Polymerase chain reaction banding patterns of the 5S rDNA gene as a diagnostic tool for the discrimination of South American mullets of the genus Mugil

The mullets of the family Mugilidae, and in particular the species of the genus Mugil, are characterized by highly conserved morphological features, which hinder taxonomic classification considerably. Given this, the present study aimed to evaluate the efficiency of the 5S rDNA gene – a rapid and low cost genotyping marker – for the discrimination of Mugil species. Variations in the banding pattern permitted the reliable differentiation of four species –Mugil curema, Mugil incilis, Mugil sp. and Mugil hospes– although those of three others (Mugil liza, Mugil platanus and Mugil cephalus) were identical. This suggests that these three forms have diverged very recently, on a time scale that has limited changes in the length of the fragments of the 5S rDNA gene. The results of the study confirm the efficiency of this marker as a diagnostic tool for the identification of Mugil species, and its usefulness for population management and pisciculture, an important economic activity involving this group.     

Effect of diet on composition of cecal contents and on excretion and composition of soft and hard feces of rabbits

A total of 260 New Zealand White growing rabbits were used to study the effect of diet on chemical composition of cecal contents and on production and composition of soft and hard feces. Eight diets varying in their acid detergent fiber (9.8% to 32.7%) and starch (13% to 30%) levels were evaluated. The diet affected (P less than .01) all the variables studied, except dry matter (DM) and molar proportions of volatile fatty acids on cecal contents. An increase of dietary crude fiber increased crude fiber level in cecal contents (from 11.58% to 26.53%). However, a relatively lower proportion of fibrous material was found in the cecal contents when rabbits were fed the more fibrous diets. This suggests that dietary fiber has a direct influence on the efficiency of particle separation in the digestive tract. Crude protein and volatile fatty acid concentrations of cecal contents decreased (from 30.14% to 19.65% and from 47.8 to 36.7 mmol/liter, respectively) when dietary crude fiber increased. This could be related to availability of energy to cecal microorganisms. Ammonia concentration of cecal contents was not affected by dietary crude fiber. Daily production of soft feces varied from 14.98 to 29.59 g DM/d, and the contribution of soft feces to total DM and to crude protein intake ranged from 10.6% to 15.0% and from 12.8% to 20.5%, respectively; these values were the smallest and the largest for the least and the most fibrous diets, respectively. From this study we conclude that dietary fiber has a major effect on the digestive processes in the rabbit and that dietary starch level has no influence on any of the variables studied.     

Effect of type of fiber on the rate of passage and on the contribution of soft feces to nutrient intake of finishing rabbits.

One hundred thirty New Zealand White x California rabbits were used to study the influence of the source of dietary fiber on several digestive criteria. Five diets were formulated to provide 10% crude fiber (CF) on a DM basis. Sixty percent of this dietary fiber was supplied by alfalfa hay, citrus pulp, beet pulp, grape marc, or rice hulls in the diets. Weights of cecal contents, cecal ammonia concentration, and molar proportion of acetic acid in diets including pulps were higher and cecal levels of DM, CP, and molar proportion of butyric acid were lower than those of the alfalfa diet. Cecal ammonia, VFA, and CF concentrations of the grape marc diet were the lowest of all the diets studied; cecal CF level and molar proportion of acetic acid were significantly higher and CP level and molar proportion of butyric acid were lower in rice hull than in alfalfa diets. Diet had no influence on daily soft feces excretion (10 g DM/d), but N contribution of soft feces to N intake was higher in the diet with citrus pulp (18.7%) than in the other diets (12%). Mean retention time (R) was measured using fuchsin-stained feed and was found to be higher (21.3 h) in the rice hull diet and lower (9.3 h) in the grape marc diet than in the other diets. When coprophagy was prevented, R decreased by 0 to 7 h. In conclusion, the use of byproducts to substitute for traditional sources of fiber in rabbit diets influenced the retention time of the digesta in some segments of the gut and thus altered several digestive criteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Qualitative and quantitative polymerase chain reaction (PCR) for detection of Leishmania in spleen samples from naturally infected dogs

Because infected dogs are widely considered to be the main domestic reservoir for Leishmania infantum (syn Leishmania chagasi) parasites in Brazil, the diagnosis of canine visceral leishmaniasis (CVL) must be made both accurately and promptly. The present study attempted to standardize a conventional polymerase chain reaction (cPCR) protocol for the detection of L. infantum DNA in canine spleen samples. Quantitative PCR (qPCR) technique was used to confirm the presence of Leishmania DNA in the canine spleen fragments. A comparison was made between the efficacies of these molecular diagnostic techniques and conventional parasitological and serological methods. cPCR protocols for spleen samples were standardized using primers that amplify a 145 bp fragment, located at the parasite kinetoplast minicircle. The genus specificity of the cPCR protocol was assessed by its inability to amplify the DNA of other common canine pathogens, such as Ehrlichia canis, Babesia canis, Toxoplasma gondii and Trypanosoma cruzi. cPCR protocol sensitivity was tested by assessing the reaction detection limit, determined to be 10 fg of L. infantum reference strain DNA, which corresponds to a range of 0.03-0.1 parasites per fragment. Standardized cPCR protocol was used to detect the presence of Leishmania in 45 dog spleen samples. Our results showed that 40% of the spleen fragment cultures were positive for Leishmania parasites, 58% of the dog serum samples tested positive using ELISA, and parasite DNA was detected in 44% using qPCR, while 47% of the spleen samples using cPCR. Diagnostic methods performance was assessed and revealed a better degree of ascertainment for cPCR when compared to other diagnostic methods. The sensitivity of ELISA was 83.3%, qPCR was 83.3%, and cPCR was 88.9%; PPV for ELISA was 57.7%, qPCR was 75% and cPCR was 76.2%; the Kappa coefficients were found to be 0.40 (fair) for ELISA, 0.64 (substantial) for qPCR and 0.68 (substantial) for cPCR. In both oligosymptomatic and polysymptomatic dogs, cPCR revealed the better performance analysis when compared to other diagnostic methods. The findings presented herein establish cPCR as the most indicated test to detect Leishmania when compared to the other two diagnostic methods evaluated. Despite the fact that the qPCR protocol provides a highly accurate quantification of parasites when targeting the SSU rRNA gene, this technique does not significantly improve the diagnosis of CVL when compared with the performance of the cPCR protocol, which focused on the kinetoplast minicircle.     

Development of molecular assays for the identification of the 11 Eimeria species of the domestic rabbit (Oryctolagus cuniculus)

Coccidiosis are the major parasitic diseases in poultry and other domestic animals including the domestic rabbit (Oryctolagus cuniculus). Eleven distinct Eimeria species have been identified in this host, but no PCR-based method has been developed so far for unequivocal species differentiation. In this work, we describe the development of molecular diagnostic assays that allow for the detection and discrimination of the 11 Eimeria species that infect rabbits. We determined the nucleotide sequences of the ITS1 ribosomal DNAs and designed species-specific primers for each species. We performed specificity tests of the assays using heterologous sets of primers and DNA samples, and no cross-specific bands were observed. We obtained a detection limit varying from 500fg to 1pg, which corresponds approximately to 0.8-1.7 sporulated oocysts, respectively. The test reported here showed good reproducibility and presented a consistent sensitivity with three different brands of amplification enzymes. These novel diagnostic assays will permit population surveys to be performed with high sensitivity and specificity, thus contributing to a better understanding of the epidemiology of this important group of coccidian parasites.     

Differentiation of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> strains from Bulgaria by PCR-RFLP analysis

Fifty strains of Erwinia amylovora isolated in Bulgaria from different host plants and locations as well as in different years were analysed by RFLP analysis of the pEA29 PstI amplified fragment with HpaII. All the strains formed three well-resolved fragments (large—from 365 to 440 bp, medium—about 341 bp and small—about 180 bp).The strains were classified into three RFLP groups based on the polymorphism in the length of the largest fragment. This fragment was of intermediate size for 63% of the strains, and it was the longest (from 410 to 440 bp) for 29% of the strains. The variable region was sequenced for five strains. The DNA sequence analysis confirmed the different size of the largest fragment. Ten or more than ten SSRs were found for the strains in the group with the largest size of the largest fragment. Some correlation between the RFLP profiles and the origin of the strains was revealed. The RFLP profiles displayed stability in certain strains isolated from the same trees and orchards, but in different years. The number of SSRs was different in strains isolated from one and the same host plant, orchard and year, and also in strains isolated from the same host plant and orchard, but in different years. This could indicate that under natural conditions the fire blight symptoms might be caused by a mixture of E. amylovora strains with different SSR numbers, and so coexistence of distinguishable strains or a change in the population could be assumed.     

Aluminium Toxicity Risk for Pinus pinaster in Acid Soils (Galicia,NW Spain)

The influence of bedrock on aluminium toxicity and aluminium speciation in the soil solution was studied in four Pinus pinaster plots. Growth and biomass parameters in the acidic soils were also evaluated in relation to different Al toxicity indices. The plots were developed over slate, biotitic schist, mica schist and granite. Samples of rhizospheric and non‐rhizospheric soil, 1‐year‐old needles and roots were collected in each study plot. Total Al, reactive Al, acid‐soluble Al, non‐labile and labile Al and Al species (Al³⁺, Al‐OH, Al‐F and Al‐SO₄) were determined in soil solution. Reactive Al dominated over the acid‐soluble Al, and the non‐labile Al predominated over the labile Al in all soils, but particularly over mica schist. In the biotitic schist soil, the Al forms and total Al were lower, whereas concentrations were always higher over mica schist. The Al forms considered most toxic were Al³⁺ and Al‐OH, and Al concentrations were highest over slate and mica schist. Al toxicity indices in soil, needle and roots showed a risk of toxicity in mica schist, slate and granite. The stand site indices over slate and mica schist were lower, consistent with the high labile Al and Al³⁺ + Al‐OH in soil solution. Despite the high stand site index over granite, the growth efficiency was low, in accordance with very low ratios of Ca/Al in needles or fine roots. This confirmed the adaptation of maritime pine to granitic substrates. Copyright © 2016 John Wiley & Sons, Ltd.     

Buttressed trees of Brosimum alicatrum Sw. affect mycelial mat abundance and indirectly the composition of soil meso-fauna

Mycelial mats in a tropical rainforest in southern Mexico were more abundant under large buttressed trees than small trees of Brosimum alicastrum. P and K, though not N, concentrations were lower in fine roots from mats than in control samples and complex changes in functional groups of arthropods associated with mats were also evident.     

Vaginal lactic acid bacteria in healthy and ill bitches and evaluation of in vitro probiotic activity of selected isolates

The concentrations of lactic acid bacteria (LAB) in 42 vaginal samples from healthy and ill bitches were determined. Eight isolates belonging to the genera Lactobacillus and Enterococcus were selected and identified and their in vitro antimicrobial activity against canine pathogens and their ability to adhere to canine vaginal epithelial cells were determined. There was no correlation between the concentrations of vaginal LAB and clinical status, body temperature, vaginal pH, or age. Although the animals were either well or suffering from various illnesses, LAB were found in almost every sample, and the selected isolates showed promising probiotic-related features. These findings are significant for the design of strategies for the modulation of vaginal microbiota by vaginal LAB isolates.