Metagenomics in animal gastrointestinal ecosystem: a microbiological and biotechnological perspective.

Metagenomics- the application of the genomics technologies to nonculturable microbial communities, is coming of age. These approaches can be used for the screening and selection of nonculturable rumen microbiota for assessing their role in gastrointestinal (GI) nutrition, plant material fermentation and the health of the host. The technologies designed to access this wealth of genetic information through environmental nucleic acid extraction have provided a means of overcoming the limitations of culture-dependent microbial genetic exploitation. The molecular procedures and techniques will result in reliable insights into the GI microbial structure and activity of the livestock gut microbes in relation to functional interactions, temporal and spatial relationships among different microbial consortia and dietary ingredients. Future developments and applications of these methods promise to provide the first opportunity to link distribution and identity of rumen microbes in their natural habitats with their genetic potential and in situ activities.

Biotransformation of lantadenes, the pentacyclic triterpenoid hepatotoxins of lantana plant, in guinea pig.

Oral administration of lantana (Lantana camara var. aculeata) leaf powder to guinea pigs at a dose of 6 g/ kg body weight elicited cholestasis. The animals were euthanized 48 h after dosing. Liver homogenates, bile, gall bladder, blood, urine, contents of gastrointestinal tract (GIT) and faeces were analysed for the principal hepatotoxin in lantana leaves viz. lantadene A (LA), its congeners and biotransformation products, using high performance liquid chromatographic technique. Lantadenes could not be detected in liver, bile, gall bladder, blood and urine samples. LA and lantadene B (LB), their derivatives reduced lantadene A (RLA), reduced lantadene B (RLB) and two unidentified metabolites could be detected in the contents of lower GIT and faeces. In vitro incubation of lantana leaf powder with guinea pig caecal contents under anaerobic conditions elicited biotransformation of LA and LB to RLA and RLB, respectively. On the other hand, incubation of lantana leaf powder with cattle rumen liquor under anaerobic conditions did not elicit biotransformation of lantadenes.

A spectrophotometric method for assay of tannase using rhodanine.

A method for assay of microbial tannase (tannin acyl hydrolase) based on the formation of chromogen between gallic acid and rhodanine is reported. Unlike the previous protocols, this method is sensitive up to gallic acid concentration of 5 nmol and has a precision of 1.7% (relative standard deviation). The assay is complete in a short time, very convenient, and reproducible.

Epidemiology of parasitoses in dairy animals in the North West Humid Himalayan Region of India with particular reference to gastrointestinal nematodes.

A systematic survey was conducted during two spells of 5 years each (1986-1990 and 1993-1997) to study the prevalence of parasitoses with particular reference to gastrointestinal (GI) nematodes and flukes in dairy animals (crossbred and native cattle, and buffaloes) belonging to 12 villages in the Kangra valley (Himachal Pradesh). Flukes (Fasciola, amphistomes and Dicrocoelium) and strongyles were the most important parasitic infections. Fasciola was endemic throughout the year, with a higher percentage infection in buffaloes than in cattle. Other fluke and nematode infections showed a seasonal pattern in prevalence, with a small peak in March April followed by a high peak in July September. The faecal egg counts (eggs per gram, epg) of flukes (Fasciola/amphistomes) ranged from 50 to 300 in cattle and 50 to 400 in buffaloes, with high loads during the rainy and post-rainy seasons. The GI nematode egg counts (excluding Toxocara) revealed a similar trend, with the overall monthly mean epg ranging from 85 to 1720 in cattle and 90 to 1625 in buffaloes, with a high peak during the months of July to September. On coproculture of positive samples, the nematode infections in order of prevalence were: Strongyloides, Trichostrongylus, Haemonchus, Oesophagostomum, Bunostomum and Mecistocirrus. The prevalence of most of the parasites was lower during the second 5-year period.

Disposition of lantadene A, the pentacyclic triterpenoid hepatotoxin, orally administered to guinea pigs.

Lantadene A (LA) administered orally to guinea pigs elicited cholestasis. LA could not be detected in liver, bile, gall bladder, blood and urine. LA and its biotransformation product reduced lantadene A (RLA) could be detected in caecum, large intestine, and faeces. In vitro incubation of LA with liver homogenates under aerobic and anaerobic conditions did not elicit its biotransformation to RLA. On the other hand, in vitro incubation of LA with guinea pig caecal and large intestinal contents under anaerobic conditions elicited conversion of LA to RLA. This is the first report of the biotransformation of LA in the animal system.

Microbial degradation of tannins--a current perspective.

Tannins are water-soluble polyphenolic compounds having wide prevalence in plants. Hydrolysable and condensed tannins are the two major classes of tannins. These compounds have a range of effects on various organisms--from toxic effects on animals to growth inhibition of microorganisms. Some microbes are, however, resistant to tannins, and have developed various mechanisms and pathways for tannin degradation in their natural milieu. The microbial degradation of condensed tannins is, however, less than hydrolysable tannins in both aerobic and anaerobic environments. A number of microbes have also been isolated from the gastrointestinal tract of animals, which have the ability to break tannin-protein complexes and degrade tannins, especially hydrolysable tannins. Tannase, a key enzyme in the degradation of hydrolysable tannins, is present in a diverse group of microorganisms, including rumen bacteria. This enzyme is being increasingly used in a number of processes. Presently, there is a need for increased understanding of the biodegradation of condensed tannins, particularly in ruminants.

Preliminary studies on tannin degradation by Aspergillus niger van Tieghem MTCC 2425.

A tannin-degrading strain of Aspergillus niger van Tieghem was grown at pH 5.0 and 30 degrees C in a defined medium where tannins were the sole source of carbon and energy. The fungus had variable growth in tannic acid- and quebracho tannin-medium and could tolerate these tannins even up to 150 g-1 without showing any growth inhibition.

Isolation of a tannin-protein complex-degrading fungus from faeces of hill cattle.

Faecal samples of 52 hill cattle fed largely on oak leaves were screened for tannin-protein complex-degrading micro-organisms under different culture conditions using tannin-treated brain heart infusion agar medium. None of the animals were found to harbour tannin-protein complex-degrading enterobacteria. However, a fungus identified as Aspergillus niger van Tieghem having tannin-protein complex-degrading activity, was consistently isolated from the faeces of such animals. Optimum growth and sporulation was noticed under aerobic conditions at 30 degrees C on Czapek Yeast Agar medium.

Prevalence of dicrocoeliosis in sheep and goats in Himachal Pradesh, India.

A survey incorporating field and abattoir studies was carried out on the prevalence of Dicrocoelium dendriticum in sheep and goats in the Kangra valley of Himachal Pradesh (India). Coprological studies on these migratory animals revealed that 8.1% of sheep and 4.1% of goats were positive for dicrocoeliosis, with a mean number of eggs per gram of faeces (EPG) of 702.1 for sheep and 566.7 for goats. The necropsy liver examination of slaughtered animals revealed that 24.1% of sheep and 12.3% of goats were infected with D. dendriticum and they had mean fluke burdens of 247.2 (sheep) and 298.1 (goats). No significant differences in fluke burden, liver lesion score and EPG count were observed between sheep and goats. The seasonal distribution of dicrocoeliosis indicated a higher percentage of infection in autumn and winter as compared with spring and summer. The potential role of migratory sheep and goats in the epizootiology of dicrocoeliosis is discussed.

Anthelmintic activity of ivermectin against experimental Ascaridia galli infection in chickens.

The efficacy of ivermectin against Ascaridia galli infection was evaluated in chickens under controlled laboratory conditions in two separate experiments. In each experiment 22 White Leghorn chicks were randomly assigned to three groups of 10 (infected-treated), 9 (infected-control) and 3 (uninfected-control) birds. Each bird in the former two groups was orally infected with 1,500 embryonated A. galli eggs. The chicks in the treated group were subcutaneously injected with ivermectin at a dose of 0.3 mg kg-1 body weight on Day 10 (Experiment 1) and Day 35 post-infection (Experiment 2) for immature and adult infections, respectively. The treated birds had 0.9% (Experiment 1) and 0.4% (Experiment 2) worm recovery compared with 8.7 and 8% in the infected-untreated controls of the respective experiments. The fall in post-treatment faecal egg counts was 81 and 92% in birds treated on Days 10 and 35, respectively. The drug was found to be 90 and 95% effective against immature and adult worms, respectively. The lower lesion score and post-treatment near-normal haematobiochemical picture in treated birds confirmed these observations. The treated birds also had a better growth rate than the untreated chickens. The mature worms in the intestinal lumen of the host were more sensitive to the treatment than the immature stages of the parasite in the tissue phase.

Effect of bronchodilator and intravascular oxygen releaser on the course of Dictyocaulus filaria infection in lambs.

The effect of a bronchodilator (or ciprenaline sulphate) and intravascular oxygen releaser (sodium dihydrogen orthophosphate) on the host in experimental Dictyocaulus filaria infection was studied. Fifteen male lambs of Dorset-Muzaffarnagri breed, aged 4-6 months, were divided into four groups of four (infected bronchodilator), four (infected i.v. O2 releaser), four (infected untreated controls) and three (uninfected controls). The administration of i.v. O2 releaser helped in increasing the length of useful patency, estimated total larval production and survival rate of D. filaria producer lambs. The administration of I.V. O2 releaser and bronchodilator helped in efficiently restoring the altered values of blood pH, haemoglobin, packed cell volume, erythrocyte sedimentation rate and osmotic fragility of erythrocytes to near normal levels. However, the blood clotting time and level of lactate dehydrogenase activity remained altered and followed a course typical of ovine dictyocauliosis.

Osmotic fragility of sheep erythrocytes and alterations in their membrane constituents in Dictyocaulus filaria infection.

Alterations in the sheep erythrocyte membrane constituents during the course of Dictyocaulus filaria infection were studied in 4-6 month old Nali lambs. During the acute course of infection, plasma cholesterol, membrane cholesterol, cholesterol: phospholipid ratio and acetylcholinesterase activity fell significantly when compared with uninfected controls. The onset of the fall in the values of these parameters was observed 1-2 weeks prior to an increase in the osmotic fragility of erythrocytes. The altered values persistently remained at sub-normal levels during the chronic stage of the infection. However, the membrane proteins and phospholipids of sheep erythrocytes remained unaffected during the entire period of study. The clinico-parasitological picture of the disease, as judged by the clinical course of disease, faecal larval output and necropsy worm recovery, was typical of ovine dictyocauliosis.

Experimental studies on anaemia in sheep infected with Dictyocaulus filaria.

The pathogenesis of Dictyocaulus filaria induced anaemia was experimentally investigated. Nineteen Dorset-Muzaffarnagri male lambs were divided into two groups of 13 and six. The lambs in the former group received a primary infection dose of 2000 D. filaria infective larvae, whereas animals in the latter group were kept as uninfected controls. The haematological alterations and faecal larval output were monitored weekly until day 220 post-infection. The infection caused an increase in erythrocyte sedimentation rate (ESR) and total leukocyte count (TLC). However, a decrease in packed cell volume (PCV), haemoglobin, total erythrocyte count (TEC) and blood pH was observed in acute infection. The shape and size of the erythrocytes, serum bilirubin level, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) remained unaffected In carriers, except for TEC, the values of these parameters returned to near-normal levels. The analysis of the results suggests that the infected animals developed a normocytic normochromic anaemia which persisted during the later stage of infection.

Effect of Dictyocaulus filaria infection on the osmotic fragility of sheep erythrocytes.

The osmotic fragility of erythrocytes in lambs experimentally infected with Dictyocaulus filaria was studied weekly for 71 weeks. In acute infection, the erythrocytic fragility increased from the third week of infection onwards, reached its peak by the eleventh week and declined thereafter. However, in the chronic immune-carrier stage, this increase in the fragility did not return to normal until the end of the experiment. This enhanced fragility showed a positive correlation with the faecal larval count, worm burden and the extent of lung damage in lambs.

Control of sheep lungworm in India.

Lungworm infections (parasitic bronchitis) of sheep and goats are widely prevalent in hilly regions of India and neighbouring countries. Several species of strongyloid nematodes are involved but the most prevalent, and most pathogenic, is Dictyocaulus filaria - responsible for heavy mortality in young animals and severe morbidity in survivors. Control of these parasites now relies on a gamma-attenuated D. filaria vaccine developed in India in 1971. Vaccination of 6-week-old lambs has since become an accepted part of the regular sheep husbandry practice in India, and trials are now underway to extend use of the vaccine to goats, and to set up similar vaccination procedures in other countries. In this review, the authors trace the development of the D. filaria vaccine, highlighting its production and application. Although imperfect, the vaccine is now making a substantial contribution to improved sheep rearing by pastoral in the northern hill areas of India.

Preliminary observations on the effect of Dictyocaulus filaria on the blood clotting time of sheep.

The whole blood clotting time progressively increased from the second to the eighth week in lambs receiving a primary infection of Dictyocaulus filaria larvae. However, in vaccinated lambs it remained unaffected. Levamisole hydrochloride was ineffective when given 4 days after infection but restored the blood clotting time to near normal soon after treatment when it was given 30 days after infection.

Serum lactate dehydrogenase: clinical significance of alterations in its activity in lambs infected with Dictyocaulus filaria.

Alterations in the serum lactate dehydrogenase activity of Dictyocaulus filaria infected lambs were studied. The significant increase in its activity during patency correlated well with the progress of the disease and lung damage caused by the parasite. The enzyme may be of use in assessing the potency of D filaria vaccine and the chemotherapeutic value of an anthelmintic. Its use as a non-invasive method for earlier diagnosis and prognosis of the disease under experimental conditions is suggested.