Pathogens in Milk: Enterobacter Species. Reference Module in Food Sciences.

Enterobacter species commonly occur in the environment and are recognized as opportunistic human pathogens in clinical settings. However, with the exception of Enterobacter sakazakii (Cronobacter), Enterobacter species are not normally considered foodborne pathogens. Cronobacter are particularly associated with illness in infants, particularly within the first 3 months after birth. Therefore, although Cronobacter are found in a wide range of fresh and dried food materials, it is their contamination of the infant formula production chain that is the major cause for concern. Cronobacter are noted for their ability to survive during desiccation and their persistence in dried infant food for at least 2 years.

Investigation of the impact of simulated commercial centrifugation and microfiltration conditions on levels of Mycobacterium avium subsp. paratuberculosis in milk

The potential for physical removal of Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) from milk by centrifugation and microfiltration was investigated by simulating commercial processing conditions in the laboratory by means of a microcentrifuge and syringe filters, respectively. Results indicated that both centrifugation of preheated milk (60 degrees C) at 7000 x g for 10 s, and microfiltration through a filter of pore size 1.2 mu m, were capable of removing up to 95-99.9% of M. paratuberculosis cells from spiked whole milk and Middlebrook 7H9 broth suspensions, respectively. Centrifugation and microfiltration may therefore have potential application within the dairy industry as pretreatments to reduce M. paratuberculosis contamination of raw milk.

Inactivation of Mycobacterium avium subsp. paratuberculosis in milk during commercial pasteurisation

Four studies have been published relating to the inactivation of Mycobacterium avium subsp. paratuberculosis (Map) by commercial HTST pasteurization. Three of these were large surveys of commercially pasteurized milk at processing/retail level in the UK and Ontario, Canada, and the fourth a pasteurization study involving naturally infected milk and commercial-scale pasteurizing plant. Evidence that Map is capable of surviving commercial pasteurization was obtained in two of the studies: viable Map was cultured from 50 ml aliquots of commercially pasteurized milk after decontamination with 0.75% cetylpyridinium chloride for 5 h and then culture on Herrold's egg-yolk medium without antibiotics. In both studies culture did not commence until 24-72 h post-pasteurization and samples were stored at 4 degrees C in the interim period. In the other two milk surveys smaller volumes of milk were tested (1-5 ml and 15 ml) and no firm evidence of surviving Map was obtained. The three milk surveys differed in other respects - chemical decontamination, culture media used and use of antibiotics. Recent findings suggest that sub-lethally heat-injured Map in pasteurized milk have the potential to recover viability if stored at 4 degrees C for 48 h between heating and testing.

Dried blood spot liquid chromatography assay for therapeutic drug monitoring of metformin

The use of blood spot collection cards is a simple way to obtain specimens for analysis of drugs for the purpose of therapeutic drug monitoring, assessing adherence to medications and preventing toxicity in routine clinical setting. We describe the development and validation of a microanalytical technique for the determination of metformin from dried blood spots. The method is based on reversed phase high-performance liquid chromatography with ultraviolet detection. Drug recovery in the developed method was found to be more than 84%. The limits of detection and quantification were calculated to be to be 90 and 150 ng/ml, respectively. The intraday and interday precision (measured by CV%) was always less than 9%. The accuracy (measured by relative error, %) was always less than 12%. Stability analysis showed that metformin is stable for at least 2 months when stored at -70 degrees C. The small volume of blood required (10 mu L), combined with the simplicity of the analytical technique makes this a useful procedure for monitoring metformin concentrations in routine clinical settings. The method is currently being applied to the analysis of blood spots taken from diabetic patients to assess adherence to medications and relationship between metformin level and metabolic control of diabetes. (c) 2006 Elsevier B.V. All rights reserved.

Components of the peptidome and transcriptome persist in lin wa pi: the dried skin of the Heilongjiang brown frog Rana amurensis as used in Traditional Chinese Medicine

Although the ancient practice of traditional Chinese medicine (TCM) utilizes predominantly herbal ingredients, many of which are now the subject of intense scientific scrutiny, significant quantities of animal tissue-derived materials are also employed. Here we have used contemporary molecular techniques to study the material known as lin wa pi, the dried skin of the Heilongjiang brown frog, Rana amurensis, that is used commonly as an ingredient of many medicines, as a general tonic and as a topical antimicrobial/wound dressing. Using a simple technology that has been developed and validated over several years, we have demonstrated that components of both the skin granular gland peptidome and transcriptome persist in this material. Interrogation of the cDNA library constructed from the dried skin by entrapment and amplification of polyadenylated mRNA, using a "shotgun" primer approach and 3'-RACE, resulted in the cloning of cDNAs encoding the precursors of five putative antimicrobial peptides. Two (ranatuerin-2AMa and ranatuerin-2AMb) were obvious homologs of a previously described frog skin peptide family, whereas the remaining three were of sufficient structural novelty to be named amurins 1-3. Mature peptides were each identified in reverse phase HPLC fractions of boiling water extracts of skin and their structures confirmed by MS/MS fragmentation sequencing. Components of traditional Chinese medicines of animal tissue origin may thus contain biologically active peptides that survive the preparation procedures and that may contribute to therapeutic efficacy.

Effects of the stockperson on dairy cow behaviour and milk yield

The relationship between stockperson behaviour, measured as verbal and physical interactions with the dairy cows (no. ¼ 210), during milking and the subsequent milk yield obtained was examined. The numbers of steps and kicks made by the cows during milking was recorded. The behaviour of two stockteams, each consisting of two stockmen, were recorded over 10 weekend sessions. The two teams varied in the types of interactions and when the stockteam that performed more positive interactions worked with the cows (team A), the cows had a significantly higher milk yield (P , 0·05) although this difference was small (17·54 v. 17·44 kg). When team A was milking the cows also stepped and kicked on the platform significantly more ( P , 0·05) compared with team B. The results also indicated that while each stockteam tended to interact with the same cows each session, different stockpersons interacted with different cows. These findings highlight the importance of the role of the stockperson in milk output and dairy cow behaviour in a commercial setting.

Prevalence of Mycobacterium avium subspecies paratuberculosis in Swiss raw milk cheeses collected at the retail level.

A total of 143 raw milk cheese samples (soft cheese, n = 9; semihard cheese, n = 133; hard cheese, n = 1), collected at the retail level throughout Switzerland, were tested for Mycobacterium avium ssp. paratuberculosis (MAP) by immunomagnetic capture plus culture on 7H10-PANTA medium and in supplemented BAC-TEC 12B medium, as well as by an F57-based real-time PCR system. Furthermore, pH and water activity values were determined for each sample. Although no viable MAP cells could be cultured, 4.2% of the raw milk cheese samples tested positive with the F57-based real-time PCR system, providing evidence for the presence of MAP in the raw material. As long as the link between MAP and Crohn’s disease in humans remains unclear, measures designed to minimize public exposure should also include a focus on milk products.

Elements of the granular gland peptidome and transcriptome persist in air-dried skin of the South American orange-legged leaf frog, Phyllomedusa hypocondrialis.

The defensive strategy of amphibians against predator attack relies heavily on the secretion of noxious/toxic chemical cocktails from specialized skin granular glands. Bioactive peptides constitute a major component of secretions in many species and the most complex are produced by neotropical leaf frogs of the sub-family Phyllomedusinae. We recently reported that these skin secretions contain elements of both the granular gland peptidome and transcriptome and that polyadenylated mRNAs constituting the latter are protected from degradation by interactions with endogenous amphipathic peptides. This thus permits parallel amino acid sequencing of peptides and nucleic acid sequencing of cloned precursor transcripts from single lyophilized samples of secretion. Here we report that the protection afforded is sufficiently robust to permit transcriptome studies by cloning of full-length polyadenylated peptide precursor encoding mRNAs from libraries constructed using ambient temperature air-dried skin from recently deceased specimens as source material. The technique was sufficiently sensitive to permit the identification of cDNAs encoding antimicrobial peptides constituted by six different isoforms of phylloseptin and two dermaseptins. Also, for the first time, establishment of the nucleic acid and amino acid sequence of the precursor encoding the phyllomedusine frog skin bradykinin-related peptide, phyllokinin, from cloned cDNA, was achieved. These data unequivocally demonstrate that the granular gland transcriptome persists in air-dried amphibian skin—a finding that may have fundamental implications in the study of archived materials but also in the wider field of molecular biology.