Mycoplasma species in vaginas of dairy cows before and after exposure to bulls and their association with conception.

Mycoplasma species can colonize the urogenital tract of dairy cattle. However, interrelationships between Mycoplasma spp. and reproductive performance in dairy herds are unclear. In this study, we measured apparent prevalences of Mycoplasma spp. in the vaginas of dairy cows (n = 629) pre- and post-bull exposure in dairy herds with and without Mycoplasma bovis clinical disease (n = 5 herds), and assessed associations between variables describing reproductive performance and consequent Mycoplasma spp. isolation. Mycoplasma spp. were infrequently isolated from the vagina pre- (1.9%; 12/629) and post-bull (3.2%; 20/629) exposure. Of the mycoplasmas isolated, Mycoplasma bovigenitalium was isolated most frequently (87.5%; 28/32), followed by Mycoplasma californicum (9.3%; 3/32). Mycoplasma bovis was only isolated from one cow. We were unable to provide any evidence of venereal transmission of M. bovis in cows in M. bovis-infected herds that use natural service bulls. There was an insufficient number of cows with Mycoplasma spp. in the vagina pre-bull exposure to assess effects on subsequent reproductive performance. Cows that had not conceived before post-bull exposure sampling had much greater odds (odds ratio 14.8; 95% confidence interval 4.2 to 52.3) of having a Mycoplasma sp. isolated from the vagina at this time compared with those that had conceived. Also, within those that had conceived, delayed conception increased the odds of having a Mycoplasma spp. isolated from the vagina at the post-bull exposure sampling by a factor of 1.62 for every additional week not pregnant. The likely cause of these findings is that cows that remain not pregnant for longer are more likely to be served by a bull (likely repeatedly) and subsequently become colonized with a Mycoplasma sp. (mostly M. bovigenitalium) through venereal transmission. In dairy herds that use bulls, there is a greater chance of isolating a Mycoplasma sp. (mostly M. bovigenitalium) after a period of bull breedings from the vaginas of cows that have remained nonpregnant for longer during the bull breeding period.

Mycoplasma bovis and other Mollicutes in replacement dairy heifers from Mycoplasma bovis-infected and uninfected herds: A 2-year longitudinal study.

Replacement dairy heifers exposed to Mycoplasma bovis as calves may be at risk of future clinical disease and pathogen transmission, both within and between herds; however, little information is available about these risks. We conducted a 2-yr longitudinal (panel) study starting with 450 heifer calves reared to weaning in 8 herds (7 M. bovis infected with clinical disease, 1 uninfected) under the same ownership. After weaning, heifers were commingled and managed with non-study heifers at a single heifer rearing facility. Nose, conjunctival, and vaginal swabs were collected along with a blood sample at weaning, prebreeding, precalving, and approximately 1 mo postcalving. Additionally, a colostrum sample was collected upon calving and a composite milk sample was collected 1 mo postcalving. The swabs, colostrum, and milk samples were cultured for Mycoplasma spp., and serum from the blood was evaluated for serological evidence of exposure to M. bovis using an ELISA. Despite a high M. bovis ELISA seroprevalence at weaning in the heifers from the 7 M. bovis-infected herds with clinical disease [72% (289/400); range by herd: 28-98%], M. bovis was isolated from only 4% (16/400) of the same heifers at the same time. In heifers from the uninfected herd at weaning, M. bovis seroprevalence was 2% (1/50) and M. bovis was not detected by culture. Mycoplasma bovis was isolated from 0.5% (2/414) of heifers at prebreeding, 0% (0/374) of heifers at precalving, and 0.3% (1/356) of heifers 1 mo postcalving. The nose was the predominant anatomical site of M. bovis colonization (74%; 14/19 culture positives). A single heifer (from an M. bovis-infected herd with clinical disease) was repeatedly detected with M. bovis in its nose at weaning, prebreeding, and postcalving samplings. This demonstrates the possibility, albeit rare, of a long-term M. bovis carrier state in replacement heifers exposed to M. bovis as calves, up to at least 1 mo after entry into the milking herd. No M. bovis clinical disease was detected in any heifer from weaning through to the end of the study (approximately 1 mo after calving). Acholeplasma spp. were commonly isolated throughout the study. Mycoplasma bovigenitalium, Mycoplasma bovoculi, and Mycoplasma bovirhinis were isolated infrequently. Mycoplasma bovis seroprevalences at prebreeding, precalving, and postcalving samplings were 27% (112/414), 12% (46/374), and 18% (65/356), respectively. Overall, the results show that replacement heifers from groups exposed to M. bovis preweaning can become colonized with M. bovis and that colonization can, uncommonly, be present after their first calving. For groups of 50 or more heifers exposed to M. bovis preweaning, there is at least a nontrivial probability that the group will contain at least 1 shedding heifer postcalving.

Whole transcriptome analysis of bovine mammary progenitor cells by P-Cadherin enrichment as a marker in the mammary cell hierarchy.

Adult bovine mammary stem cells possess the ability to regenerate in vivo clonal outgrowths that mimic functional alveoli. Commonly available techniques that involve immunophenotype-based cell sorting yield cell fractions that are moderately enriched, far from being highly purified. Primary bovine mammary epithelial cells segregated in four different populations according to the expression of P-Cadherin and CD49f. Sorted cells from each fraction were tested for the presence of lineage-restricted progenitors and stem cells. Only cells from the CD49fhigh/P-Cadherinneg subpopulation were able to give rise to both luminal- and myoepithelial-restricted colonies in vitro and generate organized outgrowths in vivo, which are hallmarks of stem cell activity. After whole transcriptome analysis, we found gene clusters to be differentially enriched that relate to cell-to-cell communication, metabolic processes, proliferation, migration and morphogenesis. When we analyzed only the genes that were differentially expressed in the stem cell enriched fraction, clusters of downregulated genes were related to proliferation, while among the upregulated expression, cluster of genes related to cell adhesion, migration and cytoskeleton organization were observed. Our results show that P-Cadherin separates mammary subpopulations differentially in progenitor cells or mammary stem cells. Further we provide a comprehensive observation of the gene expression differences among these cell populations which reinforces the assumption that bovine mammary stem cells are typically quiescent.

Whole dairy herd sampling to detect subclinical intramammary Mycoplasma bovis infection after clinical mastitis outbreaks.

After clinical Mycoplasma bovis mastitis outbreaks in dairy herds, M. bovis can persist as subclinical intramammary infections. Identification and culling of sub-clinically infected cows may be warranted to reduce future pathogen transmission and disease. In this study, apparent cow-level prevalences of M. bovis intramammary infection within 4 milking herds immediately following outbreaks of clinical disease due to M. bovis were determined utilising PCR and culture. All clinically affected M. bovis cows had been culled from the herds prior to herd sampling. Composite milk samples were collected once from each cow (n = 2,258) using a routine milk recording sampling technique. These samples were pooled for PCR screening; positive pools were analysed in different sized pools as needed from large to small, until individual PCR-positive animals could be identified. Despite M. bovis seroprevalences of 76% (herd 1), 40% (herd 2), 20% (herd 3) and 16% (herd 4), apparent prevalences of intramammary infection in the main milking group based on PCR in herds 1 to 4 were 0.2% (1/497), 0.0% (0/475), 0.1% (1/816) and 0.0% (0/444), respectively. Due to the low apparent prevalences of subclinical intramammary mycoplasma infections in these herds and the high expense associated with milk sample collection and testing, the return on diagnostic investment was very limited, particularly considering that additional cows are likely to have been colonised with mycoplasma in other anatomical sites. The results of this study suggest that pursuing identification of cows with subclinical intramammary mycoplasma infections following resolution of clinical M. bovis disease outbreaks in dairy herds may be of minimal benefit in programs designed to control or eradicate M. bovis.

Isolation of Mycoplasma spp. and serological responses in bulls prior to and following their introduction into Mycoplasma bovis-infected dairy herds.

With the common use of bulls for breeding following a period of artificial insemination in seasonally bred dairy herds, it is important to consider the potential role of the bull in transmission of Mycoplasma spp. within and between herds. This study aimed to assess the prevalence of Mycoplasma spp. in a population of bulls before and after use in Mycoplasma bovis-infected herds. The frequency of subclinical infection was also measured serologically postbreeding, and the association of Mycoplasma spp. on semen quality was evaluated. Mycoplasma bovis was isolated from 4 of 118 bulls after use in 4 herds infected with M. bovis. In the bulls, M. bovis seroprevalence increased from 9% prebreeding to 46% postbreeding with a total seroconversion rate of 44% across the 4 herds, with no evidence of clinical disease. There was no association of Mycoplasma spp. in the bulls' semen and abnormal palpation characteristics (enlarged or nodular) of seminal vesicular glands or poor semen quality attributes such as semen mass activity, sperm motility, and morphology. These results demonstrate a high degree of subclinical exposure of the bulls to M. bovis in infected herds and highlight the potential for bulls to be mycoplasma carriers within and between herds. Herd biosecurity protocols and control programs should take into account the potential role of bulls in the introduction and spread of Mycoplasma spp.

Short communication: Shedding of Mycoplasma bovis and antibody responses in cows recently diagnosed with clinical infection.

Mycoplasma bovis can have significant consequences when introduced into immunologically naïve dairy herds. Subclinically infected carrier animals are the most common way that M. bovis is introduced into herds. Although M. bovis udder infections can be detected by milk sampling lactating animals before their introduction, currently, no definitive way of identifying M. bovis carrier animals that are nonlactating (i.e., calves, heifers, dry cows, or bulls) is available. Understanding the prevalence of M. bovis shedding from various body sites in clinically infected animals could inform strategies for the detection of subclinical infection in nonlactating stock. The mucosal surfaces of the nose, eye, and vagina of 16 cows with recent clinical mastitis caused by M. bovis were examined for the presence of M. bovis shedding. Blood was collected for serological evaluation by a commercially available ELISA. Mycoplasma bovis was isolated from the vagina of only 3 (18.8%) of the cows and was not detected from the noses or eyes of any of the cows. Fifteen of the 16 (93.8%) cows were seropositive to the ELISA. With such low prevalence of detection of M. bovis from the vagina and no detections from the noses or eyes of recently clinically infected animals, it is very likely that sampling these sites would be ineffective for detecting subclinical infection in cattle. Serology using the ELISA may have some use when screening animals for biosecurity risk assessment. However, more information regarding time to seroconversion, antibody longevity, and test diagnostic sensitivity and specificity are required to define the appropriate use of this ELISA for biosecurity purposes.

Bulk tank milk antibody ELISA as a biosecurity tool for detecting dairy herds with past exposure to Mycoplasma bovis.

In Australia, one of the biosecurity recommendations to help prevent the introduction of Mycoplasma bovis into a dairy herd is to use a PCR assay on bulk tank milk (BTM) samples to evaluate the M. bovis infection status of potential source herds. An alternative approach is to assess the immunological status of the herd with respect to previous exposure to M. bovis via the use of an ELISA that is commercially available for use on cattle milk and serum. The objectives of this study were to (1) evaluate factors potentially associated with variation in the ELISA BTM optical density coefficient (ODC%) in previously exposed herds, (2) evaluate the association between the proportion of cows that are ELISA positive and the BTM ELISA ODC%, (3) assess agreement between the BTM ELISA and PCR and culture, and (4) compare BTM ELISA ODC% between the "hospital" herd and the main lactating herd on the same farm. Bulk tank milk samples (n = 192) were collected from 19 dairy herds with a history of clinical M. bovis disease and from 6 control herds (herds with no known clinical cases of mycoplasmosis). For 28 of the BTM samples collected, blood was also collected from 50 lactating cows contributing to that bulk tank sample. From 1 herd, concurrent paired BTM samples were collected from the main herd and the hospital herd on 16 occasions. All BTM samples were analyzed by ELISA (Bio-X Bio K 302, Bio-X Diagnostics, Rochefort, Belgium), PCR, and culture. The BTM ELISA ODC% was associated with time since initial M. bovis outbreak and time since the start of the herd's calving period. Following an initial outbreak of M. bovis, the BTM ELISA ODC% was highest in the first 8 mo. In split- and seasonal-calving herds, significantly higher BTM ELISA ODC% results were observed 5 to 8 wk after the commencement of the calving period. A significant association was observed between the within-herd seroprevalence for the lactating herd and BTM ELISA ODC%, but within-herd seroprevalence explained little of the variation in BTM ELISA ODC%. When comparing the BTM ELISA with a multiplex probe PCR and culture followed by 16S to 23S rRNA sequencing, there was virtually no agreement above that expected by chance; prevalence-adjusted bias-adjusted kappa values were 0.22 and 0.25 for ELISA category versus PCR category and culture, respectively. Finally, the hospital herd BTM ELISA ODC% mirrored that for the main herd BTM but was significantly higher. This study demonstrates that this commercially available ELISA used on BTM samples may complement the use of BTM PCR or culture in identifying herds from which purchase of animals may pose a higher biosecurity risk for introduction of M. bovis into noninfected herds.

Milk acidification to control the growth of Mycoplasma bovis and Salmonella Dublin in contaminated milk.

Bacterial contamination of milk fed to calves compromises calf health. Several bacterial pathogens that infect cows, including Mycoplasma bovis and Salmonella enterica ssp. enterica serovar Dublin, are shed in milk, providing a possible route of transmission to calves. Milk acidification lowers the milk pH so that it is unsuitable for bacterial growth and survival. The objectives of this study were to (1) determine the growth of M. bovis and Salmonella Dublin in milk, and (2) evaluate the efficacy of milk acidification using a commercially available acidification agent (Salstop, Impextraco, Heist-op-den-Berg, Belgium) to control M. bovis and Salmonella Dublin survival in milk. For the first objective, 3 treatments and a positive control were prepared in 10 mL of milk and broth, respectively, and inoculated with M. bovis or Salmonella Dublin to an approximate concentration of 104 cfu/mL. Each treatment was retained at 5, 23, or 37°C with the positive control at 37°C. Aliquots were taken at 4, 8, 24, 28, 32, 48, 52, and 56 h after inoculation and transferred onto agar medium in triplicate following a 10-fold dilution series in sterile phosphate-buffered saline. All plates were incubated and colonies counted. For the second objective, 4 treatments and a positive control were prepared with 100 mL of milk and inoculated with M. bovis or Salmonella Dublin to an approximate concentration of 106 cfu/mL. With the use of Salstop, treatments were adjusted to an approximate pH of 6, 5, 4, or 3.5. The positive control was left untreated. At 1, 2, 4, 6, 8, and 24 h after treatment, triplicate aliquots were taken, the pH measured, and then the aliquots were transferred onto agar medium and into broth for enrichment. Following incubation, agar colonies were counted, while broths were plated and incubated prior to colonies being counted. All trials were repeated. Mycoplasma bovis did not grow in milk, but Salmonella Dublin proliferated. The pH of all acidification treatments remained stable for 24 h. No viable M. bovis organisms were detected at 1 h of exposure to pH 3.5 and 4 or at 8 h of exposure to pH 5. Following 24 h of exposure to pH 6 M. bovis remained viable. No viable Salmonella Dublin organisms were detected at 2 and 6 h of exposure to pH 3.5 and 4, respectively. Salmonella Dublin remained viable following 24 h of exposure to pH 5 and 6. These results demonstrate that milk acidification using Salstop is effective at eliminating viable M. bovis and Salmonella Dublin organisms in milk if the appropriate pH and exposure time are maintained.

The influence of extracellular matrix and prolactin on global gene expression profiles of primary bovine mammary epithelial cells in vitro.

An in vitro bovine mammosphere model was characterized for use in lactational biology studies using a functional genomics approach. Primary bovine mammary epithelial cells cultured on a basement membrane, Matrigel, formed three-dimensional alveoli-like structures or mammospheres. Gene expression profiling during mammosphere formation by high-density microarray analysis indicated that mammospheres underwent similar molecular and cellular processes to developing alveoli in the mammary gland. Gene expression profiles indicated that genes involved in milk protein and fat biosynthesis were expressed, however, lactose biosynthesis may have been compromised. Investigation of factors influencing mammosphere formation revealed that extracellular matrix (ECM) was responsible for the initiation of this process and that prolactin (Prl) was necessary for high levels of milk protein expression. CSN3 (encoding kappa-casein) was the most highly expressed casein gene, followed by CSN1S1 (encoding alphaS1-casein) and CSN2 (encoding beta-casein). Eighteen Prl-responsive genes were identified, including CSN1S1, SOCS2 and CSN2, however, expression of CSN3 was not significantly increased by Prl and CSN1S2 was not expressed at detectable levels in mammospheres. A number of novel Prl responsive genes were identified, including ECM components and genes involved in differentiation and apoptosis. This mammosphere model is a useful model system for functional genomics studies of certain aspects of dairy cattle lactation.

A functional genomics approach to evaluate candidate genes located in a QTL interval for milk production traits on BTA6.

The potential genetic and economic advantage of marker-assisted selection for enhanced production in dairy cattle has provided an impetus to conduct numerous genome scans in order to identify associations between DNA markers and future productive potential. One area of focus has been a quantitative trait locus on bovine chromosome 6 (BTA6) found to be associated with milk yield, milk protein and fat percentage, which has been subsequently fine-mapped to six positional candidate genes. Subsequent investigations have yet to resolve which of the potential positional candidate genes is responsible for the observed associations with productive performance. In this study, we analysed candidate gene expression and the effects of gene knockdown on expression of beta- and kappa-casein mRNA in a small interfering RNA transfected bovine in vitro mammosphere model. From our expression studies in vivo, we observed that four of the six candidates (ABCG2, SPP1, PKD2 and LAP3) exhibited differential expression in bovine mammary tissue over the lactation cycle, but in vitro functional studies indicate that inhibition of only one gene, SPP1, had a significant impact on milk protein gene expression. These data suggest that the gene product of SPP1 (also known as osteopontin) has a significant role in the modulation of milk protein gene expression. While these findings do not exclude other positional candidates from influencing lactation, they support the hypothesis that the gene product of SPP1 is a significant lactational regulatory molecule.

In vitro replication models for the hepatitis C virus.

Soon after the discovery of the hepatitis C virus (HCV), attention turned to the development of models whereby replication of the virus could be investigated. Among the HCV replication models developed, the HCV RNA replicon model and the newly discovered infectious cell culture systems have had an immediate impact on the study of HCV replication, and will continue to lead to important advances in our understanding of HCV replication. The aim of this study is to deal with developments in HCV replication models in a chronological order from the early 1990s to the recent infectious HCV cell culture systems.

Lactational performance of Quackenbush Swiss line 5 mice.

We evaluated 2 strains of mice for their utility in the investigation of nutritional and molecular regulatory mechanisms of lactation. The lactational performance and milk composition were characterized for an inbred mouse strain, inbred Quackenbush Swiss line 5 (QSi5) selected persistently for fecundity, and a nonselected strain, CBA. The milk yield assessed by changes in BW in response to suckling of sustainable litter sizes for each strain was 3-fold greater (P < 0.001) in QSi5 mice than the CBA strain. The QSi5 mice also produced milk more efficiently (P < 0.001) than CBA mice, despite having the same quantity of mammary tissue per unit of BW. Milk composition did not vary between strains or by stage of lactation, with the exception of lactose concentration, which was greater (P = 0.003) in QSi5 mice. Expression of epsilon-casein was > or = 10-fold greater, and alpha(S1)-casein was > or = 3-fold greater, during mid and late lactation compared with early lactation in both strains, whereas kappa-casein underwent an apparent alteration in posttranslational modifications in both strains from early to mid lactation. Changes in casein composition coincided with an increased susceptibility to proteolytic degradation; hence milk from early lactation may be more readily degraded to facilitate digestion in the neonate. The greater milk synthetic capacity of QSi5 mice over the lactation cycle provides a useful model for studies of nutritional and molecular regulation of lactation.

The role of radiotracer imaging in Parkinson disease.

Radiotracer imaging (RTI) of the nigrostriatal dopaminergic system is a widely used but controversial biomarker in Parkinson disease (PD). Here the authors review the concepts of biomarker development and the evidence to support the use of four radiotracers as biomarkers in PD: [18F]fluorodopa PET, (+)-[11C]dihydrotetrabenazine PET, [123I]beta-CIT SPECT, and [18F]fluorodeoxyglucose PET. Biomarkers used to study disease biology and facilitate drug discovery and early human trials rely on evidence that they are measuring relevant biologic processes. The four tracers fulfill this criterion, although they do not measure the number or density of dopaminergic neurons. Biomarkers used as diagnostic tests, prognostic tools, or surrogate endpoints must not only have biologic relevance but also a strong linkage to the clinical outcome of interest. No radiotracers fulfill these criteria, and current evidence does not support the use of imaging as a diagnostic tool in clinical practice or as a surrogate endpoint in clinical trials. Mechanistic information added by RTI to clinical trials may be difficult to interpret because of uncertainty about the interaction between the interventions and the tracer.

Discharge-flow kinetics measurements using intracavity laser absorption spectroscopy.

Intracavity laser absorption spectroscopy ("ICLAS") has been demonstrated as a feasible detection method for trace species in a discharge flow tube. This implementation has been used to measure the rate of the reaction between atomic hydrogen and NO to form HNO in helium carrier gas. A reaction rate constant of (4.3 +/- 0.4) x 10(-32) cm(6) molecule(-2) s(-1) at 295 K was measured for the reaction H + NO + M --> HNO + M (M = He). The pressure and concentration range enabled by ICLAS detection has allowed us to limit reactive pathways that would inhibit the formation of HNO. The sensitivity of ICLAS, coupled with the versatility of the discharge flow technique, suggests that intracavity absorption spectroscopy will be a useful technique for kinetics measurements on free radicals and other reactive species.

A strategy for obtaining near full-length HCV cDNA clones (assemblicons) by assembly PCR.

Hepatitis C virus (HCV) genotype is a predictor of response, and guides the duration of antiviral therapy. However, with the exception of HCV genotype 1a, 1b and 2a, a limited number of clones from other genotypes exist. Here we report the optimization of long RT-PCR to generate three overlapping amplicons that span the near full length HCV genome from a panel of HCV genotypes (1a, 1b, 2a, 2b, 3a, 4a, 5a). Assembly-PCR (As-PCR) was used to construct near full-length cDNA clones (assemblicons) for each genotype. The optimization of the long RT-PCR on genotype 1a and 1b indicated that QIAamp Viral RNA kit (Qiagen, UK), Expand RT and Expand Long Template PCR system (Roche, UK), were the most efficient in producing the requisite three overlapping amplicons and assemblicons for each genotype. The genotype of each assemblicon was confirmed. Assemblicon generation was only possible when the overlapping amplicons were biotinylated. As-PCR obviated the need for time consuming ligations and cloning. The use of three overlapping amplicons in the construction of HCV assemblicons minimised the chimeric nature of the resultant clone. As-PCR may prove a methodological avenue through which a larger panel of consensus HCV clones could be made available for HCV in vitro investigation.

Do Taq-generated RT-PCR products from RNA viruses accurately reflect viral genetic heterogeneity?

Since the first report of genetically heterogeneous, or quasispecies, populations of RNA viruses, the genetic heterogeneity of the RNA genomes of major viral pathogens has been extensively studied. These studies aim to provide insights into the evolutionary pressures that act upon viruses, in order to define windows where anti-viral therapies will be most effective, to take prognostic values from viral genetic distributions at a given time, and to aid the development of novel therapeutic compounds that may tilt viral replication towards information loss. Many methodologies are employed to analyse genetic distributions of a virus in a given sample, but all involve the generation, and subsequent analysis, of the sequence information contained in a reverse-transcription-polymerase chain reaction (RT-PCR) product. Despite the fact that the aim of these RT-PCRs is to obtain sequence information from viral genomes, their application to this task is approached without adequate consideration of this end-goal. The establishment of an RT-PCR for a specific viral target genome generally proceeds in the same fashion as one would apply to establishing a PCR to determine the presence or absence of a specific target sequence in a given sample. However, it is becoming increasingly apparent that RT-PCR products generated by amplification with the ubiquitous thermostable DNA polymerase Taq, coupled with standard cloning and sequencing methodologies, has the potential to yield inaccurate and misleading data as pertains to the information content of populations of RNA viral genomes. This review discusses varying approaches employed to analyse heterogeneous populations of hepatitis C virus RNA genomes.

Plasma, liver and kidney folate and plasma homocysteine concentrations are poor response variables at very low dietary folate intakes, in a folate depletion/repletion rat model.

Folate depletion/repletion rat models are popular protocols for assessing the bioavailability of folate. Much of the early work carried out on folate bioavailability concentrated on foods naturally high in folate. However, foods low in folate often contribute significantly to folate intake because of their high consumption in the general population. Therefore, the assessment of the bioavailability of foods low in folate is essential to properly estimate folate intake. The present study investigated plasma, liver and kidney folate and plasma homocysteine concentrations as appropriate response variables for measuring folate bioavailability in the rat at very low dietary folate intakes. One hundred and one weanling male rats (Wistar strain) were fed a folate-deficient diet containing 1% succinyl sulfathiazole for 28 d. Following depletion, six rats were randomly assigned to each of 16 repletion diets containing folic acid, fortified white bread, unfortified wholemeal bread or unfortified rye bread calculated to provide 6.25, 12.5, 18.75 and 25 micrograms folate/kg of each diet. After a further 28 d, plasma, liver and kidney folate concentrations were determined by microbiological assay. Plasma homocysteine was measured by HPLC as a functional indicator of folate status. Only a weak correlation was found between the response variables measured and dietary folate intake, indicating that this folate depletion/repletion rat model is not suitable for testing the response of rats fed diets containing very low levels of folate.

Effects of preparation and cooking of folic acid-fortified foods on the availability of folic acid in a folate depletion/repletion rat model.

The practice of food fortification with folic acid offers the potential to increase the folate intake of the general population. To fully exploit the potential of fortification for raising folate nutriture, appropriate food vehicles need to be selected. Selection should involve determination of the availability of folic acid as affected by characteristics of the carrier food, food matrix, food preparation, and cooking. The present study investigated the effects of preparation and cooking of a range of folic acid-fortified foods on the folate status of folate-deficient rats. Fifty-six weanling male rats (Wistar strain) were fed a folate-deficient diet containing 1% succinyl sulfathiazole for 28 days. Following depletion, six rats were randomly assigned to each of eight repletion diets containing cooked or uncooked meringue mix, quick bread mix, brownie mix, or pizza base mix. The test foods were fortified with 1400 microg of folic acid/kg of food and incorporated as 19% of the repletion diets. Each of the first four groups was pair-fed a diet containing a cooked fortified food with another group fed the corresponding uncooked fortified food. After a further 28 days, plasma, liver, and kidney folate concentrations were determined by microbiological assay. Mean plasma and liver folate concentrations of rats fed diets containing cooked fortified foods were similar to those of rats fed uncooked fortified foods. Preparation and cooking did not affect the availability of folic acid from the selected cereal-based convenience foods in this rat model system, suggesting that these foods are appropriate vehicles for fortification with folic acid.

Influence of folic acid-fortified foods on folate status in a folate depletion-repletion rat model.

An increasing number of foods fortified with varying levels of folic acid are appearing in the market place, targeted either at the general population or at specific consumer groups. Although it is assumed that the folate in these products should be highly bioavailable, there is a need to carry out studies to ascertain that this is, in fact, the case. The present study investigated the ability of selected folic acid-fortified foods (targeted at different types of consumer) to increase the folate status of folate-deficient rats. Forty-two weanling male rats (Wistar strain) were fed a folate-deficient diet containing 1 % succinyl sulfathiazole (w/w) for 28 d. Following depletion, seven rats were randomly assigned to each of five repletion diets containing folic acid, Complan, Slim Fast, Opti-Fuel2 or Cola Coa calculated to provide 200 microg folate/kg of each diet. Calculations were based on folate information from the product labels. After a further 28 d, plasma, liver and kidney folate concentrations were determined by microbiological assay. Plasma homocysteine was measured by HPLC as a functional indicator of folate status. The folate content of the foods was measured by tri-enzyme extraction followed by microbiological assay. Our analyses suggest that there may be considerable inaccuracies on the part of the manufacturers in relation to the folate declarations on the product labels. Despite this, the four foods evaluated were highly effective in elevating plasma, liver and kidney folate and lowering plasma homocysteine concentrations in rats. These results lend support to the policy of food fortification with folic acid as a means of raising the folate status of the population, and in particular to the fortification of specific foods which may target areas of the population where increased folate status is most needed.

Hyperinsulinemia, hyperglycemia, and impaired hemostasis: the Framingham Offspring Study.

CONTEXT: Increased risk for cardiovascular disease in persons with glucose intolerance (impaired glucose tolerance and type 2 diabetes mellitus) is not fully explained by concomitant elevations in traditional atherosclerosis risk factors. Hyperinsulinemia associated with glucose intolerance may increase risk directly, or its effect could be mediated through impaired hemostatic function. OBJECTIVE: To evaluate associations between fasting insulin levels and hemostatic factors in subjects with normal and impaired glucose homeostasis. DESIGN: Cross-sectional analysis conducted between January 1991 and June 1995. SETTING: The population-based Framingham Offspring Study. SUBJECTS: A total of 1331 men and 1631 women aged 26 to 82 years, without diagnosed diabetes or cardiovascular disease and classified as having normal glucose tolerance (80.2%) or glucose intolerance (impaired glucose tolerance and impaired fasting glucose combined, 15.2%; previously undiagnosed diabetes, 4.7%) using an oral glucose tolerance test. MAIN OUTCOME MEASURES: Trends across quintiles of fasting insulin in levels of plasminogen activator inhibitor 1 (PAI-1) antigen, tissue-type plasminogen activator (tPA) antigen, von Willebrand factor (vWF) antigen, factor VII antigen, fibrinogen, and plasma viscosity. We stratified analyses by sex and glucose tolerance status and adjusted hemostatic factor levels for obesity, lipid levels, and traditional cardiovascular disease risk factors. RESULTS: Mean levels of all hemostatic factors (except for fibrinogen in men) increased across fasting insulin quintiles among subjects with normal glucose tolerance (P<.001 for trend). Levels of PAI-1 and tPA antigens, but not other hemostatic factors, were higher comparing subjects with glucose intolerance with those with normal glucose tolerance (P<.001). Among subjects with glucose intolerance, levels of PAI-1 and tPA antigen in men and women (P<.01 for trend) and vWF antigen in men (P<.05 for trend) increased significantly across insulin quintiles, but levels of factor VII antigen, fibrinogen, and plasma viscosity did not increase. CONCLUSIONS: Elevated levels of fasting insulin are associated with impaired fibrinolysis and hypercoagulability in subjects with normal glucose tolerance. Hyperinsulinemia is associated primarily with impaired fibrinolysis in subjects with glucose intolerance. Excess risk for cardiovascular disease associated with hyperinsulinemia and glucose intolerance may be mediated in part by enhanced potential for acute thrombosis.