Monitoring and identification of residues of anabolic preparations in slaughtered cattle by HPLC with diode array detection

Summary

The new combination of isocratic high performance liquid chromatography (HPLC) with on line UV spectrum detection via a diode array configuration has been applied to the detection and identification of anabolics present in application sites of cattle.

Combination of the characteristic retention time in the HPLC chromatogram and a comparison of the full spectrum between 190–400 nm of the anabolic components with that of a standard resulted in a very reliable identification. By means of this method 117 samples of application sites were investigated for the presence of anabolic residues. Of the xenobiotic anabolics, 19‐nortestosterone (NT) was found most frequently (in 96 cases), whereas diethylstilbestrol (DES) was found in only 11 cases.

In all samples the identification of NT and DES was confirmed by high resolution gas chromatography‐mass spectrometry (GCMS).     

Molecular and biological characterisation of Cryptosporidium in pigs

OBJECTIVE: Genetic and biological characterisation of 12 isolates of Cryptosporidium from pigs and comparing them with Cryptosporidium isolates from humans and cattle. DESIGN: Cryptosporidium isolates from pigs were compared with those obtained from human and cattle using rDNA sequence analysis. The infectivity of two of the porcine isolates was determined in neonatal mice and the clinical history of the infected pigs recorded. RESULTS: Pig-derived isolates of Cryptosporidium exhibited two distinct genotypes; a porcine genotype and a bovine genotype, which is common to cattle and other livestock. The porcine genotype did not produce any infection in neonatal mice whereas the bovine genotype did. CONCLUSION: Two distinct genetically and biologically differing strains of Cryptosporidium appeared to be associated with acute diarrhoea in pigs. Whether Cryptosporidium was a primary or secondary pathogen is unclear but warrants further investigation. As the bovine genotype is known to infect humans, the results suggest that pigs can act as reservoirs of cryptosporidial infections for humans and other live-stock. The zoonotic potential of the pig-adapted genotype is uncertain and requires further study.     

Effect of chicken egg yolk immunoglobulins on serum biochemical profiles and intestinal bacterial populations in early‐weaned piglets

This study was conducted to test the hypothesis that dietary supplementation with anti‐E. coli, chicken egg yolk immunoglobulins (IgY), may affect early weaned piglet (EWP) intestinal functions and enteric micro‐organisms. One hundred and forty‐eight ([Landrace × Yorkshire] × Duroc) piglets, weaned at age day 21, were randomly assigned to receive one of three diets for 14 days. Treatment group one (control group) was fed the base diet. Treatment group two (antibiotics group) was fed the base diet which was supplemented with 100 ppm colistin sulphate and 15 ppm enramycin; treatment group three (IgY group) was fed the base diet which was supplemented with 500 mg/kg anti‐E. coli IgY. The study evaluated the effects on EWPs of IgY on growth, serum biochemical, inflammatory profiles and also digestion content intestinal bacterial populations. Results showed no significant difference in diarrhoea rates between IgY‐fed EWPs and antibiotic‐treated EWPs. Serum biochemical analysis showed that EWPs fed an IgY‐containing diet had both lower (p < 0.05) cholesterol and low‐density lipoprotein compared to antibiotic‐treated EWPs. Escherichia coli populations measured in IgY‐fed EWP ileal contents, compared to the control group, were significantly reduced (p < 0.05). Enterococcus, Lactobacillus, Clostridium and Bifidobacterium populations were unaffected by the IgY treatment. Larger (p < 0.05) Enterococcus populations and lower (p < 0.05) expression levels of heat‐stable enterotoxin b (STb) were observed in IgY‐fed EWP caecal digesta compared to the control group. Enteric Lactobacillus significantly decreased (p < 0.05) in EWPs fed antibiotics while it was unaffected by IgY treatment. Dietary supplementation with anti‐E. coli IgY has the potential to suppress enteric E. coli growth, but not Lactobacillus, Clostridium and Bifidobacterium. This promotes and maintains a healthy EWP intestinal environment. These findings suggest that IgY may be used as an alternative to antibiotics in EWP diets.     

A Universal `One-tube' RT-PCR Protocol for Amplifying Isolates of Bovine Viral Diarrhoea Virus

The increase in the knowledge of the genetic variability of BVDV and the identification of some of the genetic determinants of its pathogenicity require robust and practical tools for rapid molecular characterization of the various genotypes of this virus. This study was undertaken to develop a standard protocol for RT-PCR that allows the amplification of various parts of the genome of BVDV without the need for optimizing each individual reaction. The reaction set-up is very flexible because it consists of two pre-mixes. These are a master mix, with all the required reagents except the desired primers, which are the components of the second pre-mix and are therefore easily interchangeable between the different reactions. After adding any primer-containing pre-mix to the fixed master mix, a non-interrupted cycling protocol led to the generation of amplicons of up to 4 kbp in size in amounts sufficient for subsequent sequencing reactions. The method was applied to five different regions of the BVDV genome: (i) the well-known 5-UTR to differentiate genotypes I and II; (ii) the entire E2 gene, or an approximately 550 bp region within the E2 gene, in order to find the molecular equivalent of antigenic varieties; (iii) the entire structural protein coding region covering the Npro, capsid, E ^RNS, E1 and E2 genes; (iv) a 2.1 kbp region embracing the NS2/3 junction which is known to be cleaved in cytopathic biotypes of BVDV; and (v) the region covering the entire NS4B and NS5A/B genes. All six RT-PCRs were successfully applied using (i) primers with lengths of between 20 and 52 nucleotides, (ii) an aliquot of RNA extracted from either 10⁶ infected bovine embryonal lung cells or the same number of leukocytes from viraemic cattle, and (iii) all the genotype I and II strains of BVDV tested. The technique described was used to generate various Sindbis virus/BVDV recombinants. The correct processing of the amplicon-derived E2 glycoprotein of BVDV strain PT810 was demonstrated by its reaction with a monoclonal antibody in an immunofluorescence assay. Given the variety of RT-PCRs tested, we conclude that this universal protocol may be useful with other RNA viruses.     

Detrimental effects on villus form during conventional oralrehydration therapy for diarrhoea in calves; alleviation by a nutrient oral rehydration solution containing glutamine

This paper examines the possibility that treatment of diarrhoea with conventional oral rehydration solutions (ORSs) may be detrimental to villus structure by imposing nutrient deprivation and that such detrimental effects may be reduced or avoided by using a nutrient ORS. A conventional WHO-type ORS (W) was compared with two nutrient solutions (N and G) both containing high glucose concentrations and the latter containing glutamine; their effects on enteric structure were assessed by morphometric analysis of samples obtained from diarrhoeic calves after 96h treatment. Comparisons were also made with samples from controls and diarrhoeic calves at the stage where oral rehydration would have begun in the treated groups. As in our previous ORS studies, diarrhoea was induced with enterotoxigenic Escherichia coli (09:K30:K99). We measured villus length and width, crypt depth and width and calculated villus surface area in proximal, mid and distal small intestine (PSI, MSI, DSI), using standard morphometric techniques. Proximal and distal spiral colon samples (PC, DC) were examined for crypt depth and width; mitoses per crypt were counted in samples from all regions. Non-diarrhoeic calves showed the expected gradient of villus length through PSI, MSI and DSI, hence data for each region are normalized as a percentage of the control value for that region. PSI showed the greatest loss of villus length and surface area (50%) with diarrhoea. In MSI and DSI the villus loss was greater with solution W and N or G, as were increased mitoses and crypt depth. Crypt depth and mitoses also increased in the colon with solution W. Colonic crypt width increased with diarrhoea and conventional oral rehydration but less so with G; there is reason to believe that such changes have functional significance. Crypt changes in colon, MSI and DSI were least with solution G. The changes developing in diarrhoeic calves prior to treatment were thus less apparent in those treated with a nutritional ORS, particularly if it contained glutamine.     

Feed-and feed additives-related aspects of gut health and development in weanling pigs

The development of new/different management and feeding strategies to stimulate gut development and health in newly-weaned pigs, in order to improve growth performance while minimizing the use of antimicrobial compounds such as antibiotic growth promotants (AGP) and heavy mineral compounds, is essential for the long-term sustainability of the pig industry. Factors including the sub-optimal intake of nutrients and energy, inappropriate microbiota biomass and (or) balance, immature and compromised immune function, and psychosomatic factors caused by weaning can compromise both the efficiency of digestion and absorption and intestinal barrier function through mucosal damage and alteration of tight junction integrity. As a consequence, pigs at weaning are highly susceptible to pathogenic enteric conditions such as post-weaning diarrhea that may be caused by serotypes of enterotoxigenic Escherichia coli. Many dietary components, e.g., protein, fiber, feed additives and minerals, are known to influence microbial growth in the gastrointestinal tract that in turn can impact upon pig growth and health, although the relationships between these are sometimes not necessarily apparent or obvious. In a world climate of increased scrutiny over the use of antibiotics per se in pig production, certain feed additives are seen as alternatives/replacements to antibiotics, and have evolved in some cases to have important roles in everyday commercial pig nutrition. Nevertheless and in general, there remains inconsistency and variability in the efficacy of some feed additives and in cases of severe disease outbreaks, for example, therapeutic antibiotics and/or heavy minerals such as zinc oxide (ZnO) are generally relied upon. If feed ingredients and (or) feed additives are to be used with greater regularity and reliability, then it is necessary to better understand the mechanisms whereby antibiotics and minerals such as ZnO influence animal physiology, in conjunction with the use of appropriate challenge models and in vitro and in vivo techniques.     

Black tea reduces diarrhoea prevalence but decreases growth performance in enterotoxigenic Escherichia coli-infected post-weaning piglets

Enterotoxigenic Escherichia coli (ETEC) is a main cause of diarrhoea in humans and piglets. In vitro, black tea extract (BTE) has anti-pathogenic properties. Anti-diarrhoeal properties of BTE were assessed in a pig model of gastrointestinal infection. At weaning (day 0), piglets (n = 96) were randomly assigned to a diet containing 0% (control), 0.4% or 0.8% (wt/wt) BTE during 27 days. Piglets were orally infected with 6.4 × 10(6) cfu of ETEC on day 6. Faecal consistency, feed intake and body weight were measured. In a sub-study (n = 30 piglets), the effect of BTE palatability on feed intake was assessed. Additionally, the effect of BTE on ETEC growth in the presence or absence of iron was studied in vitro. The 0.8% BTE diet reduced diarrhoea prevalence by 20% but also decreased feed intake by 16% and feed efficiency by 12% over the total period. The 0.4% BTE diet decreased feed efficiency and weight gain from day 13 onwards. The palatability study demonstrated that piglets preferred the control to the BTE diets. In vitro, BTE delayed ETEC exponential growth, which was reversed by iron addition. Although BTE had anti-diarrhoeal properties, this effect was accompanied by impaired performance. The absence of a correlation between diarrhoea prevalence and feed intake suggests that reduced diarrhoea directly results from BTE rather than from reduced feed intake caused by BTE astringency.     

低蛋白质氨基酸平衡饲粮对早期断奶仔猪腹泻和生产性能的影响

本研究选用１８头２８±１日龄断奶的杂交仔猪（平均体重约５．５ｋｇ），研究低蛋白质氨基酸平衡饲粮对早期断奶仔猪腹泻和生产性能的影响。试验结果显示：（１）低蛋白质氨基酸平衡饲粮使仔猪血浆挥发性盐基氨含量、血浆尿素氨含量和腹泻指数降低（Ｐ＜０．０５或０．０１）。（２）采食全植物蛋白型氨基酸平衡饲粮（ＣＰ１８．３％）的仔猪，其平均日增重（ＡＤＧ）、平均日采食量（ＡＤＦＩ）和料肉比（Ｐ／Ｇ）与采食复合蛋白型对照饲粮（ＣＰ１９．７％）的仔猪差异不显著（Ｐ＞０．０５），但前者的增重成本比后者降低（Ｐ＜０．０５）３１％。（３）采食复合蛋白型氨基酸平衡饲粮（ＣＰ１８．４％）的仔猪的生产性能优于采食复合蛋白型对照饲粮（ＣＰ１９．７％）的仔猪，前者的ＡＤＧ和ＡＤＦＩ分别提高（Ｐ＜０．０５）６１％和３２％，Ｆ／Ｇ和增重成本分别下降（Ｐ＜０．０５）２４％和３７％。（４）采食全植物蛋白型氨基酸平衡饲粮的仔猪，其ＡＤＧ和ＡＤＦＩ低于（Ｐ＜０．０１）采食复合蛋白型氨基酸平衡饲粮的仔猪。前者的Ｆ／Ｇ和增重成本趋于高于后者，但差异不显著（Ｐ＞０．０５）。以上结果表明，低蛋白质氨基酸平衡饲粮可显著降低仔猪断奶后腹泻和提高仔猪生产性能。复合蛋白型?