Quantification of mucosa-adhered microbiota of lambs and calves by the use of culture methods and fluorescent in situ hybridization coupled with flow cytometry techniques

The intestinal mucosa-associated microbiota could play important biological roles due to its close proximity with the animal host, but knowledge on its composition is still limited. The aim of this study was to characterize the microbial communities tightly associated with different parts (rumen, duodenum and colon) of the gastrointestinal tract (GIT) of healthy lambs and calves by using both cultural, and fluorescent in situ hybridization-flow cytometry (FCM-FISH) techniques. Lactic acid bacteria genera were one of the predominant bacteria detected in lambs and calves by both methodologies, possibly constituting an index of their healthy status. The levels of Lactobacillus were significantly higher (p<0.05) in the rumen and duodenum of lambs, and in the rumen of calves. The levels of Bifidobacterium were significantly higher (p<0.05) in the colon of both animal species and the rumen of lambs. Significant differences (p<0.05) were found in counts of other microbial groups (yeast, Enterococcus, Propionibacterium, Bacteroides, Clostridium and Enterobacteriaceae) at diverse GI sections depending on the animal species. In general, microbial counts follow the same trends regardless the applied technique. The most remarkable differences were found in detection levels of Bacteroides and Clostridium, which tended to be significantly higher (p<0.05) when analysed by FCM-FISH. This technique also allowed the detection of quantitatively important bacteria (sulphate-reducing bacteria, Atopobium and Coriobacterium), which are difficult to cultivate in selective medium. Therefore, FCM-FISH has been proven to be a sensitive high throughput approach that provides additional information to that obtained by traditional culture techniques about the complexity of the GI ecosystem of these animal species.     

Determination of in vitro digestibility of forage species used in ruminant feeding

Within the evaluation of the quality of forage resources, the main parameter that defines it is the digestibility of dry matter, which together with the amount of neutral and acidic detergent fibers and crude protein constitutes the basic information to assess forages which are supplied in the diet of the cattle. This research was carried out at the University of Los Llanos (Villavicencio, Colombia), and its objective was to determine the digestibility of three forages in cattle through three different in vitro techniques: inoculation with ruminal fluid and with feces and enzymatic digestibility technique, making the comparison with the in situ technique in order to validate the techniques and equipment that are being used for these procedures. The following species were evaluated: Pennisetum purpureum (PP), Tithonia diversifolia (TD), and Bauhinia variegata (BV), assessing the curve and rate of degradation of dry matter (DM), neutral detergent fiber (NDF), and total protein (TP) (0 to 72 h). A design of repeated measures was used, under which the analysis of variance was carried out to determine the ranges of deviation between the techniques and thus establish the trend of the data; the variables evaluated were the DM, NDF, and TP digestibilities of the three forages using the four techniques (three in vitro and one in situ). After verifying the differences between the variances of the digestibilities and checking the sphericity assumption with the Mauchly test, multiple comparisons were made with the Bonferroni test with a significance of 5%. The digestibility of DM, NDF, and TP varied between 38.62 and 44.22, 54.18 and 66.97, and 47.54 and 57.05%; 49.07 and 70.70, 72.52 and 75.44, and 62.61 and 74.02%; 29.93 and 34.84, 26.21 and 70.88, and 25.67 and 50.60% respectively in forages PP, TD, and BV, depending on the technique used for their estimation. Despite finding statistically significant differences between several of the comparisons made in the digestibility techniques, a high coefficient of determination and a high correlation between the in vitro estimations with respect to the in situ estimation were found; therefore, it is possible to use these techniques routinely thus avoiding the need to have cattle with fistulae to perform digestibility tests, with enzymatic digestibility technique being the most practical one.

     

Pharmacokinetics of ciprofloxacin in sheep after single intravenous or intramuscular administration

Summary

The disposition and urinary excretion of ciprofloxacin (CIP) following intravenous (IV) or intramuscular (IM) administration of 7.5 mg/kg body weight in sheep (n = 5) was studied. The intravenous plasma concentration curve was best described pharmacokinetically by a two‐compartment open model, while the intramuscular administration data fitted better to a one‐compartment open model. Mean elimination half‐lives after IV and IM administration were 72 and 184 minutes, respectively. The absorption of intramuscularly administered CIP in sheep was fast: maximal plasma concentration (Cmax) was reached quickly (tmax 31.93 min) and attained values of 0.69 ± 0.27 mg/l. The bioavailability was 49%. The urinary data showed a significant decrease in the elimination rate constant of CIP when CIP was administered intramuscularly. The other parameters calculated did not display differences between the two routes of administration. The results obtained suggest that when CIP was administered by the IM route in the assayed dose, it was able to maintain serum concentrations above the MIC of most common pathogens over an 8‐hour period.     

Soybean cultivar performance in the presence of soybean Asian rust,in relation to chemical control programs

Soybean rust is caused by an obligate parasite (Phakopsora pachyrhizi) which has spread in Brazil in each new season since 2001 and, despite the efforts to control the disease, losses have occurred every year. Its control demands several tactics amongst which chemical control with fungicides is the main method and remains indispensable. Control strategies such as the use of cultivars with partial resistance are desirable, but are not yet commercially available. The present study analyzed the existing differences in the reactions of short, medium and long cycle soybean cultivars against Asian rust and their responses to fungicide sprays. The experiment was conducted at Uberlandia-MG, Brazil, under field conditions from December 2007 to May 2008, in the Syngenta Seeds Experimental Station. The high pressure of the disease in the experiment simulated the natural pressure that the disease often reaches in Brazil. The studied variables were: visual severity (percentage of infected leaf area), percentage defoliation and productivity (kg ha⁻¹). Disease severity was expressed as AUDPC (area under disease progress curve). Variance analysis and comparison of means by the Tukey test (5% significance) were done for all variables studied. Significant differences were observed between cultivar effects and chemical control programs. The results obtained here indicate that the cultivars M-Soy 8199RR and Emgopa 315RR were less susceptible to disease, and that a control program termed “monitoring” (in which the appearance of new pustules of the pathogen were monitored to make the decision at each fungicide spray) was the most effective.     

Effect of breed and gender on bovine liver cytochrome P450 3A (CYP3A) expression and inter-species comparison with other domestic ruminants

The cytochrome P450 (P450) superfamily represents a group of relevant enzymes in the field of drug metabolism and several exogenous or constitutional factors contribute to regulate its expression. Cattle represent an important source of animal-derived food-products and studies concerning the P450 expression are needed for the extrapolation of pharmacotoxicological data from one species to another and for the evaluation of the consumer's risk associated with the consumption of harmful residues found in foodstuffs. In the present study, possible breed-, gender- and species-differences in P4503A (the P450 subfamily more expressed in the human liver) expression were studied in vitro in Piedmontese (PDM) and Limousin (LIM) meat cattle breeds of both sexes and in domestic Ruminants (cattle, sheep and goats). Cytochrome P450 and P4503A contents as well as CYP3A-dependent drug metabolising enzymes (DME) were measured in liver microsomes. Significant lower levels of P450 (P < 0.001) and P4503A (P < 0.05) contents were observed in PDM vs. LIM of both sexes; the P4503A-dependent DME activities were significantly (P values ranging from 0.05 up to 0.001) higher in PDM cattle, particularly in males. A gender-effect in DME activities was noticed (P < 0.05) only in PDM male cattle. With regards to the species, the expression of both P4503A apoprotein and some of the related DME activities were more pronounced in sheep (P < 0.01 vs. cattle) and in goats (P < 0.05 vs. sheep; P < 0.01 vs. cattle) than in cattle. The significant differences in P4503A expression observed in LIM and PDM cattle are consistent with previously published data on strain- and breed-differences pointed out in rats and men. As far as a possible sex-effect is concerned, no clear-cut evidence is likely to be drawn. Finally, P4503A expression was more relevant in small ruminants.     

Use of touch-down polymerase chain reaction to enhance the sensitivity of Mycobacterium bovis detection.

The confirmatory diagnosis of Mycobacterium bovis (M. bovis) in animal samples is carried out by culture in Stonebrink media. However, culture is very slow because of the extremely long duplication time of the bacillus and difficult because of the scarcity of bacilli in diagnostic samples. This study describes the development of a single-tube touch-down polymerase chain reaction (PCR) protocol for the detection of M. bovis using primers that target the IS6110 element. Spiked water and milk as well as routine diagnostic samples (milk and nasal swabs) from M. bovis-positive cattle were tested. This protocol allows the rapid and sensitive detection of M. bovis in bovine samples by enhancing the sensitivity of standard PCR amplification.     

Evaluation of the API 50CH and API ZYM systems for rapid characterization of Clavibacter michiganensis subsp. sepedonicus, causal agent of potato ring rot

API 50CH and API ZYM systems were used to characterize fifty-three strains of Clavibacter michiganensis subsp. sepedonicus from different geographic locations and several reference strains of the same and different species, including other potato pathogens. Clavibacter michiganensis subsp. sepedonicus strains displayed a high level of homogeneity, both in carbohydrate utilization and in enzymatic activity. Using API 50CH and API ZYM it was possible to differentiate C. michiganensis subsp. sepedonicus strains from the remaining taxa analysed in this study, which included representative strains of the other subspecies of C. michiganensis as well as other bacterial pathogens affecting potatoes. Therefore, these systems could be used as an effective method to characterize C. michiganensis subsp. sepedonicus. Such a procedure would constitute an alternative system to the conventional nutritional and physiological identification tests currently included in the official methods employed in the European Union to detect and identify this bacterium. The results obtained with the API systems agreed with the current taxonomic classification of C. michiganensis, clearly separating sepedonicus from the other subspecies belonging to this species.