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Genetic characterization of ruminant pestiviruses: sequence analysis of viral genotypes isolated from sheep.

Historically, the genus pestivirus was believed to contain three species of viruses; bovine viral diarrhea virus (BVDV), border disease virus (BDV) and classical swine fever virus (CSFV). However, based on limited sequence analysis of a small number of pestiviral isolates from domestic livestock, evidence has recently emerged indicating that at least four distinct genotypes exist. In an attempt to gain a better understanding of the degree of viral variation among ruminant pestiviruses, the entire structural gene coding region of an ovine pestivirus. BD31, genome encompassing 3358 nucleotides was cloned and sequenced. Sequence analysis revealed that BD31 shares less than 71% nucleotide similarity with other pestiviruses, suggesting, that BD31 is distinct from BVDV, CSFV as well as other ovine and bovine pestiviruses currently referred to as BVDV type II. Based on this data, BD31 is the first North American pestivirus isolate that falls under the category true BDV. Results from the analysis of the nucleotide sequence of the E0-E1 coding region of six additional ruminant pestiviruses identified the existence of three distinct virus genotypes in North America. Thus, among ruminent pestiviruses, bovine isolates can be grouped into two genotypes, namely types 1 and 4, whereas ovine isolates fall into genotypes 1, 3 and 4.

Amino Acid Sequence↗

Metabolism of pyrrolizidine alkaloids by Peptostreptococcus heliotrinreducens and a mixed culture derived from ovine ruminal fluid.

A mixed culture of ovine ruminal microbes metabolizes the macrocyclic pyrrolizidine alkaloids present in the plant Senecio jacobaea, including jacobine and seneciphylline. Previous attempts to identify metabolites of these alkaloids have not been successful. The objective of this study was to compare the metabolism of pyrrolizidine alkaloids by a mixed culture of ovine ruminal microbes to the metabolism of pyrrolizidine alkaloids by the known organism Peptostreptococcus heliotrinreducens. P. heliotrinreducens metabolizes the pyrrolizidine alkaloids heliotrine and lasiocarpine to 7alpha-hydroxy-1-methylene-8alpha-pyrrolizidine and 7alpha-angelyl-1-methylene-8alpha-pyrrolizidine, respectively. This organism does not metabolize the pyrrolizidine alkaloids jacobine or seneciphylline. A mixed culture of ovine ruminal microbes also metabolized heliotrine and lasiocarpine to identical methylene compounds. This mixed culture also metabolized jacobine and seneciphylline, with the production of very low levels of the corresponding 1-methylene compounds. Samples were analyzed by TLC and GC/MS.

Animals↗

Limited role of lagomorphs (Oryctolagus cuniculus and Lepus capensis) in the dispersion of parasite nematodes of ruminants.

Rabbits (Oryctolagus cuniculus) and hares (Lepus capensis) were investigated under natural conditions for infection with ruminant nematodes, either in the condition of close contact with sheep (south-east of France) or unknown contact with ruminants (central region and French Champaign). Rabbits and hares were poorly infected with sheep digestive tract nematodes: Trichostrongylus colubriformis (second record) was encountered in one site and Trichostrongylus capricola (a first record) in another site, at very low intensity. Small sheep lungworms (Neostrongylus linearis and Cystocaulus ocreatus) were not recorded in rabbits grazing common pasture with sheep. Although transmission remained limited between lagomorphs and ruminants, it could play a role in the transfer of nematode isolates resistant to anthelmintics from one farm to another.

Animals↗

Simultaneous detection of Anaplasma and Ehrlichia species in ruminants and detection of Ehrlichia ruminantium in Amblyomma variegatum ticks by reverse line blot hybridization.

The detection of Anaplasma and Ehrlichia species is usually based on species-specific PCR assays, since no assay is yet available which can detect and identify these species simultaneously. To this end, we developed a reverse line blot (RLB) assay for simultaneous detection and identification of Anaplasma and Ehrlichia species in domestic ruminants and ticks. In a PCR the hypervariable V1 region of the 16S ribosomal RNA (rRNA) gene was amplified with a set of primers unique for members of the genera Anaplasma and Ehrlichia [Int. J. Syst. Evol. Microbiol. 51 (2001) 2145]. Amplified PCR products from blood of domestic ruminants or Amblyomma variegatum tick samples were hybridized onto a membrane to which eight species-specific oligonucleotide probes and one Ehrlichia and Anaplasma catch-all oligonucleotide probe were covalently linked. No DNA was amplified from uninfected blood, nor from other hemoparasites such as Theileria annulata, or Babesia bigemina. The species-specific probes did not cross-react with DNA amplified from other species. E. ruminantium, A. ovis and another Ehrlichia were identified by RLB in blood samples collected from small ruminants in Mozambique. Finally, A. variegatum ticks were tested after feeding on E. ruminantium infected sheep. E. ruminantium could be detected in adult ticks even if feeding of nymphs was carried out 3.5 years post-infection. In conclusion, the developed species-specific oligonucleotide probes used in an RLB assay can simultaneously detect and identify several Ehrlichia and Anaplasma species. However, as no quantitative data for the detection limit are available yet, only positive results are interpretable at this stage.

Anaplasma↗

Economic sustainability of small ruminants production in semi-arid areas of Lebanon.

Small ruminant production in the near east region is facing serious constraint of feed availability. A study was conducted in marginal areas of Lebanon, using a cost-benefit analysis technique (CBA), to assess the feasibility of four small ruminant production systems ranging from semi-nomadic to settled. When the owned labor cost was included as an opportunity cost in the economic analysis, CBA revealed negative returns in all four systems. Only the settled system was profitable according to the financial analysis excluding labor cost. Moreover, feed expenses if coupled with grazing costs represented a major constraint to profitability. To remedy to the feed deficit problem, the potential of using agro-industrial by-products as feed block supplements was investigated. Simulated feed block diets, using the most available by-products, provided a better nutritive value per unit cost than hand-fed diets used in the systems studied. Preliminary analysis showed that the use of feed block diets could improve the economic sustainability of small ruminant production systems.

Journal Article↗

Ownership pattern and management practices of small ruminants in The Gambia - implications for a breeding programme.

In the framework of a genetic improvement scheme a countrywide survey of small ruminant owners was conducted in The Gambia to obtain information about the ownership pattern, reasons for keeping and especially about breeding and management practices including housing and feeding. The main results were the following: women play a major role in small ruminant production, representing 52% of the owners of sheep, 67% of the owners of goats and 43% of the owners of both sheep and goats. The average number of animals owned is quite low (about six head of sheep and goats each, out of which about three are breeding females). Most of the breeding males are born in their respective flock and there are fewer breeding bucks than breeding rams. However, there are sufficient numbers of breeding males around. Animals are mainly left free to roam around during the dry season, and are either tethered or herded in the rainy season. During night animals are housed or tied under a shelter. Supplementary feeding is not common and vaccination against Pest des Petit Ruminants (PPR) and Pasteurellosis is only partly carried out. Implications for the establishment and structure of a multiplication tier within a three tier breeding scheme, nucleus-->multipliers-->farmers are discussed.

Journal Article↗

Preparation and characteristics of rumen-bypass microcapsules for improvement of productivity in ruminants.

Rumen-bypass microcapsules were prepared by a spray-dry method for protection against microbial hydrogenation in the rumen (neutral pH). Porous starch was used as the core material, and the microcapsules were prepared by a triple coating of Eudragit E100, AS-HF, and shellac. Capsules were generated with yield of about 48% and a mean particle diameter of 20-30 microm. The microcapsules had high stability in a neutral solution that mimicked a ruminal pH (pH 6.5). Moreover, when microcapsules were incubated in the presence of ruminal microorganisms, about 65% of the microcapsules were resistant to digestion in ruminal fluids, and protection of the inclusion substance was observed. In addition, the efficiency of release of these microcapsules was about 85% within only 30 min in the abomasal environment (pH 3.0).

Animals↗

In vitro metabolism of fumonisin B1 by ruminal microflora.

Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium moniliforme and F. proliferatum. Little is known of its metabolic fate after oral ingestion in ruminants, but these animals are reported to be tolerant towards FB1. The metabolism of this mycotoxin was evaluated following incubation (1 microg/ml) in ruminal fluid for up to 72 h, in the presence or absence of alfalfa as a substrate for microbial growth, using a model rumen (sealed flask, anaerobic conditions, exclusion of light, gentle agitation, 39 degrees C). The decrease in FB1 concentration and the production of short-chain fatty acids were determined. FB1 had no effect on SCFA production. After 72 h incubation, FB1 depletion was 12% and 18% in samples with and without alfalfa, respectively. No hydrolysed metabolites (aminopolyols or aminopentol) were detected. These results indicate that FB1 is poorly metabolized in the rumen and suggest that such metabolism is not the cause of the tolerance to this toxin displayed by ruminants.

Animals↗

Ruminal fermentation kinetics in ewes offered a maize stover basal diet supplemented with cowpea hay, groundnut hay, cotton seed meal or maize meal.

The effect on ruminal digestive kinetics of supplementing maize stover (MS) with cowpea hay (CW), groundnut hay (GN), cotton seed meal (CSM) or maize meal (MM) was evaluated in a 5 x 5 Latin square using five Merino ewes with ruminal cannulae. CW and GN were offered at 30% w/w to MS, while CSM and MM were given at 15 g/kg0.75 per day. There were significant time (p < 0.001) and time x diet (p < 0.05) effects on pH but the effect of diet alone was not significant (p > 0.05). There was a significant (p < 0.05) increase in the concentration of ammonia with time on the supplemented diets. There were no significant (p > 0.05) differences in the concentrations of total volatile fatty acids (VFAs). Butyrate increased with supplementation (p < 0.05). Supplementation increased the concentrations of branched-chain and total VFAs with time (p < 0.05). The non-glucogenic ratio was not significantly (p > 0.05) lower on supplemented diets owing to the higher butyrate concentration. The degradation curve constants were not significantly different (p > 0.05), the average effective dry matter degradation being 27.80% +/- 0.618% at 0.05 h-1 flow rate. The chemical and physical characteristics of the cell wall of MS may have presented few sites for microbial colonization, so limiting degradation. These results showed that small quantities of forage supplements can improve the ruminal environment in animals subsisting on low-quality forages and form a basis for comparison with bought-in protein and energy sources.

Ammonia↗

[TSE surveillance in small ruminants and pigs: a pilot study].

Switzerland is controlling Transmissible Spongiform Encephalopathies (TSE) in cattle (BSE) and small ruminants (scrapie). Since BSE is potentially transmissible to sheep, goats or pigs through feeding of contaminated meat and bone meal, implementation of an active surveillance programme for TSE in these species is discussed. The aim of this pilot study was to obtain preliminary data on the prevalence ofTSE and other neurological disorders in these populations. For that purpose, a total of 398 perished and 825 slaughtered adult small ruminants and pigs was examined for the presence of neuropathological changes. None of these animals revealed positive for TSE. However, the investigations demonstrated that perished sheep and goats exhibited a higher prevalence of relevant neuropathological changes when compared with slaughtered animals. From these results, it is concluded that perished small ruminants are probably a risk population for TSE and should be considered as target populations for an active surveillance programme.

Animals↗

Rumination and overgeneral memory in depression: effects of self-focus and analytic thinking.

Previous research has shown that, compared with a rumination induction, a brief distraction procedure reduces overgeneral autobiographical memory in depression. The authors investigated whether this effect depends on reductions in analytic thinking or reductions in self-focus. Focus of attention (high vs. low self-focus) and thinking style (high vs. low analytical thinking) were independently manipulated in depressed patients in a 2 x 2 design. Autobiographical recall was measured pre- and postmanipulation. Thinking style significantly affected overgeneral memory, whereas focus of attention significantly affected despondent mood. Reducing analytical self-focus reduced overgeneral memory, suggesting that high levels of naturally occurring ruminative analytic thinking may be important in the maintenance of overgeneral memory. Overgeneral memory in depression may be associated with chronic ruminative attempts to make sense of current or past difficulties.

Adolescent↗

The enrichment of a ruminal bacterium (Megasphaera elsdenii YJ-4) that produces the trans-10, cis-12 isomer of conjugated linoleic acid.

AIMS: To isolate predominant ruminal bacteria that produce trans-10, cis-12 conjugated linoleic acid (CLA) from linoleic acid (LA). METHODS AND RESULTS: Mixed bacteria from ruminal contents of a cow fed grain were enriched with DL-lactate and trypticase. They produced more trans-10, cis-12 CLA than those that were not enriched (7 vs 2 microg mg protein(-1), P < 0.05). Enrichments had an abundance of large cocci that produced trans-10, cis-12 CLA from LA. Strain YJ-4 produced the most trans-10, cis-12 CLA (approx. 7 microg mg protein(-1)) and 16S rDNA sequencing indicated that YJ-4 was a strain of Megasphaera elsdenii. Megasphaera elsdenii T81 produced approx. 4 microg trans-10, cis-12 CLA mg protein(-1) while strains B159, AW106 and JL1 produced < 0.5 microg mg protein(-1). The trans-10, cis-12 CLA production of YJ-4 was first order with respect to cell concentration (0-800 microg protein ml(-1)), but kinetics were not first order with respect to substrate concentration. CONCLUSIONS: Some M. elsdenii strains produce significant amounts of trans-10, cis-12 CLA. SIGNIFICANCE AND IMPACT OF THE STUDY: Trans-10, cis-12 CLA appears to cause milk fat depression in cattle fed diets supplemented with grain and polyunsaturated fatty acids, but predominant ruminal bacteria that produced trans-10, cis-12 CLA from LA had not previously been isolated.

Animals↗

Influence of ruminal distribution on norfloxacin pharmacokinetics in adult sheep.

Norfloxacin (NF) ruminal distribution after intravenous (i.v.), intramuscular (i.m.) and oral (p.o.) administration was determined in order to assess the influence of the rumen on the pharmacokinetic behaviour of NF in sheep. Norfloxacin concentrations in rumen were detected after i.v. and i.m. administrations between 4 and 48 h in all animals studied. The experimental distribution ratios after i.v. and i.m. administration expressed as area under the concentration-time curve ratios AUC(rumen)/AUC(plasma) were 0.24 and 0.39, respectively, and thus lower than corresponding predicted value of 5.06. Apparently, drug persisted in the rumen content longer than in plasma. The experimental mean residence time ratios MRT(rumen)/MRT(plasma) after i.v. and i.m. administrations were 2.33 and 1.66, respectively. After p.o. administration, NF concentrations in the rumen content were extremely high compared with the respective plasma concentrations, resulting in mean peak concentrations ratio C(max-rumen)/C(max-plasma) of 383.66 and AUC(rumen)/AUC(plasma) experimental ratio of 402.32. This value was considerably higher (approximately 79 fold) than the predicted distribution ratio of 5.06. Our results suggest that the limited p.o. bioavailability of NF in ruminants could not be attributed to ruminal degradation.

Administration, Oral↗

Adherence of Fusobacterium necrophorum subsp. necrophorum to ruminal cells derived from bovine rumenitis.

Fusobacterium necrophorum subsp. necrophorum strain VPI 2891 was shown to adhere to the surfaces of ruminal cells derived from bovine rumenitis. The strain also attached to bovine type 1 collagen. Treatment of the bacterium with antiserum to bacterial cells reduced attachment. The bacterial attachment was also markedly reduced when the ruminal cells had been pretreated with anticollagen serum. Fluorescence specific for the collagen was demonstrated on the surface of bovine tissue affected with rumenitis. These findings suggest that F. necrophorum subsp. necrophorum strain VPI 2891 adheres to the ruminal cells derived from rumenitis tissue and that the attachment may be mediated by cellular collagen.

Animals↗

Diversification of Cercopithifilaria species (Nematoda: Filarioidea) in Japanese wild ruminants with description of two new species.

Twelve of the 17 Cervus nippon nippon deer from Kyushu Island, Japan, that we examined were infected with one or two Cercopithifilaria species. C. longa n. sp. adults were in the subcutaneous tissues of limbs and the abdomen, and C. crassa n. sp. adults were in the skin, mainly in the anterior part of the back; the distribution of the dermal microfilariae generally matched that of the adult worms. The two new species were assigned to the group of primitive Cercopithifilaria species that parasitize ruminants (bavids and cervids), but the new species could readily be distinguished from others morphologically. C. longa was more primitive and resembled C. bulboidea, one of the five species from the serow Capricornis crispus, a Japanese member of the Caprinae, and species from Bovidae in Africa. C. crassa had a thick body and large spicules like C. rugosicauda from Capreolus capreolus in Europe, the only previously known Cercopithifilaria species from cervids, but it also had one or two hypertrophied pairs of caudal papillae, an unusual character found so far only in Japanese parasites. Among the 12 species known from ruminants, four are African, one is European and more highly evolved, and seven are Japanese, with some being primitive and some more evolved. The great diversity of Cercopithifilaria species in the two wild ruminants that live in Japan seems to have resulted from local speciation, which occurred during the Pleistocene, from a primitive form of the C. longa type derived from Eurasiatic ancestors, which has disappeared or, more probably, not yet been discovered.

Animals↗

Translational efficiency of casein transcripts in the mammary tissue of lactating ruminants.

Caseins are essentially concentrated in the colloidal fraction of ruminant milks as highly hydrated and mineralized spherical particles, termed casein micelles. They form a group of four peptide chains (alpha(s1), beta, alpha(s2) and kappa), encoded by four structural genes (CSN1S1, CSN2, CSN1S2 and CSN3, respectively) of which the expression is regulated by lactogenic hormones. These phosphoproteins are synthesized, essentially during lactation, in the mammary epithelial cells and we show, for the first time, that their regulation is also controlled at the translational level. Apparently, the four casein messenger are not translated with the same efficiency. Specific amplification systems have been developed and optimized to quantify, by real time quantitative PCR (qPCR), transcripts encoding the four caseins starting from total RNA extracted from mammary tissues taken on goats (n = 4), ewes (n = 3) and cows (n = 3), in lactation. The relative proportions of each specific messenger (% of casein mRNA) were compared to the relative amounts of the corresponding caseins (% of whole casein) in milks sampled from the same animals, determined after fractionation by reverse phase HPLC and integration of the corresponding peak areas. From qPCR data, the four casein transcripts appeared to be present approximately at the same level of abundance (ca. 25%, except for defective genotypes at the CSN1S1 locus, in the goat) whereas the amounts of the corresponding proteins in milk were ranging between 9 and 38% of the whole casein fraction. A comparison of specific translational efficiencies (% of protein in milk/% of transcript in the mammary tissue), showed that alpha(s1)- and beta-casein transcripts are translated ca. 3- to 4-fold more efficiently than alpha(s2)- and kappa-casein transcripts. This seems to be the rule in the three ruminant species studied. More or less optimal contexts for initiation of translation (Kozak recognition sequence of the start codon) as well as 3' untranslated region (UTR) sequences and length might explain, at least in part, our results. These preliminary results which have now to be confirmed with a larger number of individuals to strengthen our findings and conclusions, provides, however, a rational explanation to the unbalanced casein distribution (approximate proportions 4:1:4:1 for alpha(s1):alpha (s2):beta:kappa, respectively) reported for ruminant milks. The possible effects of specific secondary structures in the 5' and 3' UTRs of casein messengers still have to be considered.

3' Untranslated Regions↗

Gestation linked radical oxygen species fluxes and vitamins and trace mineral deficiencies in the ruminant.

In mammals, radical oxygen species (ROS) are essential factors of cell replication, differentiation and growth (oxidative signal), notably during gestation, but are also potentially damaging agents. In Women, ROS play a role in remodeling of uterine tissues, implantation of the embryo, settlement of the villi and development of blood vessels characteristic of gestation. The body stores of vitamins and minerals of gestating females are used to keep ROS fluxes at a level corresponding to oxidative signals and to prevent an imbalance between their production and scavenging (oxidative stress), which would be detrimental to the mother and fetus. There is some evidence that, although based on different regulatory mechanisms, most of the effects of ROS reported in humans also occur in pregnant ruminant females, some of which have been actually reported. Many vitamins and trace elements have dual effects in the organism of mammals: (a) they are involved in the control of metabolic pathways or/and gene expression, (b) but most of the time they also display ROS trapping activity or their deficiencies induce high rates of ROS production. Deficiencies induce different disorders of gestation and can be induced by different kinds of stress. An example is given, corresponding to the decreased contents of cobalt of forages, when exposed to sustained heavy rains, so that the supply of vitamins B12 to the organism of the ruminant that grazes them is reduced and failure of gestation is induced. Outdoor exposure of ruminants to adverse climatic conditions by itself can increase the vitamin and trace element requirements. Adaptation of production systems taking into account these interactions between gestation and sources of stress or change of the quality of feeding stuffs as well as further developments of knowledge in that field is necessary to promote sustainable agricultural practices.

Animal Nutritional Physiological Phenomena↗

Mastitis of dairy small ruminants.

Staphylococci are the main aetiological agents of small ruminants intramammary infections (IMI), the more frequent isolates being S. aureus in clinical cases and coagulase negative species in subclinical IMI. The clinical IMI, whose annual incidence is usually lower than 5%, mainly occur at the beginning of machine milking and during the first third of lactation. These features constitute small ruminant peculiarities compared to dairy cattle. Small ruminant mastitis is generally a chronic and contagious infection: the primary sources are mammary and cutaneous carriages, and spreading mainly occurs during milking. Somatic cell counts (SCC) represent a valuable tool for prevalence assessment and screening, but predictive values are better in ewes than in goats. Prevention is most often based on milking machine management, sanitation and annual control, and milking technique optimisation. Elimination mainly relies on culling animals exhibiting clinical, chronic and recurrent IMI, and on drying-off intramammary antibiotherapy; this treatment allows a good efficacy and may be used selectively by targeting infected udders only. Heritability values for lactation mean SCC scores are between 0.11 and 0.15. Effective inclusion of ewe's mastitis resistance in the breeding goal has recently been implemented in France following experimental and large scale estimations of genetic parameters for SCC scores.

Animals↗