Occurrence of naphtho-gamma-pyrones- and ochratoxin A-producing fungi in French grapes and characterization of new naphtho-gamma-pyrone polyketide (aurasperone G) isolated from Aspergillus niger C-433

A survey on the occurrence on grape of fungi species in 2001 and their capability to produce ochratoxin A (OTA) and naphtho-gamma-pyrones (NGPs) was conducted in different vineyards from several French viticulture regions. The total numbers of fungal isolates, from setting to harvest, were 732. The Aspergillus genus was essentially represented by section Nigri (98.53%) and it was predominant (74.72%) when compared to Penicillium (25.27%). Approximately one third (30.46%) of the fungal isolates were OTA producers, and 94.17% belong to black aspergilli; Aspergillus carbonarius was the main OTA producer. Moreover, 8.33% of isolates (belong to A. carbonarius and A. niger) were NGP producers. However, none of the Penicillium spp. or other Aspergillus spp. isolates can produces NGP derivatives under the conditions used. No other study on NGPs production by fungi isolated from grapes has been reported. In the second part, a novel NGP, named aurasperone G (1), was isolated from the fermentation broth of the culture extracts of Aspergillus niger C-433, strain producer of OTA, along with the known compound aurasperone F (2). The chemical structure of the new polyketide was proposed based on complete (1)H and partial (13)C, COSY, HMQC, 1D NOE NMR spectra as well as UV and MS spectra. This new NGP was not reported before in nature or prepared synthetically.     

Differentiation of bovine Staphylococcus aureus isolates by use of polymorphic tandem repeat typing

Staphylococcus aureus is a common cause of bovine mastitis. A simple and efficient typing method would be helpful in understanding S. aureus sources and spread. Ninety-six S. aureus strains, isolated between 1961 and 2003 from the milk of 90 dairy cows belonging to 75 French herds, were subjected to multiple-locus variable-number tandem repeats analysis (MLVA) by PCR. The conjunction of clfA, clfB, SAV1078 and fnb gene tandem repeats (TRs) enabled the definition of 61 types. When coa, spa, sdrC, sdrD and sspA TRs were used individually as additional markers, 63, 68, 67, 65 and 67 types were defined, respectively, versus 77 types when they were all included in the method. These additional TRs did not improve the differentiation of isolates collected in the same farm. The MLVA procedure using the tandem repeats embedded in clfA, clfB, SAV1078 and fnb loci as a basic combination at the herd level or associated with other TRs such as spa, sdrC, sdrD, sspA and coa can be a valuable tool for bovine S. aureus epidemiological studies.     

High resolution protein electrophoresis of 100 paired canine cerebrospinal fluid and serum

This study was performed to investigate the diagnostic relevance of cerebrospinal fluid (CSF) high resolution electrophoresis. The laboratory technique was applied to 100 paired samples of canine CSF and serum, with paired samples tested during the same analytical run, as recommended in human medicine. Ninety four of the dogs had a neurological disease and 6 healthy dogs served as a control group. A strong linear correlation between CSF total protein concentration and the albumin quota (AQ) was found in the control group and in the inflammatory (infectious or noninfectious), neoplastic, and miscellaneous groups: AQ = 0.015 CSF total protein--0.102, r = 0.990. This correlation suggests that an increased CSF total protein concentration can be an indicator of blood brain barrier dysfunction. The highest median AQ value was found in the aseptic suppurative meningitis group, but no statistical differences were found between this and the other groups. The AQ, calculated with this technique, did not provide any additional information. Moreover, although unexpected, the electrophoretic profiles were not characteristic of any particular disease. In conclusion, this study did not confirm high resolution electrophoresis of paired CSF and serum samples to be a valuable ancillary diagnostic tool for canine neurological diseases.     

Effect of repeated freeze-thaw cycles on routine plasma biochemical constituents in canine plasma

BACKGROUND: The effect of repeated freeze-thaw cycles on plasma constituents has not been assessed in dogs, although such a procedure is not uncommon to use in routine laboratory practice. OBJECTIVE: To assess the effect of freeze-thaw cycles on routine plasma constituents in healthy dogs. METHODS: Six healthy adult dogs were used. Blood was sampled and placed in heparinized tubes. After centrifugation, plasma was separated into 5 aliquots. One aliquot was considered as the reference aliquot and used immediately for the assay of all of the biochemical constituents. All of the other aliquots were stored at 20 degrees C. Three aliquots underwent 1, 2, or 3 freeze-thaw cycles during a 1- to 3-day period. The last aliquot remained at 20 degrees C throughout the study and was thawed on the third day. The following biochemical constituents were assayed: glucose, urea, creatinine, total proteins, sodium, potassium, chloride, calcium, phosphates, aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), and alkaline phosphatase (ALP). RESULTS: No clinically relevant change was observed between the different aliquots for all of the constituents. CONCLUSION: Repeated freeze-thaw cycles do not cause changes in the biochemical constituents studied in canine plasma.     

Species and staphylococcal cassette chromosome mec (SCCmec) diversity among methicillin-resistant non-Staphylococcus aureus staphylococci isolated from pigs

While methicillin-resistant Staphylococcus aureus (MRSA) ST398 is known to be widespread in pig farms, few studies have investigated the species diversity and SCCmec types of methicillin-resistant non-S. aureus staphylococci (MRNAS) residing in the nose of pigs. We examined nasal swab samples of 200 pigs originating from 10 Belgian pig farms previously found positive for MRSA ST398. Suspected staphylococcal isolates were subjected to a 16S rRNA-mecA-nuc PCR. Confirmed MRNAS were genotypically identified to the species level and investigated with a SCCmec typing PCR. MRNAS (n=72) were detected on all 10 farms and were carried by 29.5% of the pigs. Seven MRNAS species were found: Staphylococcus epidermidis (38.9%), Staphylococcus sciuri (18.1%), Staphylococcus pasteuri (18.1%), Staphylococcus rostri (12.5%), Staphylococcus warneri (8.3%), Staphylococcus haemolyticus (2.7%) and Staphylococcus hominis (1.4%). SCCmec cassettes were of type IVa (29.2%), type IVc (25%), type III (22.2%), type V (5.6%) or could not be assigned to any of the known types (NT types) (18.1%). Five distinct NT types were found. The predominance of methicillin-resistant S. epidermidis (MRSE) in our samples is remarkable, as MRSE is mainly associated with humans. The finding of three different SCCmec elements (IVa, V, NT type 1) in MRNAS that also prevail or predominate in MRSA ST398 shows that MRNAS might be an important SCCmec reservoir for MRSA in pigs. Yet, the occurrence of multiple other SCCmec types illustrates that further studies are required to understand the presence and spread of SCCmec in methicillin-resistant staphylococci from animals.     

Microbiologic findings in feedlot cattle with acute interstitial pneumonia

OBJECTIVE: To test the hypothesis that feedlot cattle with acute interstitial pneumonia (AIP) have bacterial infection of the lung or liver and concurrent bovine respiratory syncytial virus (BRSV) infection significantly more often than pen mates without AIP ANIMALS: 39 feedlot cattle with signs consistent with AIP and no history of treatment with antimicrobials and 32 healthy control cattle from the same pens. PROCEDURES: Lung and liver specimens were obtained postmortem for bacterial or mycoplasmal culture and histologic examination; lung tissue was assessed for BRSV infection immunohistochemically. RESULTS: Among affected cattle, 26 had AIP confirmed histologically. Lung tissue from 11 cattle with AIP yielded microbial respiratory tract pathogens on culture; tissues from control animals yielded no microbial growth. In 4 cattle with AIP and 2 control animals, liver abscesses were detected; bacteria were isolated from abscessed tissue in 3 and 1 of those animals, respectively. Immunohistochemically, 9 cattle with AIP and no control animals were BRSV-positive. Histologically, 9 AIP-affected cattle had only acute alveolar damage with exudation, and the other 17 had acute exudation with type II pneumocyte hyperplasia. No lesions of AIP were detected in control animals. Only 4 AIP-affected cattle had bacterial infection of the lung with concurrent BRSV infection. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that microbial respiratory tract pathogens are more common in cattle with AIP than in healthy pen mates. Control of bacterial pneumonia late in the feeding period may reduce the incidence of AIP at feedlots where AIP is a problem.     

Reliable ELISAs showing differences between resistant and susceptible lines in hens orally inoculated with Salmonella Enteritidis

Reliable ELISAs were investigated with the aim to select hen lines resistant to Salmonella Enteritidis and producing high levels of antibodies. In the first experiment, the relation between the humoral response and the bacteriological results was assessed on hens from the Y11 resistant line and the L2 susceptible line, orally inoculated with 10(8) CFU S. Enteritidis per animal. Anti-lipopolysaccharide (LPS) IgG titres were higher but the liver and spleen were less contaminated in hens from the Y11 line than in hens from the L2 line (p = 0.013, 0.031 and 0.026 respectively). In the second experiment, the hens were inoculated orally with 1.7 x 10(8) CFU S. Enteritidis per animal in order to select the ELISA methods showing the more significant differences. ELISAs were based on LPS, flagella, LPS from rough (LPS-R) and smooth strains (LPS-S) and detected IgG and IgM antibodies from sera and yolks. No between-line host response variation was observed in the yolk, with LPS-S and R antigens nor with anti-LPS IgM in the sera. Otherwise, significant differences were encountered between hen lines with the ELISAs performed on the sera detecting anti-LPS IgG, anti-flagella IgG or IgM (p = 0.017, 0.017 and p < 0.001 respectively). When comparing the kinetics of the selected ELISAs, the IgG antibodies against LPS detected between-line variations as early as 1 to 4 weeks pi, whereas with IgG against flagella, the differences were only detected at 1 and 2 weeks pi and with IgM against flagella, the differences were significant at 1, 2, 4 and 8 weeks pi. In conclusion, resistant hen lines producing higher levels of antibodies than the susceptible hen lines may be selected with these ELISAs.     

Synthesis and regulation of 17α-hydroxy-20β-dihydroprogesterone in immature males of Oncorhynchus mykiss

Three experimental approaches were chosen to study the question if the progestin 17-hydroxy-20-dihydroprogesterone (1720OHP) is synthesised in testes of young Oncorhynchus mykiss, in which the absence of spermatozoa was verified histologically: first, in order to detect 20-hydroxysteroid dehydrogenase activity (20HSD), testes homogenates were incubated with ³H-labeled 17OHP.Metabolites were analysed by TLC, HPLC, and repeated crystallization to constant isotope ratios. One of the metabolites was identified as 1720OHP-³H, indicating that already immature testes contain 20HSD activity and are able to produce 20-reduced steroids. Second, 1720OHP was quantified by radioimmunoassay in incubates of testes fragments. The sensitivity of the gonads to gonadotropin II (GtH II) became evident when comparing incubations in the absence and presence of GtH II. Third, plasma levels of 1720OHP were significantly higher in animals injected with partially purified salmon gonadotropin, compared to controls. Thus, for the first time, it could be shown that 20HSD is present in testicular cells other than spermatozoa. Furthermore, 1720OHP is indeed secreted at a very early stage of testicular development; 1720OHP secretion is also responsive to GtH II. Future studies will have to show if the functions of this progestin include the stimulation of spermatogenesis.