ASD restricted and repetitive behaviors associated at 17q21.33: genes prioritized by expression in fetal brains.

Autism spectrum disorder (ASD) is a behaviorally defined condition that manifests in infancy or early childhood as deficits in communication skills and social interactions. Often, restricted and repetitive behaviors (RRBs) accompany this disorder. ASD is polygenic and genetically complex, so we hypothesized that focusing analyses on intermediate core component phenotypes, such as RRBs, can reduce genetic heterogeneity and improve statistical power. Applying this approach, we mined Caucasian genome-wide association studies (GWAS) data from two of the largest ASD family cohorts, the Autism Genetics Resource Exchange and Autism Genome Project (AGP). Of the 12 RRBs measured by the Autism Diagnostic Interview-Revised, seven were found to be significantly familial and substantially variable, and hence, were tested for genome-wide association in 3104 ASD-affected children from 2045 families. Using a stringent significance threshold (P<7.1 × 10-9), GWAS in the AGP revealed an association between 'the degree of the repetitive use of objects or interest in parts of objects' and rs2898883 (P<6.8 × 10-9), which resides within the sixth intron of PHB. To identify the candidate target genes of the associated single-nucleotide polymorphisms at that locus, we applied chromosome conformation studies in developing human brains and implicated three additional genes: SLC35B1, CALCOCO2 and DLX3. Gene expression, brain imaging and fetal brain expression quantitative trait locus studies prioritize SLC35B1 and PHB. These analyses indicate that GWAS of single heritable features of genetically complex disorders followed by chromosome conformation studies in relevant tissues can be successful in revealing novel risk genes for single core features of ASD.

TNF-α modulates genome-wide redistribution of ΔNp63α/TAp73 and NF-κB cREL interactive binding on TP53 and AP-1 motifs to promote an oncogenic gene program in squamous cancer.

The Cancer Genome Atlas (TCGA) network study of 12 cancer types (PanCancer 12) revealed frequent mutation of TP53, and amplification and expression of related TP63 isoform ΔNp63 in squamous cancers. Further, aberrant expression of inflammatory genes and TP53/p63/p73 targets were detected in the PanCancer 12 project, reminiscent of gene programs comodulated by cREL/ΔNp63/TAp73 transcription factors we uncovered in head and neck squamous cell carcinomas (HNSCCs). However, how inflammatory gene signatures and cREL/p63/p73 targets are comodulated genome wide is unclear. Here, we examined how the inflammatory factor tumor necrosis factor-α (TNF-α) broadly modulates redistribution of cREL with ΔNp63α/TAp73 complexes and signatures genome wide in the HNSCC model UM-SCC46 using chromatin immunoprecipitation sequencing (ChIP-seq). TNF-α enhanced genome-wide co-occupancy of cREL with ΔNp63α on TP53/p63 sites, while unexpectedly promoting redistribution of TAp73 from TP53 to activator protein-1 (AP-1) sites. cREL, ΔNp63α and TAp73 binding and oligomerization on NF-κB-, TP53- or AP-1-specific sequences were independently validated by ChIP-qPCR (quantitative PCR), oligonucleotide-binding assays and analytical ultracentrifugation. Function of the binding activity was confirmed using TP53-, AP-1- and NF-κB-specific REs or p21, SERPINE1 and IL-6 promoter luciferase reporter activities. Concurrently, TNF-α regulated a broad gene network with cobinding activities for cREL, ΔNp63α and TAp73 observed upon array profiling and reverse transcription-PCR. Overlapping target gene signatures were observed in squamous cancer subsets and in inflamed skin of transgenic mice overexpressing ΔNp63α. Furthermore, multiple target genes identified in this study were linked to TP63 and TP73 activity and increased gene expression in large squamous cancer samples from PanCancer 12 TCGA by CircleMap. PARADIGM inferred pathway analysis revealed the network connection of TP63 and NF-κB complexes through an AP-1 hub, further supporting our findings. Thus, inflammatory cytokine TNF-α mediates genome-wide redistribution of the cREL/p63/p73, and AP-1 interactome, to diminish TAp73 tumor suppressor function and reciprocally activate NF-κB and AP-1 gene programs implicated in malignancy.

A mutant p53/let-7i-axis-regulated gene network drives cell migration, invasion and metastasis.

Most p53 mutations in human cancers are missense mutations resulting in a full-length mutant p53 protein. Besides losing tumor suppressor activity, some hotspot p53 mutants gain oncogenic functions. This effect is mediated in part, through gene expression changes due to inhibition of p63 and p73 by mutant p53 at their target gene promoters. Here, we report that the tumor suppressor microRNA let-7i is downregulated by mutant p53 in multiple cell lines expressing endogenous mutant p53. In breast cancer patients, significantly decreased let-7i levels were associated with missense mutations in p53. Chromatin immunoprecipitation and promoter luciferase assays established let-7i as a transcriptional target of mutant p53 through p63. Introduction of let-7i to mutant p53 cells significantly inhibited migration, invasion and metastasis by repressing a network of oncogenes including E2F5, LIN28B, MYC and NRAS. Our findings demonstrate that repression of let-7i expression by mutant p53 has a key role in enhancing migration, invasion and metastasis.

Methodology of the 2009 Survey on Living with Chronic Diseases in Canada--hypertension component.

INTRODUCTION: The Survey on Living with Chronic Diseases in Canada--hypertension component (SLCDC-H) is a 20-minute cross-sectional telephone survey on hypertension diagnosis and management. Sampled from the 2008 Canadian Community Health Survey (CCHS), the SLCDC-H includes Canadians (aged ≥ 20 years) with self-reported hypertension from the ten provinces. METHODS: The questionnaire was developed by Delphi technique, externally reviewed and qualitatively tested. Statistics Canada performed sampling strategies, recruitment, data collection and processing. Proportions were weighted to represent the Canadian population, and 95% confidence intervals (CIs) were derived by bootstrap method. RESULTS: Compared with the CCHS population reporting hypertension, the SLCDC-H sample (n = 6142) is slightly younger (SLCDC-H mean age: 61.2 years, 95% CI: 60.8-61.6; CCHS mean age: 62.2 years, 95% CI: 61.8-62.5), has more post-secondary school graduates (SLCDC-H: 52.0%, 95% CI: 49.7%-54.2%; CCHS: 47.5%, 95% CI: 46.1%-48.9%) and has fewer respondents on hypertension medication (SLCDC-H: 82.5%, 95% CI: 80.9%-84.1%; CCHS: 88.6%, 95% CI: 87.7%-89.6%). CONCLUSION: Overall, the 2009 SLCDC-H represents its source population and provides novel, comprehensive data on the diagnosis and management of hypertension. The survey has been adapted to other chronic conditions--diabetes, asthma/chronic obstructive pulmonary disease and neurological conditions. The questionnaire is available on the Statistics Canada website; descriptive results have been disseminated by the Public Health Agency of Canada.

TITRE: Méthodologie de l'Enquête sur les personnes ayant une maladie chronique au Canada ­ composante de l'hypertension de 2009. INTRODUCTION: L'Enquête sur les personnes ayant une maladie chronique au Canada ­ composante de l'hypertension (EPMCC-H) est une enquête téléphonique transversale de 20 minutes sur le diagnostic et la prise en charge de l'hypertension. L'échantillon de l'EPMCC-H, sélectionné à partir des répondants à l'Enquête sur la santé dans les collectivités canadiennes (ESCC) de 2008, était composé de Canadiens (de 20 ans et plus) des dix provinces ayant déclaré avoir reçu un diagnostic d'hypertension. MÉTHODOLOGIE: Le questionnaire a été élaboré au moyen de la technique Delphi et a fait l'objet d'un examen externe ainsi que de tests qualitatifs. Statistique Canada s'est chargé des stratégies d'échantillonnage, du recrutement, de la collecte et du traitement des données. Les proportions ont été pondérées afin de représenter la population canadienne et les intervalles de confiance (IC) à 95 % ont été calculés au moyen de la méthode de rééchantillonnage bootstrap. RÉSULTATS: Si on le compare à la population de l'ESCC ayant déclaré souffrir d'hypertension, l'échantillon de l'EPMCC-H (n = 6 142) est légèrement plus jeune (âge moyen des répondants à l'EPMCC-H : 61,2 ans, IC à 95 % : 60,8 à 61,6; âge moyen des répondants à l'ESCC : 62,2 ans, IC à 95 % : 61,8 à 62,5), comporte plus de détenteurs d'un diplôme d'études postsecondaires (EPMCC-H : 52,0 %, IC à 95 %: 49,7 % à 54,2 %; ESCC : 47,5 %, IC à 95 % : 46,1 % à 48,9 %) et moins de répondants prenant un médicament pour l'hypertension (EPMCC-H : 82,5 %, IC à 95 % : 80,9 % à 84,1 %; ESCC : 88,6 %, IC à 95 % : 87,7 % à 89,6 %). CONCLUSION: Dans l'ensemble, l'EPMCC-H de 2009 est représentatif de sa population source et fournit des données nouvelles et exhaustives sur le diagnostic et la prise en charge de l'hypertension. L'enquête a été adaptée à d'autres maladies chroniques ­ diabète, asthme/maladie pulmonaire obstructive chronique et troubles neurologiques. Le questionnaire est accessible à partir du site Web de Statistique Canada; des résultats descriptifs ont été publiés par l'Agence de la santé publique du Canada.

Perception of uncontrolled blood pressure and behaviours to improve blood pressure: findings from the 2009 Survey on Living with Chronic Diseases in Canada.

Individuals with hypertension should lower and maintain their blood pressure levels through lifestyle modification and/or pharmacotherapy. To determine whether perception of blood pressure control is related to behaviours and intentions for improving blood pressure, data from 6142 Canadians age 20+ years with self-reported hypertension were analysed. Relationships between perception of control, current behaviours for blood pressure control and intentions to improve these behaviours were examined. Although individuals who reported uncontrolled blood pressure were equally likely to report engaging in lifestyle behaviours for blood pressure control, they were more likely to indicate an intention to improve their health, compared with those who reported well-controlled/low blood pressure. These individuals were also less likely to report having enough information to control their blood pressure. In addition, they were less likely to report having been advised to take antihypertensive medication, and to be taking and adhering to medications. Individuals who perceive their blood pressure as uncontrolled have intentions to make health-enhancing changes but may lack the information to do so. The study highlights the potential need for programmes/services to help those with uncontrolled blood pressure make lifestyle changes and/or take appropriate medication.

Enhanced nutrient removal MBR system with chemical addition for low effluent TP.

A pilot study was conducted to test an membrane bioreactor (MBR) process for combined biological and chemical P removal to achieve a very low effluent total phosphorus (TP) concentration of 0.025 mg P/L. With the data from the pilot test, a simulation study was performed to demonstrate that: (1) the pilot system behaviour (effluent quality, MLSS, etc.) can be modelled accurately with an activated sludge model combined with a chemical precipitation model; and (2) with the calibrated model, simulation scenarios can be performed to further understand the pilot MBR process, and provide information for optimizing design and operation when applied at full-scale. Results from the pilot test indicated that the system could achieve very low effluent TP concentration through biological P removal with a limited chemical addition, and chemical addition to remove P to very low level did not affect other biological processes, i.e., organic and nitrogen removal. Simulation studies indicate that the process behaviour can be modelled accurately with an activated sludge model combined with a chemical precipitation model, and the calibrated model can be used to provide information to optimize system design and operation, e.g., chemical addition control under dynamic loading conditions is important for maintaining biological P removal.

Nonsteroidal anti-inflammatory drugs are associated with increased neuritic plaques.

OBJECTIVES: Observational and experimental studies suggest that nonsteroidal anti-inflammatory drugs (NSAIDs) may protect against Alzheimer disease (AD); however, clinical trials and other observational studies, including the Adult Changes in Thought (ACT) study, show no protection or promotion of AD. The objective of this study is to determine the relationship between common dementia-associated pathologies and mid- to late-life NSAID exposure. METHODS: We examined the association of mid- to late-life NSAID use with neuropathologic findings on 257 autopsies from ACT, a population-based study of brain aging and incident dementia. Cumulative standard daily doses (SDD) of nonselective NSAIDs were determined from ≥10 years of computerized pharmacy dispensing data. Analyses were adjusted for selection bias to broaden generalizability of results to 3,026 eligible participants in the ACT cohort. Seven pathologic indices were evaluated: intermediate or frequent score for neuritic plaques, Braak stages V or VI for neurofibrillary tangles, >2 cerebral microinfarcts, the presence of any neocortical Lewy bodies, any macroscopic infarcts, any amyloid angiopathy, and moderate or severe atherosclerosis. RESULTS: Of the neuropathologic indices evaluated, only neuritic plaque score was significantly increased in participants with greater use of nonselective NSAIDs (p = 0.065), specifically in those with high levels of cumulative use: 1,000-2,000 SDD (adjusted relative risk [RR] 2.16, 95% confidence interval [CI] 1.02-4.25, compared to light/nonuse [<60 SDD]) and >2,000 SDD (adjusted RR 2.37, 95% CI 1.24-4.67). CONCLUSIONS: Increased neuritic plaque accumulation may explain the association between heavy use of nonselective NSAIDs and increased risk of dementia among ACT participants.

High frequencies of leukemia stem cells in poor-outcome childhood precursor-B acute lymphoblastic leukemias.

In order to develop a xenograft model to determine the efficacy of new therapies against primary human precursor-B acute lymphoblastic leukemia (ALL) stem cells (LSCs), we used the highly immunodeficient non-obese diabetic (NOD).Cg-Prkdc(scid)IL2rg(tmlWjl)/SzJ (NOD-severe combined immune deficient (scid) IL2rg(-/-)) mouse strain. Intravenous transplantation of 2 of 2 ALL cell lines and 9 of 14 primary ALL cases generated leukemia-like proliferations in recipient mice by 1-7 months after transplant. Leukemias were retransplantable, and the immunophenotypes, gene rearrangements and expression profiles were identical or similar to those of the original primary samples. NOD-scid mice transplanted with the same primary samples developed similar leukemias with only a slightly longer latency than did NOD-scid-IL2Rg(-/-) mice. In this highly sensitive NOD-scid-IL2Rg(-/-)-based assay, 1-100 unsorted primary human ALL cells from five of five tested patients, four of whom eventually experienced leukemia relapse, generated leukemias in recipient mice. This very high frequency of LSCs suggests that a hierarchical LSC model is not valuable for poor-outcome ALL.

Epigenomic alterations and gene expression profiles in respiratory epithelia exposed to cigarette smoke condensate.

Limited information is available regarding epigenomic events mediating initiation and progression of tobacco-induced lung cancers. In this study, we established an in vitro system to examine epigenomic effects of cigarette smoke in respiratory epithelia. Normal human small airway epithelial cells and cdk-4/hTERT-immortalized human bronchial epithelial cells (HBEC) were cultured in normal media with or without cigarette smoke condensate (CSC) for up to 9 months under potentially relevant exposure conditions. Western blot analysis showed that CSC mediated dose- and time-dependent diminution of H4K16Ac and H4K20Me3, while increasing relative levels of H3K27Me3; these histone alterations coincided with decreased DNA methyltransferase 1 (DNMT1) and increased DNMT3b expression. Pyrosequencing and quantitative RT-PCR experiments revealed time-dependent hypomethylation of D4Z4, NBL2, and LINE-1 repetitive DNA sequences; up-regulation of H19, IGF2, MAGE-A1, and MAGE-A3; activation of Wnt signaling; and hypermethylation of tumor suppressor genes such as RASSF1A and RAR-beta, which are frequently silenced in human lung cancers. Array-based DNA methylation profiling identified additional novel DNA methylation targets in soft-agar clones derived from CSC-exposed HBEC; a CSC gene expression signature was also identified in these cells. Progressive genomic hypomethylation and locoregional DNA hypermethylation induced by CSC coincided with a dramatic increase in soft-agar clonogenicity. Collectively, these data indicate that cigarette smoke induces 'cancer-associated' epigenomic alterations in cultured respiratory epithelia. This in vitro model may prove useful for delineating early epigenetic mechanisms regulating gene expression during pulmonary carcinogenesis.

Natural attenuation of nitrogen loading from septic effluents: Spatial and environmental controls.

We assess the role of septic systems as potential nitrogen (N) sources to coastal open water bodies. To quantify the potential role of septic tanks, we document the distribution pattern and functionality of septic tanks in McIntosh County in Georgia, USA, and examine factors governing the mitigation of septic N loading in coastal groundwater. Employing a field calibrated 2D variable-density reaction-transport model, we focus on the role of setback distance of a leaky septic source from the receiving surface waters, on transport and biogeochemical characteristics of the subsurface environment, and on leachate composition and reactivity. We conclude that the removal of bioavailable nitrogen via denitrification may be increased by increasing the septic system setback distance, in particular in brackish and saline coastal settings where sulfide produced in sulfate reduction can limit N(2) production.

Aeromonas species associated with necrotizing enteritis and septicemia in an adult male ostrich (Struthio camelus).

A deceased 10-yr-old male ostrich was diagnosed with severe necrotizing enteritis and septicemia. The bird was inappetent for 3 wk and had neurologic signs 2 days prior to death. Macroscopically, no significant lesions were noted aside from congestion of the liver, kidneys, and spleen. Histopathology revealed severe fibrinonecrotic enteritis,associated with large numbers of gram-negative bacteria, multifocal fibrinoid necrosis in portal arteries, accumulation of fibrin in hepatic sinusoids, myocardial degeneration, and necrosis. There was also squamous metaplasia in the glands of the esophagus and external ears. A gram-negative rod was isolated in pure culture from intestine, liver, lungs, and trachea and identified as an Aeromonas species. The concentration of vitamin A in the liver was extremely low. The lesions seen in the intestine and liver and the isolation of an Aeromonas sp. from various tissues strongly suggest that this bacterium was the cause of the necrotizing enteritis, septicemia, and death of this ostrich. Vitamin A deficiency might have predisposed the bird to the Aeromonas infection.

Short communication: Attempts to identify Clostridium botulinum toxin in milk from three experimentally intoxicated Holstein cows.

Three adult lactating Holstein cows were injected in the subcutaneous abdominal vein with 175 ng/kg of body weight of Clostridium botulinum type C toxin (451 cow median toxic doses) to determine if this botulinum toxin crosses the blood-milk barrier. Whole blood (in sodium heparin) and clotted blood serum samples were taken at 0 min, 10 min, and 3, 6, 9, and 12 h postinoculation. Milk samples were taken at 0 min and at 3, 6, 9 and 12 h postinoculation. All samples were tested for the presence of the toxin using the mouse bioassay and immunostick ELISA test. The immunostick ELISA identified the toxin in whole blood and the mouse bioassay identified the toxin in serum at all times examined in all 3 animals. Toxin was not identified by either detection method in milk samples collected from the 3 animals. From these results, it appears that Clostridium botulinum type C toxin does not cross from the blood to the milk in detectable concentrations.

Genetic diversity of bovine ulcerative mammary dermatitis-associated Treponema.

The etiology of bovine ulcerative mammary dermatitis (UMD) is poorly characterized. The goal of this study was to genetically analyze spirochetes present in UMD lesions. DNA prepared from UMD lesion biopsies and from spirochetes cultured from the corresponding lesion biopsies was PCR amplified using primers for the 16S rDNA-tRNA(ile) intergenic spacer region (ISR) of Treponema 16S-23S rDNA. Analysis of cloned ISR amplicons from three cultivable UMD-associated spirochetes indicated that two isolates cluster closely with cultivable papillomatous digital dermatitis (PDD)-associated and human-associated Treponema phylotypes, while the remaining isolate is unique. Analysis of ISR amplicons from UMD lesion biopsies identified additional not-yet-cultivable Treponema phylotypes. Our results revealed the presence of a genetically diverse Treponema population in an UMD lesion.

Cutaneous pythiosis in a nestling white-faced ibis.

A nestling white-faced ibis (Plegadis chihi) with multifocal skin ulcerations on the wings, neck, head, and limbs was found in a wetland agricultural region of the central valley in California. Pathologic, microbiologic, and molecular findings were consistent with restricted, cutaneous infection by the oomycete Pythium insidiosum. The microscopic features of the disease, including intense, necrotizing eosinophilic and granulomatous inflammation, are similar to those previously described in mammals. Pythiosis, which is most typical in tropical and subtropical climates, has recently emerged in California as a cause of cutaneous and enteric disease in horses and dogs, respectively. Environmental stability and persistence of a "water-mold" in the arid central valley of California could be associated with agricultural and community watering practices. To the best of the authors' knowledge, this is the first published report of pythiosis in birds.

Listeriosis in a cockatiel (Nymphicus hollandicus).

Listeriosis was diagnosed in a 4-yr-old female cockatiel (Nymphicus hollandicus) that died after exhibiting clinical signs that included a fluffed-up appearance, weakness, and loss of weight of several days duration. Grossly, the bird was moderately emaciated, and the liver and spleen were enlarged. Microscopically, there was mild-to-moderate inflammation associated with rod-shaped, gram-positive bacteria in the liver, spleen, kidneys, adrenal glands, bone marrow, and esophagus. Listeria monocytogenes was isolated from the liver, trachea, and intestine. The isolate was identified as type 1 by agglutination with specific antisera, and it further identified as belonging to serovar group 1/2a, 3a by multiplex polymerase chain reaction assay. Listeria monocytogenes also was detected in affected tissues by immunohistochemistry.

Histochemical and immunohistochemical evidence of a bacterium associated with lesions of epizootic bovine abortion.

Epizootic bovine abortion (EBA), a tick-transmitted disease of pregnant cattle grazing foothill pastures, is a major cause of reproductive failure in California and adjacent states. Affected fetuses develop a chronic disease, resulting in late-term abortion or premature calving. Despite investigations spanning 50 years, to the authors' knowledge, the etiologic agent of EBA has not yet been isolated from affected fetuses or the tick vector. The diagnosis of EBA is based on gross and microscopic lesions. Recently, documentation that the etiologic agent is susceptible to antibiotics and identification of a unique 16S deltaproteobacterial rDNA gene sequence in 90% of thymus tissues from aborted fetuses have supported the role of a bacterial infection as the cause of EBA. To determine whether bacteria could be detected in the tissues, histochemical staining and immunohistochemical procedures were used on formalin-fixed, paraffin-embedded tissues. Use of a modified Steiner silver stain revealed small numbers of intracytoplasmic bacterial rods in 37 of 42 thymic samples from EBA-affected fetuses. Improved detection was achieved by use of immunohistochemical staining with serum from EBA-affected fetuses that resulted in detection of numerous bacterial rods in the cytoplasm of histiocytic cells in the thymus from all 42 EBA-affected fetuses. Immunohistochemical examination of additional tissues from 21 field and experimental EBA cases revealed positively stained intracytoplasmic bacterial rods in many organs with inflammatory lesions. Use of the modified Steiner stain and immunohistochemical staining of tissues from negative-control fetuses failed to reveal organisms. To the authors' knowledge, this is the first report to document morphologic evidence of a bacterium associated with the lesions of EBA.

Immunohistochemical identification of Campylobacter fetus in natural cases of bovine and ovine abortions.

An immunohistochemistry (IHC) procedure for the detection of Campylobacter fetus antigens using an avidin-biotin complex technique was performed on formalin fixed bovine and ovine fetal tissues from 26 natural cases of Campylobacter spp. abortion (four ovine and 22 bovine). The species of Campylobacter isolated included C. fetus ssp. venerealis from 13 bovine fetuses, C. fetus ssp. fetus from two ovine and one bovine fetus, Campylobacter jejuni from seven bovine fetuses, Campylobacter lari from two ovine fetuses and an unspeciated Campylobacter species in one bovine fetus. Histologic lesions identified in the aborted fetuses included placentitis, serositis, pneumonia, gastroenteritis, hepatitis and encephalitis. Campylobacter fetus antigens were identified by IHC in 13 of 13 bovine fetuses from which C. fetus ssp. venerealis was isolated and in two of two ovine fetuses from which C. fetus ssp. fetus was isolated. The IHC stains were negative in tissues from seven bovine fetuses from which C. jejuni was isolated, one bovine fetus infected with C. fetus ssp. fetus, one bovine fetus infected with the unspeciated Campylobacter and two ovine fetuses infected with C. lari. In positive cases, the IHC stain most frequently identified bacteria in the lung and gastrointestinal tract. The C. fetus IHC procedure performed on formalin fixed tissues is a practical tool for the diagnosis of natural cases of ovine and bovine abortion caused by C. fetus.

An improved molecular assay for Tritrichomonas foetus.

Tritrichomonas foetus (T. foetus) is the causative agent of bovine trichomonosis, a sexually transmitted disease leading to abortion (from 1 to 8 months gestation), infertility, and occasional pyometra. The annual losses to the U.S. beef industry are estimated to be in the hundreds of millions of dollars. Currently, the "gold standard" diagnostic test for trichomonosis in most countries is the cultivation of live organisms from reproductive secretions. The cultured organisms can then be followed by PCR assays with primers that amplify T. foetus to the exclusion of all other trichomonad species. Thus, negative results present as null data, indistinguishable from failed PCR amplification during T. foetus specific amplification. Our newly developed assay improves previously developed PCR based techniques by using diagnostic size variants from within the internal transcribed spacer 1 (ITS1) region that is between the 18S rRNA and 5.8S rRNA subunits. This new PCR assay amplifies trichomonad DNA from a variety of genera and positively identifies the causative agent in the bovine trichomonad infection. This approach eliminates false negatives found in some current assays as well as identifying the causative agent of trichomonad infection. Additionally, our assay incorporates a fluorescently labeled primer enabling high sensitivity and rapid assessment of the specific trichomonad species. Moreover, electrophoretic separation of amplified samples can be outsourced, thus eliminating the need for diagnostic laboratories to purchase expensive analysis equipment.

Effects of growth hormone on leucine absorption, intestinal morphology, and ultrastructure of the goldish intestine.

The mechanisms whereby exogenous growth hormone modulates intestinal structure and function in fish were investigated. Goldfish (Carassius auratus) were fed commercial flake diet sprayed with recombinant carp growth hormone (cGH) daily for 1 month. Control animals received food sprayed with the vehicle. After 1 month of daily feedings, body mass and length were determined, and animals were sacrificed to study intestinal characteristics. Sections of foregut were removed after determination of total gut length for measurement of leucine uptake, histology, and epithelial ultrastructure. Oral administration of cGH for 1 month resulted in a 40% increase in body mass and an 8% increase in body length above controls. Gut length was 43% greater and the gut length to body length ratio was 32% greater as a result of the cGH treatment. Feeding with cGH also resulted in a significant increase in leucine uptake and increased gut mucosal thickness. Analysis of transmission electron micrographs revealed significant increases in the microvillous height and density and epithelial surface area. The findings indicate that growth hormone added to feed may increase growth in fish, in part by significantly increasing gut length, mucosal thickness, and epithelial brush border surface area, leading to enhanced epithelial absorption.

Comparison of the 5.8S rRNA gene and internal transcribed spacer regions of trichomonadid protozoa recovered from the bovine preputial cavity.

Sequence analysis of the 5.8S rRNA gene and the internal transcribed spacer regions (ITSRs) was used to compare trichomonadid protozoa (n = 39) of varying morphologies isolated from the bovine preputial cavity. A multiple sequence alignment was performed with bovine isolate sequences and other trichomonadid protozoa sequences available in GenBank. As a group, Tritrichomonasfoetus isolates (n = 7) had nearly complete homology. A similarity matrix showed low homology between the T. foetus isolates and other trichomonads recovered from cattle (<70%). Two clusters of trichomonads other than T. foetus were identified. Eighteen isolates comprised 1 group. These isolates shared >99% homology among themselves and with Pentatrichomonas hominis. The other non-T. foetus cluster (n = 14) did not exhibit a high degree of homology (<87%) with other bovine isolates or any of the trichomonad sequences available in GenBank. The sequence homology among isolates in that cluster was >99%, except for 1 isolate that varied from the others in both ITSRs (approximately 2% dissimilarity). Sequence analysis of the 5.8S rRNA gene and ITSRs was useful for comparing trichomonadid protozoa isolated from the bovine preputial cavity and demonstrated that 2 distinct types of trichomonads constituted the non-T. foetus isolates recovered from the bovine preputial cavity.