Validation of the ultrasonographic assessment of the femoral trochlea epiphyseal cartilage in foals at osteochondrosis predilected sites with magnetic resonance imaging and histology.

BACKGROUND: Noninvasive imaging tools are needed to screen foal femoropatellar joints to detect subclinical osteochondrosis lesions due to focal failure of endochondral ossification to enhance early management to optimise intrinsic healing events. Recently investigations employing 3T susceptibility-weighted magnetic resonance imaging (3T SWI MRI) and CT have demonstrated their capacity for early osteochondrosis diagnosis, but these technologies are not practical for field screening. We postulate that ultrasonography is a valuable field tool for the detection of subclinical osteochondrosis lesions. OBJECTIVES: The goals were to 1) describe the ultrasonographic features of the femoral trochlea of healthy and osteochondrosis-predisposed neonatal foals, 2) validate the capacity of ultrasound to assess cartilage canal vascular archictecture and the ossification front and 3) evaluate field feasibility in a pilot study. STUDY DESIGN: Experimental study. METHODS: Ultrasonographic evaluation of osteochondrosis predisposed (n = 10) and control (n = 6) femoral trochleas was performed ex vivo and compared with site-matched histological sections and 3T SWI MRI. The articular and epiphyseal cartilage thickness, ossification front indentation and cartilage canal vascular archictecture were assessed at each ROI. Femoral trochleae of foals (n = 3) aged ≈ 1, 3 and 6 months were also evaluated with ultrasonography in field. RESULTS: Ultrasonographic measurements strongly correlated with the histological measurements. There was no difference in the cartilage thickness or ossification front indentation between control and osteochondrosis-predisposed specimens. The cartilage canal vascular archictecture on ultrasonograms corresponded with the vessel pattern observed on site matched histology and 3T SWI MRI. MAIN LIMITATIONS: The number of specimens for study was limited and no early osteochondrosis lesions were present within the predilected group, but a field study is now underway. CONCLUSION: Ultrasonographic examination of the femoral trochlea permitted accurate evaluation of cartilage thickness, cartilage canal vascular archictecture and ossification front indentation in young foals and is a promising, practical tool for screening subclinical osteochondrosis and monitoring and managing lesions at important clinical sites.

Critical roles for the netrin receptor deleted in colorectal cancer in dopaminergic neuronal precursor migration, axon guidance, and axon arborization.

DCC (deleted in colorectal cancer), a receptor for the axon guidance cue netrin-1, is highly expressed by mesencephalic dopaminergic (DA) neurons during development; however, the contribution of DCC to DA development remains largely uncharacterized. DA neurons in ventral mesencephalic nuclei also express UNC5 homologue netrin receptors from late embryogenesis to adulthood, raising the possibility that DA axons could be attracted or repelled by netrins. Examining newborn dcc null mice, we report that loss of DCC function results in profound alterations of DA circuitry, including DA progenitor cell migration defects, reduced numbers of DA cells in midbrain nuclei, an anomalous DA ventral commissure, malformed DA innervation of the ventral striatum, and reduced DA innervation of the cerebral cortex. Caspase-3 activation was detected in inappropriately localized DA cells, consistent with apoptosis contributing to reduced cell numbers. Dcc heterozygous mice express reduced levels of DCC protein. Although less severely disrupted than dcc nulls, newborn and adult dcc heterozygotes also have fewer DA neurons in ventral mesenscephalic nuclei. Despite the reduced numbers of DA neurons, newborn dcc heterozygotes and nulls exhibit similar DA innervation density as wild-type littermates in the nucleus accumbens core, and adult dcc heterozygotes exhibit increased DA innervation in medial prefrontal cortex. A trend towards increased innervation of medial prefrontal cortex was detected in newborn dcc heterozygotes, but did not reach statistical significance, suggesting that the increase in adult heterozygotes results from enhanced DA arborization during postnatal development. Consistent with the hypothesis that DCC regulates DA axonal projections, disrupting DCC function in culture inhibits netrin-1 induced DA axon extension and axon branching. Furthermore, disrupting DCC function in isolated DA neurons grown as micro-island cultures reduces the number of autaptic synapses per cell. We conclude that DCC regulates appropriate precursor cell migration, axon guidance, and terminal arborization by DA neurons.

Tissue HHV6 and 7 determination in pediatric solid organ recipients--a pilot study.

Herpes virus infections remain a major challenge in solid organ transplantation. HHV6 and 7 blood viral load was associated with pathology after renal transplantation. Little is known about the significance of tissue HHV6 and 7 infections. A total of 18 tissue biopsies (13 kidney, three GI and two BAL) from nine pediatric transplant patients (five kidney, two liver, one combined liver and kidney and one bone marrow transplant) were subjected to blood HHV6 IgG and IgM testing. In addition, tissue HHV6 and 7 semi-quantitative PCR analysis with subsequent detection by ELISA and quantitative methods were applied to the same samples. We also studied four native kidney biopsies of children with other kidney disease. The results of the biopsies were correlated with clinical data. Of the transplant patients, 78% were HHV6 IgG positive. Six of nine had a positive IgM on at least one occasion, however, only two of nine transplant patients were symptomatic with a mixed CMV/EBV septic picture of multi-organ failure. Only these two patients had a significant tissue viral load for HHV6. Additionally, a very significant tissue viral load for HHV6 was detected in an immunocompromised patient 3 wk after a roseola-like febrile illness. The HHV6 copies were 31, 88 and 206 per 10 microL of DNA, respectively. In the patient who also had the fourth positive ELISA for HHV6 PCR product, the Multiplex PCR and restriction enzyme assay on its PCR product revealed a significant contribution by HHV7, while the HHV6-B signal was rather weak. Significant tissue HHV6 loads can be found in tissue biopsies from organ recipients with significant illness and also in native kidneys after primary infection. This may explain the high prevalence of HHV6 in transplanted kidneys. Further studies on HHV6 and 7 using molecular techniques should be supported.

Phosphorus removal from trout farm effluents by constructed wetlands.

Freshwater trout farms need a high and continuous clean water flow to keep fish exposed to a non-toxic ammonium concentration. As a result, the concentration of effluents from these farms are even below standard effluent criteria for municipal wastewater effluent for solids, nitrogen and phosphorus. Nevertheless, the mass of pollutants discharged, originating mostly from excreta and undigested fish food, must be reduced by simple and economical treatment processes. We designed and operated a three-stage system aimed at retaining solids by a 60 pm nylon rotating microscreen followed by treatment with a phosphorus-retaining constructed wetland system. Washwater from the microscreen was pumped to a series of two horizontal flow beds of 100 m3 each (0.6 m deep). Coarse (2 mm) and finer (< 2 mm) crushed limestone were used in each bed, respectively, with the first one being planted with reeds (Phragmites australis) and the second one designed to remove even more phosphorus by adsorption and precipitation. Preliminary results indicated that the microscreen captured about 60% of the suspended solids and that greater than 95% of the suspended solids and greater than 80% of the total phosphorus mass loads were retained by the beds. The potential of constructed wetlands as an ecologically attractive and economical method for treating fish farm effluents to reduce solids and phosphorus discharge appears promising.

Atypical Actinobacillus pleuropneumoniae isolates that share antigenic determinants with both serotypes 1 and 7.

In the present study, the characterization of 3 atypical isolates of Actinobacillus pleuropneumoniae is presented. Two isolates (1B and 27E) showed positive reactions in coagglutination, immunodiffusion, and indirect hemagglutination tests for serotypes 1 and 7, whereas the third isolate (26B) reacted with antisera to serotypes 1, 4, and 7. These atypical isolates of A. pleuropneumoniae possessed a capsular polysaccharide (CPS) antigenically related to serotype 1 as well as an O-chain lipopolysaccharide antigenically related to serotype 7 or to serotypes 4 and 7, as shown by the use of monoclonal antibodies. Results of toxin profile and virulence assays for mice and pigs showed them to be more related to A. pleuropneumoniae serotype 7 field isolates. All 3 isolates induced antibodies mainly against serotype 7/4 O-long-chain lipopolysaccharide (LC-LPS) and, to a lesser extent, to the CPS of serotype 1, in experimentally infected pigs. Diagnostic laboratories that use a LC-LPS-based enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of A. pleuropneumoniae infection in swine would probably diagnose herds infected with these atypical isolates as being infected by A. pleuropneumoniae serotypes 7 or 4, whereas those that use a CPS-based ELISA would probably consider them as infected by A. pleuropneumoniae serotype 1.

Effects of global regulatory proteins and environmental conditions on fimbrial gene expression of F165(1) and F165(2) produced by Escherichia coli causing septicaemia in pigs.

Escherichia coli O115:F165 strains are associated with septicaemia in young pigs and possess at least two types of fimbriae. F165(1) fimbriae belong to the P fimbrial family and F165(2) fimbriae belong to the S fimbrial family. Regulatory regions of foo (F165(1)) and fot (F165(2)) fimbrial gene clusters from wild-type strain 4787 were sequenced and characterised. Expression of F165(1) and F165(2) fimbrial genes was analysed by using lacZ and/or luxAB as reporter genes under the control of the native fimbrial promoters. Differential expression of fimbrial genes was observed. Global regulatory mechanisms such as catabolite repression, leucine-responsive regulatory protein (Lrp), methylation and DNA supercoiling were demonstrated to influence foo and fot expression. foo and fot expression was optimal at 37 degrees C and under aerobic conditions. Expression of foo was higher on minimal medium, whereas fot expression was higher on complex Luria-Bertani medium. This could reflect an in vivo differential expression.

[Lyme disease in Upper Normandy: report of a hospital survey]. / Maladie de Lyme chez l'enfant en Haute-Normandie: à propos d'une enquête hospitalière.

This study presents the results of a hospital survey on Lyme disease in children living in upper Normandy, a region that is quite densely wooded (with 18% forest areas and woods). The aim of this survey was to assess the prevalence of this disease in children from the Seine-Maritime and L'Eure, hospitalized in pediatric wards in the Seine-Maritime department, which includes Rouen, Dieppe, Fécamp, Elbeuf, and Le Havre. Fifteen cases of Lyme disease were diagnosed between September 1988 and June 1997. The children (6 girls and 9 boys) were aged between 5 and 14 years old. Only 7 subjects showed primary symptoms, while secondary symptoms were observed in 12 children. In the study population, a high prevalence (11 out of the 15 children) of neurological disorders was found. The following secondary symptoms were noted: 5 cases of erythema migrans, 2 cases of non-malignant cutaneous lymphocytoma, and 4 cases which in fact had previously displayed primary clinical signs (3 subjects with erythema migrans and 1 subject with non-malignant cutaneous lymphocytoma); 7 cases of uni- or bilateral facial paralysis, the most frequent neurological manifestation with or without lymphocytic meningitis; 1 case of central vestibular syndrome with a hyperalgesic meningoradicular reaction in the vicinity of the tick bite; 1 case of peripheral radicular involvement and intense pain in the left lower limb; 4 cases of ocular disorders (3 diplopias, 1 bilateral conjunctivitis complicated by kerato-uveitis, 1 bilateral complete cecitis). Only 10 child had rheumatological symptoms, i.e., Lyme arthritis of the right knee. Treatment consisted of amoxicillin (10 children) administered at a dosage of 50 to 100 mg/kg/d over a period ranging from 10 days to 1 month, or ceftriaxone (7 children) at a dosage of 50 to 100 mg/kg/d administered intravenously over a period ranging from 8 days to 3 weeks. Two of the children received combined antibiotic therapy, and 5 subjects had adjunct corticotherapy.

Distinct roles for Galpha(i)2 and Gbetagamma in signaling to DNA synthesis and Galpha(i)3 in cellular transformation by dopamine D2S receptor activation in BALB/c 3T3 cells.

Control of cell proliferation depends on intracellular mediators that determine the cellular response to external cues. In neuroendocrine cells, the dopamine D2 receptor short form (D2S receptor) inhibits cell proliferation, whereas in mesenchymal cells the same receptor enhances cell proliferation. Nontransformed BALB/c 3T3 fibroblast cells were stably transfected with the D2S receptor cDNA to study the G proteins that direct D2S signaling to stimulate cell proliferation. Pertussis toxin inactivates G(i) and G(o) proteins and blocks signaling of the D2S receptor in these cells. D2S receptor signaling was reconstituted by individually transfecting pertussis toxin-resistant Galpha(i/o) subunit mutants and measuring D2-induced responses in pertussis toxin-treated cells. This approach identified Galpha(i)2 and Galpha(i)3 as mediators of the D2S receptor-mediated inhibition of forskolin-stimulated adenylyl cyclase activity; Galpha(i)2-mediated D2S-induced stimulation of p42 and p44 mitogen-activated kinase (MAPK) and DNA synthesis, whereas Galpha(i)3 was required for formation of transformed foci. Transfection of toxin-resistant Galpha(i)1 cDNA induced abnormal cell growth independent of D2S receptor activation, while Galpha(o) inhibited dopamine-induced transformation. The role of Gbetagamma subunits was assessed by ectopic expression of the carboxyl-terminal domain of G protein receptor kinase to selectively antagonize Gbetagamma activity. Mobilization of Gbetagamma subunits was required for D2S-induced calcium mobilization, MAPK activation, and DNA synthesis. These findings reveal a remarkable and distinct G protein specificity for D2S receptor-mediated signaling to initiate DNA synthesis (Galpha(i)2 and Gbetagamma) and oncogenic transformation (Galpha(i)3), and they indicate that acute activation of MAPK correlates with enhanced DNA synthesis but not with transformation.

Phase variation of F165(1) (Prs-like) fimbriae from Escherichia coli causing septicaemia in animals.

Escherichia coli O115:F165 strains are associated with septicaemia in young pigs and synthesize fimbriae involved in virulence, designated as F165(1). F165(1) fimbriae belong to the P fimbrial family and are encoded by the foo gene cluster. The foo regulatory region of strain 5131 possesses characteristics similar to that of members of the P regulatory family, including papI and papB homologues, and two GATC sites separated by 102 bp, targets of differential Dam methylation. In wild-type strains, the synthesis of F165(1) is repressed by leucine and the fimbriae undergo phase variation. Immunofluorescence staining showed that phase variation of F165(1) results in a majority of cells (98%) in the ON phase, in contrast with phase variation of other members of this regulatory family, for which the majority of the cells are in the OFF state. Using a translational fusion in strain 5131 between phoA and fooA, encoding for the major structural subunit of F165(1), it was shown that leucine inhibits the OFF to ON switch and modulates the basal transcription of the foo operon.

Use of polymerase chain reaction and immunohistochemistry for detection of canine adenovirus type 1 in formalin-fixed, paraffin-embedded liver of dogs with chronic hepatitis or cirrhosis.

To assess the possible involvement of canine adenovirus type 1 (CAV-1) in naturally occurring cases of canine chronic liver disease, a polymerase chain reaction (PCR)-based assay was developed to detect a conserved region of the major core protein gene (pVII) of CAV-1 in formalin-fixed, paraffin-embedded liver sections. Results were compared with a standard avidin-biotin immunoperoxidase complex technique that detected CAV-1 antigens using a commercial monoclonal anti-adenovirus antibody. Seventeen cases of cirrhosis and 28 cases of chronic hepatitis with piecemeal necrosis and progressive fibrosis were selected for the study. Formalin-fixed, paraffin-embedded liver sections of 2 cases of infectious canine hepatitis (ICH) and crude DNA extract from CAV-1 (ATCC VR 293 Utrecht strain) served as positive controls. A 411-base-pair viral region was amplified and sequenced as CAV-1 pVII in both cases of infectious canine hepatitis and in the CAV-1 crude DNA extract. The 2 ICH cases were positive for CAV-1 antigens by the immunoperoxidase method. CAV-1 DNA or antigens were not detected by either technique in any of the 45 cases of chronic liver disease selected for the study. These results indicate that both PCR and immunohistochemistry are reliable and rapid techniques for detecting CAV-1 in formalin-fixed, paraffin-embedded liver sections of dogs with ICH. Several possibilities may explain the negative results obtained with both techniques in this study, including the noninvolvement of CAV-1 in canine chronic hepatitis and cirrhosis and the possibility that the virus causes initial damage, provokes a self-perpetuating chronic liver disease, and disappears.

DNA probe and polymerase chain reaction procedure for the specific detection of Serpulina hyodysenteriae.

Serpulina (Treponema) hyodysenteriae, a Gram-negative anaerobic spirochete, is the causative agent of swine dysentery, a mucohaemorrhagic diarrheal disease in which lesions are confined to the large intestine of pigs. A DNA probe and polymerase chain reaction (PCR) amplification procedures which are specific, rapid , and sensitive for the detection of S.hyodysenteriae have been developed. Clone pF12 from a plasmid library of S.hyodysenteriae B204 genomic DNA was identified as a clone specific for S.hyodysenteriae but not for S.innocens by differential hybridization screening with S.hyodysenteriae and S.innocens genomic DNA probes. A DNA probe consisting of a 1.3 kb restriction fragment from pF12 was found to be highly specific for S. hyodysenteriae and detected 10(5) bacterial cells. A PCR procedure using primers derived from this fragment yielded a single product which was specifically generated for S.hyodysenteriae template DNA and not for other control cells DNA. PCR provided increased sensitivity with the direct detection of as few as 10 S.hyrodysenteriae cells. The PCR procedure could detect S.hyodysenteriae cells in seeded faecal matter. Moreover the PCR assay was able to detect most S. hyodysenteriae field isolates of serotypes 8 and 9. These tools have diagnostic application in veterinary microbiology.

Cloning of determinants encoding F165(2) fimbriae from porcine septicaemic Escherichia coli confirms their identity as F1C fimbriae.

Cloning of the f165(2) operon that encodes F1C-like fimbriae in Escherichia coli indicates that this operon is a member of the S/Foc family. The genetic determinant coding for the F165(2) fimbriae was cloned from the chromosome of the porcine E. coli wild-type strain 4787 (O115:K-1:H51:F165). The cloned F165(2) and the wild-type operon expressed a major fimbrial protein subunit of molecular mass 17.2 kDa that was detected by anti-F165 and anti-F1C polyclonal sera. The sequences of the f165(2)A and f165(2)FGH genes are reported. Major subunit gene f165(2)A encodes a mature protein of 156 amino acids. Minor subunit genes F, G and H encode mature proteins of 148, 145 and 276 amino acids, respectively. The amino acid sequences of the four proteins share similarities with those of the known S and F1C fimbrial antigens that are produced by extraintestinal E. coli which is associated with sepsis, urinary tract infections and newborn meningitis. The F165(2)A protein was identical to the major subunit protein of F1C, with a difference only at the first position. It was also similar, to a lesser extent, to the major subunit proteins of SfaI and SfaII fimbriae. F165(2)F was identical to FocF and SfaG/SfaIIS, and F165(2)H was more closely related to FocG than to SfaIS/SfaIIS, and F165(2)H was more closely related to FocH than to SfaIH/SfaIIH.

Characterization of Serpulina hyodysenteriae isolates of serotypes 8 and 9 from Quebec by restriction endonuclease fingerprinting and ribotyping.

This study was undertaken to assess the discriminatory value of restriction endonuclease fingerprinting (REF) analysis and ribotyping of 21 Serpulina hyodysenteriae isolates of serotypes 8 and 9. For REF analysis, DNAs were digested with the BglII restriction enzyme and the resultant fragments were separated by polyacrylamide gel electrophoresis. For ribotyping, hybridization of BglII genomic fragments with a probe of rrnB operon using an Escherichia coli rDNA probe was performed on all isolates. Although many isolates shared a common pattern by BglII REF and BglII ribotyping analysis, differences among some S. hyodysenteriae isolates were observed. REF and ribotyping using BglII restriction enzyme, were not specific for serotypes. The predominance of an REF and a ribotype pattern among S. hyodysenteriae isolates from Quebec suggested that epidemiologically important S. hyodysenteriae types occur in different swine herds.

Virulence factors associated with F165-positive Escherichia coli strains isolated from piglets and calves.

In this study, 91 F165-positive Escherichia coli isolated from calves and piglets with diarrhea or septicemia were characterized with respect to receptor binding specificity, presence of the aerobactin system, production of colicin V, resistance to the bactericidal effects of serum. Although most F165-positive isolates shared similar DNA sequences with pap operon sequences, less than half of these isolates demonstrated MRHA to P antigen of human red blood cells and Forssman antigen of sheep red blood cells recognized by P and F (or Prs) adhesins respectively. Certain F165-positive isolates sharing similar DNA sequences with both pap and sfa operon sequences demonstrated mannose-resistant hemagglutination of sheep erythrocytes, as observed in human uropathogenic E. coli possessing the prs operon. Most isolates caused mannose-resistant, neuraminidase-resistant hemagglutination of human, equine, feline, and bovine erythrocytes. Thus, F165-positive isolates express one or more adhesins with different receptor binding specificities. An association was observed between the various receptor binding specificities and serogroup. Most F165-positive isolates possessed the aerobactin system and were resistant to the bactericidal effects of serum, but only 38.5% isolates produced colicin V.

Receptor binding specificity and pathogenicity of Escherichia coli F165-positive strains isolated from piglets and calves and possessing pap related sequences.

Most of 82 F165-positive Escherichia coli isolated from calves and piglets with diarrhea or septicemia and possessing pap related sequences caused mannose-resistant, neuraminidase-resistant hemagglutination of human and bovine erythrocytes. Less than half of these isolates demonstrated binding specificity for the alpha-D-galactosyl-(1-4)-beta-D-galactopyranose or galactose-N-acetyl-alpha-(1-3) galactose-N-acetyl moieties recognized by P and F (or Prs) adhesins respectively. Binding specificity for the galactose-N-acetyl-alpha-(1-3) galactose-N-acetyl moiety was associated with isolates causing septicemia in newborn piglets.

Isolation and characterization of adhesin-defective TnphoA mutants of septicaemic porcine Escherichia coli of serotype O115:K-:F165.

Non-enterotoxigenic porcine Escherichia coli strains belonging to the serogroup O115 have been associated with septicaemia and diarrhoea. Putative factors important in the pathogenicity of E. coli of serogroup O115 include fimbrial antigen F165, haemagglutination (MRHA), lipopolysaccharide, serum resistance, capsule and production of aerobactin. Using TnphoA transposon insertion mutagenesis, two classes of mutants were obtained from E. coli of serotype O115:F165 with respect to the phenotypic expression of fimbrial antigen F165 and MRHA of sheep erythrocytes: class I, F165-MRHA-, serum resistant; class II, F165+MRHA-, serum resistant. In a chicken lethality model, class I mutants were either virulent or of intermediate virulence, while class II mutants were of intermediate virulence. Alkaline phosphatase activity of class I and class II TnphoA mutants showed similar environmental regulation to that of fimbrial antigen F165. Moreover, class I and class II mutants were mutated in the prs-like locus, and lacked a 18.5 kDa and/or a 17.5 kDa fimbrial band.

Molecular cloning of a determinant coding for fimbrial antigen F165(1), a Prs-like fimbrial antigen from porcine septicaemic Escherichia coli.

The genetic determinant coding for F165(1) fimbriae was cloned from the chromosome of the porcine Escherichia coli wild-type strain 4787 (O115:K-:H51:F165). The fimbrial determinant was further subcloned into the BamHI site of pACYC184 and a restriction map was established. On Southern hybridization, identity between the chromosomally encoded prs-like determinant of strain 4787 and its cloned counterparts was demonstrated. The cloned F165(1) fimbriae and those of the wild-type strain possessed a major protein subunit of molecular mass 18.5 kDa. Strains expressing F165(1) fimbriae were detected using an F165-specific polyclonal antiserum and caused mannose-resistant haemagglutination and agglutination of Forssman latex beads. Antiserum against the cloned F165(1) fimbriae recognized a 18.5 kDa band in the parent strain 4787.