiomyopathy in Dobermann dogs.

Dilated cardiomyopathy (DCM) is a common disease of the myocardium recognized in human, dog and experimental animals. Genetic factors are responsible for a large proportion of cases in humans, and 17 genes with DCM causing mutations have been identified. The genetic origin of DCM in the Dobermann dogs has been suggested, but no disease genes have been identified to date. In this paper, we describe the characterization and evaluation of the canine sarcoglycan delta (SGCD), a gene implicated in DCM in human and hamster. Bacterial artificial chromosomes (BACs) containing the canine SGCD gene were isolated with probes for exon 3 and exons 4-8 and were characterized by Southern blot analysis. BAC end sequences were obtained for four BACs. Three of the BACs overlapped and could be ordered relative to each other and the end sequences of all four BACs could be anchored on the preliminary assembly of the dog genome sequence (www. ensembl.org). One of the BACs of the partial contig was localized by fluorescent in situ hybridization to canine chromosome 4q22, in agreement with the dog genome sequence. Two highly informative polymorphic microsatellite markers in intron 7 of the SGCD gene were identified. In 25 DCM-affected and 13 non DCM-affected dogs seven different haplotypes could be distinguished. However, no association between any of the SGCD variants and the disease locus was apparent.

Comparison of real-time PCR methods for detection of Salmonella enterica and Escherichia coli O157:H7, and introduction of a general internal amplification control.

The objectives of this study were to compare different real-time PCR-based methods for detection of either Salmonella spp. or E. coli O157:H7 with respect to sensitivity, precision and accuracy. In addition, a general internal amplification control (IAC) is presented, allowing prevention of false negative results. The IAC allows insight in amplification efficiency and enables a more accurate quantification with the evaluated real-time PCR methods. Implementation of the IAC with the different PCR methods did not affect the precision of the methods, but the sensitivity was reduced 10-fold. Introduction of an IAC with the Salmonella enterica specific detection method showed a shift in Ct-value (increase of target Ct-value with 0.45+/-0.17 cycles), while with the method to detect E. coli O157:H7 no influence of IAC co-amplification was observed. The quantification threshold of the methods in which the IAC was included was determined at 1 pg of target DNA (equal to 200 CFU) per reaction. Qualitative detection was feasible down to 10 fg of target DNA per reaction using both methods in which the IAC was incorporated. The adjusted methods have the potential to provide fast and sensitive detection of Salmonella spp. or E. coli O157:H7, enabling accurate quantification and preventing false negative results by using the general IAC.

Comparative chromosome painting between the domestic pig (Sus scrofa) and two species of peccary, the collared peccary (Tayassu tajacu) and the white-lipped peccary (T. pecari): a phylogenetic perspective.

The Suidae and the Dicotylidae (or Tayassuidae) are related mammalian families, both belonging to the artiodactyl suborder Suiformes, which diverged more than 37 million years ago. Cross-species chromosome painting was performed between the domestic pig (Sus scrofa; 2n = 38), a representative of the Suidae, and two species of the Dicotylidae: the collared peccary (Tayassu tajacu; 2n = 30) and the white-lipped peccary (T. pecari; 2n = 26). G-banded metaphase chromosomes of the two peccaries were hybridized with whole chromosome painting probes derived from domestic pig chromosomes 1-18 and X. For both peccary species, a total of 31 autosomal segments that are conserved between pig and peccary could be identified. The painting results confirm conclusions inferred from G-band analyses that the karyotypes of the collared peccary and the white-lipped peccary are largely different. The karyotypic heterogeneity of the Dicotylidae contrasts with the relative homogeneity among the karyotypes of the Suidae. For this difference between the Dicotylidae and the Suidae, a number of explanations are being postulated: 1) the extant peccaries are phylogenetically less closely related than is usually assumed; 2) the peccary genome is less stable than the genome of the pigs; and 3) special (e.g. biogeographical or biosocial) circumstances have facilitated the fixation of chromosome rearrangements in ancestral dicotylid populations.

Fourteen chromosomal localizations and an update of the cytogenetic map of the rabbit.

In order to improve the informativeness of the cytogenetic map of the rabbit genome, fourteen markers were regionally mapped to individual chromosomes. The localizations comprise eleven gene loci (PRLR, GHR, HK1, ACE, TF, 18S+28S rDNA, CYP2C4, PMP2, TCRB, ALOX15 and MT1) and three microsatellite loci (Sat13, Sol33 and D1Utr6). Five of the genes contain known microsatellite sequences. To achieve these localizations, homologous and heterologous small insert clones, and clones from a rabbit Bacterial Artificial Chromosome (BAC) library were used as probes for fluorescence in situ hybridization experiments. Results indicate that especially BAC clones are a valuable tool for cytogenetic mapping. Some of the genes were selected for mapping on the basis of human- rabbit comparative painting data, to achieve localizations on gene-poor rabbit chromosomes. Our data are, in general, in agreement with the human-rabbit comparative painting data. By mapping microsatellite sequences that have also been used in linkage studies, links are provided between the genetic and physical maps of the rabbit genome. Linkage groups I, VI and XI could be assigned to chromosomes 1, 5 and 3 respectively. Moreover, in this paper we give an overview of the current status of the rabbit cytogenetic map. This map now comprises 62 physically mapped genes, which are scattered over all autosomes, except chromosome 2, and the X chromosome.

A radiation hybrid map of the X-chromosome of the dog (Canis familiaris).

The dog serves as an animal model for several human diseases including X-chromosome diseases. Although the canine X-chromosome is one of the largest chromosomes in the dog, only a few markers have been mapped to it to date. Using a commercially available canine whole genome radiation hybrid (RH) panel we have localized 14 microsatellite markers, 18 genes and 13 STSs on the canine X-chromosome, extending the total number of mapped markers to 45 covering an estimated 830 cR. Out of these 45 markers, seven distinct groups of markers could be established with an average spacing of 18.8 cR(3000) and ten markers remained unlinked. Using FISH analysis, six markers could be mapped physically to the p- or q-arm of the X-chromosome. Combined with the FISH mapping, three RH groups could be assigned to the p-arm and two RH groups to the q-arm. Comparison with the human X-chromosome map revealed conserved synteny up to 234 cR (TIMP1-ALAS2-AR-IL2RG-XIST). We show here that the similarity of the canine and human X-chromosomes is the largest for any mammalian species beyond the primates.

Establishment of an R-banded rabbit karyotype nomenclature by FISH localization of 23 chromosome-specific genes on both G- and R-banded chromosomes.

Direct detection of fluorescent in situ hybridization signals on R-banded chromosomes stained with propidium iodide is a rapid and efficient method for constructing cytogenetic maps for species with R-banded standard karyotypes. In this paper, our aim is to establish an R-banded rabbit karyotype nomenclature that is in total agreement with the 1981 G-banded standard nomenclature. For this purpose, we have produced new GTG- and RBG-banded mid-metaphase karyotypes and an updated version of ideograms of R-banded rabbit chromosomes. In addition, to confirm correlations between G- and R-banded chromosomes, we have defined a set of 23 rabbit BAC clones, each containing a specific gene, one marker gene per rabbit chromosome, and we have localized precisely each BAC clone by FISH on both G- and R-banded chromosomes.