Methylation methods for the quantitative analysis of conjugated linoleic acid (CLA) isomers in various lipid samples

Precise methylation methods for various chemical forms of conjugated linoleic acid (CLA), which minimize the formation of t,t isomers and allylmethoxy derivatives (AMD) with the completion of methylation, were developed using a 50 mg lipid sample, 3 mL of 1.0 N H(2)SO(4)/methanol, and/or 3 mL of 20% tetramethylguanidine (TMG)/methanol solution(s). Free CLA (FCLA) was methylated with 1.0 N H(2)SO(4)/methanol (55 degrees C, 5 min). CLA esterified in safflower oil (CLA-SO) was methylated with 20% TMG/methanol (100 degrees C, 5 min), whereas CLA esterified in phospholipid (CLA-PL) was methylated with 20% TMG/methanol (100 degrees C, 10 min), followed by an additional reaction with 1.0 N H(2)SO(4)/methanol (55 degrees C, 5 min). Similarly, CLA esterified in egg yolk lipid (CLA-EYL) was methylated by base hydrolysis, followed by reaction with 1.0 N H(2)SO(4)/methanol (55 degrees C, 5 min). These results suggest that for the quantitative analysis of CLA in lipid samples by GC, proper methylation methods should be chosen on the basis of the chemical forms of CLA in samples.     

Effects of illite supplementation on in vitro and in vivo rumen fermentation,microbial population and methane emission of Hanwoo steers fed high concentrate diets

This study was conducted to evaluate the effects of feeding supplemental illite to Hanwoo steers on methane (CH₄) emission and rumen fermentation parameters. An in vitro ruminal fermentation technique was conducted using a commercial concentrate as substrate and illite was added at different concentrations as treatments: 0%, 0.5%, 1.0%, and 2.0% illite. Total volatile fatty acids (VFA) were different (P < 0.05) at 24 h of incubation where the highest total VFA was observed at 1.0% of illite. Conversely, lowest CH₄ production (P < 0.01) was found at 1.0% of illite. In the in vivo experiment, two diets were provided, without illite and with addition of 1% illite. An automated head chamber (GreenFeed) system was used to measure enteric CH₄ production. Cattle received illite supplemented feed increased (P < 0.05) total VFA concentrations in the rumen compared with those fed control. Feeding illite numerically decreased CH₄ production (g/day) and yield (g/kg dry matter intake). Rumen microbial population analysis indicated that the population of total bacteria, protozoa and methanogens were lower (P < 0.05) for illite compared with the control. Accordingly, overall results suggested that feeding a diet supplemented with 1% illite can have positive effects on feed fermentation in the rumen and enteric CH₄ mitigation in beef cattle.     

Production of conjugated linoleic acid (CLA) by Bifidobacterium breve LMC520 and its compatibility with CLA-producing rumen bacteria

This study was performed to characterize the ability of an active Bifidobacterium strain to produce conjugated linoleic acid (CLA) and to test its possible utilization as a probiotic compatible to the ruminal condition. Bifidobacterium breve LMC520 can actively convert linoleic acid (LA) to cis-9,trans-11-CLA, which is a major isomer derived from microbial conversion. LMC520 showed reasonable tolerance under acidic conditions (pH 2.5 with 1% pepsin) and in the presence of oxgall (0-3%). The growth and CLA production of LMC520 were tested under ruminal conditions and compared with those of Butyrivibrio fibrisolvens A38, which is a major CLA producer in the rumen as an intermediate in the biohydrogenation (BH) process. LMC520 converted 15% of LA to CLA under ruminal conditions, which was 2 times higher activity than that of A38, and there was no decline in CLA level during prolonged incubation of 48 h. The BH activity of LMC520 was comparable to that of A38. When LMC520 was cocultured with A38, even with slight decrease of CLA due to high BH activity by A38, but the level of CLA was maintained by the high CLA-producing activity of LMC520. This comparative study shows the potential of this strain to be applied as a functional probiotic not only for humans but also for ruminants as well as to increase CLA production.     

Production of silkworms with conjugated linoleic acid (CLA) incorporated into their lipids by dietary CLA

Silkworms with conjugated linoleic acid (CLA) incorporated into their lipids (designated CLA silkworms) were produced to enhance the quality of silkworms having a synergistic effect with CLA functions by dietary synthetic CLA. Silkworm larvae were fed fresh mulberry leaves (control diet) until the third instar stage and were then subjected to various levels (0%, 0.1%, 1%, 5%, and 10%) of CLA-sprayed mulberry leaves (designated CLA diet) beginning on the first day of the fourth instar stage and continuing to the third day of the fifth instar stage. CLA contents in CLA silkworms increased proportionally with increasing CLA levels of CLA diets. CLA silkworms on a 1% CLA diet contained 2.2 g CLA/100 g lipid without body weight reduction, whereas CLA silkworms on a 10% CLA diet contained 14.8 g CLA/100 g lipid with a significant reduction of body weight, relative to the control silkworms. The CLA content in the lipids of CLA silkworms on a 10% CLA diet was significantly higher than that of CLA silkworms on a 5% CLA diet. A 0.1% CLA diet was not sufficient to accumulate CLA in the silkworms. Most of the CLA (approximately 99%) of silkworm lipids was present in triglyceride (TG) with a similar ratio of c9,t11 and t10,c12 CLA isomers. These results suggest that a 1% CLA diet was suitable for the production of CLA silkworms.     

Inclusion complex of conjugated linoleic acid (CLA) with cyclodextrins

Conjugated linoleic acid (CLA) inclusion complexes with alpha-cyclodextrin (alpha-CD), beta-cyclodextrin (beta-CD), and gamma-cyclodextrin (gamma-CD) (designated CLA/CDs inclusion complexes) were prepared to determine the mole ratio of CLA complexed with CDs and the oxidative stability of CLA in the CLA/CDs inclusion complexes. When measured by GC, (1)H NMR, and T(1) value analyses, 1 mole of CLA was complexed with 5 mol of alpha-CD, 4 mol of beta-CD, and 2 mol of gamma-CD. The oxidation of CLA induced at 35 degrees C for 80 h was completely prevented by the formation of CLA/CDs inclusion complexes.     

Preparation of a t,t conjugated linoleic acid methylester (CLA-Me) isomers mixture from synthetic CLA by methylation with BF3/methanol

Mixtures of t,t conjugated linoleic acid methylester (t,t CLA-Me) isomers were prepared from synthetic CLA, consisting of 47.8% t10,c12 CLA; 45.5% c9,t11 CLA; 2.0% t,t CLA; and 4.7% others, by methylation with BF(3)/methanol (designated TT-TC/CT) in conjunction with purification at -68 degrees C for 24 h. The amount or composition of the TT-TC/CT was greatly affected by the concentration of BF(3) in methanol and the duration of methylation. The methylation of 50 mg of synthetic CLA for 30 min with 1 mL of 7.0% BF(3)/methanol produced a TT-TC/CT (21.54 mg) with the composition of 1.3% t12,t14; 5.9% t11,t13; 42.7% t10,t12; 44.0% t9,t11; 5.0% t8,t10; and 1.1% t 7,t9 CLA, whereas the methylation for 60 min with 14.0% BF(3)/methanol produced a TT-TC/CT (28.62 mg) with the composition of t,t CLA isomers different from that of TT-TC/CT by methylation for 30 min with 7.0% BF(3)/methanol. A large quantity of TT-TC/CT (14.15 g) with the composition similar to that of TT-TC/CT prepared from 50 mg of synthetic CLA was also prepared from 25 g of synthetic CLA. The purity of TT-TC/CT samples was greater than 98%. These results suggest that TT-TC/CT with a purity greater than 98% was easily prepared from synthetic CLA by BF(3)-catalyzed methylation, and the amount and composition of t,t CLA isomers of TT-TC/CT samples could be controlled by methylation conditions.     

Analysis of microbiological and chemical quality of poultry meat in the vicinity of the Mbeubeuss landfill in Malika (Senegal)

A total of 100 samples of poultry meat were collected in poultry farms in the vicinity of the Mbeubeuss landfill in the Niayes (Senegal) for microbiological and chemical analysis. Fifty-four (54) samples were collected in farms located less than 1 km from the landfill and 46 samples were collected in farms located a bit further (more than 1 km from the landfill). Microbiological quality was determined using techniques recommended by Association Française de Normalisation (AFNOR). Lead and cadmium concentration in poultry meat was measured by flame spectrometry while total mercury was determined by atomic absorption spectrometry. Three percent (3%) of the samples’ quality were unsatisfactory for E. coli, 1% for Staphylococci and 7% for Salmonella spp. Poor meat quality was found either in farms located less than 1 km of the landfill or in farms located at more than 1 km of the landfill. Except for Salmonella, only meat samples from poultry receiving drinking water from well showed unsatisfactory microbiological quality. The samples were free of cadmium and lead but were contaminated by mercury. Sixty-eight percent (68%) of the samples contained mercury with a high contamination level (>0.011 mg/kg) in 20% of the samples. No significant difference was found between the farms that were nearest to and further away from the landfill while the source of drinking water seemed to be the main cause of contamination of poultry meat by mercury.     

Survey of Anaplasmataceae bacteria in sheep from Senegal

Purpose

The authors studied the role of bacteria belonging to Anaplasmataceae family as the causes of acute illnesses of sheep in West Africa.

Methods

We examined and sampled 120 febrile sheep in two regions of Senegal for this study. The DNA extracted from these blood samples was tested by PCR using two pairs of primers (groEL-based and 16S rRNA gene-based).

Results

In 52/120 samples, the microscopic examination revealed intraerythrocytic and/or intraphagocytic spherical inclusions. In 48/52 cases, we succeeded in identifying the bacterial agent: in 38 cases, it was Anaplasma ovis; in six cases, it was Ehrlichia ruminantium; in two cases, Anaplasma phagocytophilum; in one case, Anaplasma platys; and in one case, a yet uncultured Anaplasma sp. closely related to A. phagocytophilum.

Conclusions

Our studies demonstrated the great variety of pathogenic bacteria from the Anaplasmataceae family in the blood of clinically ill sheep. A. ovis was identified unexpectedly often. For the first time, A. phagocytophilum was found in sub-Saharan Africa, and its further epidemiology may be now reconsidered. The roles of canine pathogen, A. platys, and yet undescribed Anaplasma sp. “Badiouré” in ovine pathology should be more closely studied.     

Impact of irrigation water quality on soil nitrifying and total bacterial communities

Disturbance induced by two contrasting irrigation regimes (groundwater versus urban wastewater) was evaluated on a sandy agricultural soil through chemical and microbial analyses. Contrary to wastewater, groundwater displayed very high nitrate contents but small amounts of ammonium and organic matter. Despite these strong compositional shifts, soil organic carbon and nitrogen, nitrate and ammonium contents were not significantly different in both types of irrigated plot. Moreover, neither microbial biomass nor its activity, determined as fluorescein diacetate hydrolysis activity, was influenced by irrigation regimes. Bacterial community structure, assessed by denaturing gradient gel electrophoresis (DGGE) of 16S ribosomal DNA fragments, was also weakly impacted as molecular fingerprints shared an overall similarity of 85%. Ammonia-oxidizing bacterial community (AOB) was monitored by DGGE of the functional molecular marker amoA gene (alpha subunit of the ammonia monooxygenase). Surprisingly, no amoA signals were obtained from plots irrigated with groundwater, whereas signal intensities were high in all plots under wastewater. Among the last, compositional shifts of the AOB community were weak. Overall, impact of irrigation water quality on soil chemistry could not be evidenced, whereas effects were low on the total bacterial compartment but marked on the AOB community.     

Performance of enzyme-linked immunosorbent assays for detection of antibodies against T. congolense and T. vivax in goats

Indirect ELISAs using denatured antigen preparations of Trypanosoma (T.) congolense (TcAGd) and T. vivax (TvAGd) for detection of anti-trypanosome antibodies in bovine serum (I-TAB ELISAs), were adapted for serodiagnosis in goats. The diagnostic proficiency, the cross-reactivity with sera from heterologous trypanosome infections and the operational performance of the assays were evaluated on experimentally trypanosome-infected goats. The I-TAB ELISA (TcAGd) detected antibodies in all T. congolense infected goats (100% overall sensitivity) from 2 to 4 weeks post-infection (p.i.) until the end of the experiments. Specificity tested on 92 uninfected goats was 96.7%. Extensive cross-reactions of I-TAB ELISA (TcAGd) with sera from T. vivax or T. brucei infected goats were observed. The I-TAB ELISA (TvAGd) detected antibodies in 5 of the 6 T. vivax infected goats, specificity tested on uninfected goats was 100%. Cross-reactivity with sera from T. congolense or T. brucei infected goats remained limited. Infecting species identification based on the highest percent positivity (PP) in both systems, correctly identified all T. congolense infections, but misidentified in 2/19 occasions a T. vivax infection as a T. congolense infection. In the absence of T. brucei specific antigen coated plates, T. brucei infections were identified in, respectively, 7/9 and 2/9 occasions as T. congolense or T. vivax infections. Acceptable inter-plate repeatability was observed. The implications of results and technical requirements for ongoing applied research are discussed.