Expression of the Major Outer Membrane Protein (MOMP) of Chlamydophila abortus,Chlamydophila pecorum,and Chlamydia suis in Escherichia coli using an Arabinose‐inducible Plasmid Vector

The ompA genes encoding the 40 kDa major outer membrane protein (MOMP) of Chlamydophila (Ch.) abortus, Ch. pecorum, and Chlamydia (C.) suis were cloned into the arabinose‐inducible plasmid vector pBADMycHis, and recombinant MOMPs (rMOMP) from the three chlamydial species were expressed at high levels in Escherichia (E.) coli. The proteins lacking the 22 aa N‐terminal signal peptide were expressed as insoluble cytoplasmic inclusion bodies which were readily purified using immobilized metal‐affinity chromatography. The rMOMPs including the N‐terminal signal peptide were expressed and translocated as a surface‐exposed immunoaccessible protein into the outer membrane of E. coli. Transformants expressing this full‐length rMOMP were significantly reduced in viability. Purified native elementary bodies (EB) and rMOMPs of the three chlamydial species purified from the E. coli cytoplasm were used for immunization of rabbits. The resulting sera were analysed for their ability to recognize homologous and heterologous rMOMP and native EB. When testing rMOMP antisera against rMOMP and EB antigens, marked cross‐reactivities were detected between the three species. Using EB antisera and rMOMPs as antigens, a significant species‐specific reactivity was measured.     

Selection of useful probiotic lactic acid bacteria from the Lactobacillus acidophilus group and their applications to functional foods

In the present review, a new mass screening system for selecting probiotic strains from Lactobacillus (L) acidophilus group lactic acid bacteria (LAB) with strong adhesion to the human intestinal tract is described. Characteristics of antimicrobial peptides (bacteriocin), lactose‐hydrolyzing enzymes and immunostimulative oligo DNA motifs in L. gasseri strains are also described. Finally, the use of L. acidophilus LAB, selected by our screening method, that have strong adhesion to the human colonic mucosa in functional yogurt products is described. Adhesiveness to the human intestine is one of the most important characteristics of probiotic LAB. A new screening system that involves a combination of three methods is proposed: rat colonic mucin (RCM)‐micro plate assay, Carnoy's histochemical staining method and carbohydrate probe binding assay. By using an RCM‐coated poly‐vinylidene‐diflouride membrane that mimics the human colonic mucous layer, a new lectin was isolated and its structure was clarified by gene cloning. Furthermore, the structures and functions of a new cyclic bacteriocin (gassericin A), new lactose‐hydrolyzing enzymes, and new immunostimulating oligo DNA motifs from Lactobacillus gasseri (B₁ subgroup) were clarified. A new functional yogurt ‘Fit down’ is proposed, that is fermented by an adhesive strain of L. acidophilus LA67 selected by our screening and contains antihypertensive peptides derived from whey proteins by protease digestion. In the future, superior functional foods containing more effective probiotic LAB are expected to be developed by the use of the proposed mass screening system.     

Food preservative potential of gassericin A‐containing concentrate prepared from cheese whey culture supernatant of Lactobacillus gasseri LA39

Gassericin A (GA) is a circular bacteriocin produced by Lactobacillus gasseri LA39. In this study, GA‐containing concentrate was prepared using a cross‐flow membrane filtration device (30 kDa cut‐off) from the culture supernatant of Lb. gasseri LA39 cultivated in a cheese whey‐based food‐grade medium. The bacteriocin activity titer in the concentrate was 16 times as high as that of the culture supernatant and was completely maintained through each incubation at 4°C for 3 months, 37°C for 2 months, 60°C for 5 h, and 100°C for 30 min. The GA‐containing concentrate was used with glycine powder to make custard creams, and then four representative strains of custard cream spoilage bacteria (Bacillus cereus, Lactococcus lactis subsp. lactis, Achromobacter denitrificans and Pseudomonas fluorescens) were individually inoculated at c. 10³ colony forming units/g in the custard creams. Throughout 30 days of incubation at 30°C, all of the inoculated bacteria were completely inhibited by the combination of 5% (w/w) of the GA‐containing concentrate and 0.5% (w/w) glycine. This is the first highly practical application of GA to foods as a biopreservative, and the concentration method and the bacteriocin concentrate would contribute to biopreservation of several foods.     

Enhancement of mucosal immune response against the M2eHBc+ antigen in mice with the fusion expression products of LTB and M2eHBc+ through mucosal immunization route

M2e is the external domain of M2 protein, a conservative transmembrane protein of the avian influenza A virus. Previous research had shown that the vaccine of the formation particle of M2e and hepatitis B virus core antigen (HBcAg) can fully protect mice against a lethal H5N1 subtype avian influenza virus (AIV) infection. As an effective approach against mucosal tissue infectious agent, mucosal vaccination requires effective and safe adjuvants. Here we have first fused two M2e peptide to the N terminal and the major immunodominant region (MIR) of the HBcAg protein simultaneously to create a fusion gene, named as M2eHBc+, and then inserted B subunit of Escherichia coli heat labile enterotoxin (LTB) into the N terminal of M2eHBc+ to construct the second fusion gene, named as LBM2eHBc+. These two fusion genes can be efficiently expressed in Escherichia coli cell and the yield peptide can self-assemble into virus-like particles (VLP). The mice immunization with two types of the purified particles by intranasal dropping and oral routes revealed that LTB can significantly enhance the mucosal immune responses of mice to co-expression M2eHBc+ particle form antigen.     

Partial characterization of structure and function of a xylanase gene from the rumen hemicellulolytic bacterium Eubacterium ruminantium

A gene encoding for xylanase activity in the rumen hemicellulolytic bacterium Eubacterium ruminantium was cloned into pBR322 in Escherichia coli (E. coli ). The primary clone had a 5.7 kb insert produced by Eco RI partial digestion. Subcloning followed by sequencing allowed for the discovery that this enzyme has a glycosyl‐hydrolase family 10 catalytic domain with a family 9 carbohydrate binding module at C‐terminus and a region partially homologous to a family 22 carbohydrate binding module at N‐terminus. Cloned xylanase is specifically active against xylan and oligoxyloside to produce xylobiose and xylotriose, showing optimal pH and temperature at 7.0 and 50°C, respectively. Molecular size of the xylanase (91 kDa) was confirmed by zymogram analysis of the E. coli clone, which agreed with the predicted size from the DNA sequence. Functions of the two modules at C‐ and N‐termini were evaluated by using xylanase variants with and without the respective module and the C‐terminal module was found to be functional in binding to acid‐swollen cellulose and insoluble oat‐spelt xylan, whereas the N‐terminal module was inactive for binding them.     

Antimicrobial activity of a bovine myeloid antimicrobial peptide (BMAP‐28) against methicillin‐susceptible and methicillin‐resistant Staphylococcus aureus

A bovine myeloid antimicrobial peptide (BMAP‐28) is a member of the cathelicidin family which is included in the innate immune system of mammals. Recently, there have been many studies about antimicrobial peptides. This study aims to clarify whether BMAP‐28 has bactericidal activity against methicillin‐resistant Staphylococcus aureus (MRSA) and compares its activity against methicillin‐susceptible S. aureus (MSSA) and MRSA. We found that the peptide was effective in killing MRSA (minimal inhibitory concentration (MIC) range; 5–20 µg/mL). It was also revealed that MSSA (MIC range; 1.25–20 µg/mL) had two levels of susceptibility to BMAP‐28. We also examined the effect of BMAP‐28 on bacterial shape to visually show its activity. After exposure to the peptide, both MSSA and MRSA cells showed the morphological changes on their surfaces. Our results indicate that BMAP‐28 is a promising candidate for medicine against drug‐resistant bacteria.     

Clinical Evaluation of a Peptide‐ELISA based upon N‐terminal B‐cell Epitope of the VapA Protein for Diagnosis of Rhodococcus equi Pneumonia in Foals

A total of 227 field samples from naturally exposed foals aged between 3 weeks and 6 months were used in an evaluation of a peptide‐based enzyme‐linked immunosorbent assay (ELISA) for diagnosis of Rhodococcus equi infection. A biotinylated peptide derived from the virulence‐associated protein A (VapA) of R. equi, a horse pathogen, was synthesized and designated as PN11‐14. The peptide corresponds to the N‐terminal B‐cell epitope TSLNLQKDEPNGRASDTAGQ of the VapA protein. Based upon a serum immunoglobulin (Ig)G titre of 512 as a positive cut‐off value for the R. equi infection, the ELISA provided the overall sensitivity of 47.62%, specificity of 69.67% and an accuracy of 59.47% with a positive predictive value of 57.47% for true R. equi pneumonia. The assay was improved by detecting VapA‐specific IgGb antibodies against N‐terminal B‐cell epitope of the VapA protein rather than IgG antibodies. The VapA‐IgGb ELISA showed the overall sensitivity of 70.47%, specificity of 72.13% and accuracy of 71.36% with a positive predictive value of 68.52%. Diagnosis of R. equi disease in 6‐week‐old foals showed that the VapA‐IgGb ELISA provided an increasing trend (P = 0.0572) in sensitivity of 82.4% in comparison with the VapA‐IgG ELISA which showed the sensitivity of 58.8%. However, differences in specificity of both tests were statistically insignificant (P = 0.357) as analysed by the McNemar test. These results indicated that detection of VapA‐specific IgGb antibodies may be a better predictor of R. equi disease in foals.     

Nuclear transfer using a bovine mammary epithelial cell line (BMEC)

The developmental potential of nuclei from a bovine mammary epithelial cell line (BMEC) in nuclear transfer was investigated. For nuclear transfer donors, BMEC cells (passage 15) were cultured for 4–5 days after seeding at cell densities of 1.0 × 10⁵ cells/cm² (high‐density group) or 0.8 × 10⁴ cells/cm² (low‐density group). First, the effective electric stimulation for fusion of enucleated oocytes with BMEC cells was examined. Fusion rates reached maximum with two DC pulses of 30 V/150 µm for 20 µs in the high‐density group and with two DC pulses of 25 V/150 µm for 10 µs in the low‐density group. The fusion rate (37.5%) in the high‐density group was significantly (P < 0.005) lower than in the low‐density group (71.4%). Second, the in vitro developmental potential of nuclear transfer embryos derived from BMEC cells was examined. In the high‐density and low‐density groups, 18.8% and 24.1% of fused oocytes, respectively, developed to blastocyst stage. The results of this study indicate that nuclei from BMEC cells support the development of nuclear transfer embryos to the blastocyst stage and that the efficiency of oocyte–cell fusion is affected by the culture conditions of the donor BEMC cells before nuclear transfer.     

旋毛虫新生幼虫期特异性T314全长cDNA的克隆及表达

利用PCR将旋毛虫新生幼虫期特异性全长T314cDNA的信号肽祛作后重组到原核表达载体pET-28a。将重组质粒pET28-T314转入克隆菌DH5a和表达菌DE3，分别提取质粒进行酶切，测序鉴定。用KIPTG诱导培养重组表达菌DE3，做菌体裂解物外源表达产物的SDS－PAGE分析。将重组质粒表达的融合蛋白免疫家兔制备抗血清，采用Western blot检测T314cDNA在旋毛虫不同发育时期的表达情况及其天然表达产物的相对分子质量。测序结果显示：外源T314cDNA的PCR产物在重组质粒pET28-T314中阅读框架正确；SDS－PAGE分析结果显示：经IPTG诱导后转化菌DE3的裂解产物与对照菌相比出现了1条相对分子质量约为39000的新条带，大小与外源cDNA融合蛋白的理论推导值38800相符；Western blot检测结果显示：T314cDNA的天然表达产物仅存在旋毛虫新生幼虫，不存在于成虫与肌幼虫，且相对分子质量大小与其在原核重组质粒中表达产物相同。     

Production of bacteriocin by Leuconostoc mesenteroides 406 isolated from Mongolian fermented mare's milk,airag

The purification and characterization of a bacteriocin produced by Leuconostoc mesenteroides strain 406 that was isolated from traditional Mongolian fermented mare's milk, airag, were carried out. Leuconostoc mesenteroides strain 406 was identified on the basis of its morphological and biochemical characteristics and carbohydrate fermentation profile and by API 50 CH kit and 16S ribosomal DNA analyses. The neutral‐pH cell‐free supernatant of this bacterium inhibited the growth of several lactic acid bacteria and food spoilage and pathogenic organisms, including Listeria monocytogenes and Clostridium botulinum. The bacteriocin was heat‐stable and not sensitive to acid and alkaline conditions, but was sensitive to several proteolytic enzymes such as pepsin, pronase E, proteinase K, trypsin, and α‐chymotrypsin, but not catalase. Optimum bacteriocin production (4000 activity units/mL) was achieved when the strain was cultured at 25°C for 24–36 h in Man Rogosa Sharpe medium. The bacteriocin was partially purified by ammonium sulfate precipitation (80% saturation), dialysis (cut‐off MW: 1000), and gel filtration chromatography. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis revealed that the bacteriocin had a molecular weight of approximately 3.3 kDa. To our knowledge, this is the first report of the isolation of a bacteriocin‐producing Leuconostoc strain from airag. An application to fermented milks would be desired.     

Bacteriocin production of probiotic Lactobacillus gasseri LA39 isolated from human feces in milk-based media

The use of bacteriocins from Lactobacillus gasseri , a probiotic lactic acid bacterium, as bio-preservatives in the food industry and animal formulations has been limited because few strains of Lb. gasseri are cultivated and produce a bacteriocin in natural media such as milk and milk-based media. By the determination of the growth-supplements to milk among the 47 nutrients, Lb. gasseri JCM1131^T, LA39 and LA158 isolated from human feces were successfully cultured in reconstituted skim milk and cheese whey using proteose peptone as a nutrient supplement, where Lb. gasseri LA39 produced a useful bacteriocin, gassericin A, with effective growth-inhibiting activity against Gram-positive food-borne pathogens. The data suggest these developed low-cost safe media supporting enough production of bacteriocins by the probiotic Lb. gasseri LA39 could be used to improve the safe bio-preservation of foods and therapy of bovine mastitis, and extra cheese whey produced by cheese making industry is reused in the cultivation for probiotics effectively.     

Quantitative studies of the in vitro production of pipecolic acid by rumen protozoa and its degradation by rumen bacteria

An in vitro study was conducted to quantitatively investigate the metabolism of pipecolic acid (Pip), a neuromodulator, by mixed rumen bacteria (B), mixed rumen protozoa (P), a combination of B and P (BP), species‐enriched rumen protozoal suspension (Polyplastron sp., Diploplastron sp., entodinia and Entodinium caudatum) and pure cultures of several isolates of rumen bacteria (Prevetolla bryantii, Prevetolla albensis, Streptococcus bovis, Veillonella parvula, Megasphaera elsdenii and Ruminococcus albus). Only P produced Pip from L‐lysine (1.0 mmol/L L‐Lys) at a rate of 83.5 ± 1.6 µmol/L/h and even in BP, Pip was produced from L‐Lys by P and increased at a rate of 31.2 ± 3.8 µmol/L/h. Pip production by P was highest when the substrate (L‐Lys) concentration was 6 mmol/L and then the rate was 580 ± 36 µmol/L/h. Pipecolic acid production by P suspension enriched with different species of protozoa showed that Polyplastron sp. had the highest Pip production rate of 0.907 ± 0.092 µmol/L/mg protozoal protein per h, and Diploplastron sp. had the lowest rate of 0.55 ± 0.13 µmol/L/mg protozoal protein per h. The addition of D‐Lys (1.0 mmol/L) as a substrate to the P suspension revealed that P were also able to produce Pip from D‐Lys, though at a lower rate (1/3) compared with L‐Lys (1.0 mmol/L), suggesting the presence of epimerases in P. It was confirmed that B were unable to produce Pip from L‐ or D‐Lys. Only B degraded Pip (1.0 mmol/L) after a lag phase at a rate of 56.0 ± 1.5 µmol/L/h. The B suspension was able to degrade D‐Lys, though the products were not identified. Pip degradation by pure culture of some species of rumen bacteria showed that P. bryantii and R. albus had the highest rate followed by P. albensis, S. bovis and M. elsdenii with a low rate of Pip degradation. Veillonella parvula showed no ability to degrade Pip. The results suggest that a fairly large proportion of rumen‐produced Pip is likely to be absorbed by the host animal before degradation by rumen bacteria.     

In vitro immunomodulatory effect of nisin on porcine leucocytes

Nisin, a lantibiotic bacteriocin, has been used for years as a natural food preservative. In addition to its antimicrobial activity, nisin also shows immunomodulatory properties, and the nisin‐producing Lactococcus lactis strain has been successfully tested as a probiotic in weaned piglets. However, the impact of nisin on porcine immune cells has not yet been explored. The objective of the present study was to examine the in vitro immunomodulatory effect of nisin on porcine peripheral blood leucocytes. The whole heparinized blood samples or freshly isolated peripheral blood mononuclear cells (PBMCs) were incubated with different nisin concentrations (0, 1.56, 3.125, 6.25, 12.5, 25 or 50 µg/ml) for 1, 24, 48 or 72 hr. Escherichia coli bacteria were used to stimulate blood phagocytes, while concanavalin A and lipopolysaccharide from E. coli were used as mitogens. Control cells remained unstimulated. MTT colorimetric assay was used to evaluate PBMCs viability and mitogenic response. Phagocyte activity and T‐cell proliferation were measured by flow cytometry. Flow cytometer was also used for immunophenotyping of T cells. Cytokine levels in the culture media were determined using commercial immunoassay (ELISA) kits. The highest concentration of nisin exhibited proliferative activity (p ? 0.05), stimulated interleukin‐1 beta (IL‐1β) and interleukin‐6 (IL‐6) production (both at p ? 0.001), and increased the percentage of CD4⁺CD8⁺ T cells (p ? 0.001) among unstimulated leucocytes. After cell stimulation, however, the highest nisin concentration showed antiproliferative activity (p ? 0.05), decreased phagocytic functions (p ? 0.05) and inhibited the synthesis of IL‐6 (time‐ and concentration‐dependent effect). As a typical bacterial product, nisin had a stronger impact on innate immune cells, and its effect on T cells was likely a consequence of the modulation of the activity of antigen‐presenting cells. Nisin may be a good candidate as an immunomodulator in pig breeding.     

Study of Vancomycin Resistance in Faecal Enterococci from Healthy Humans and Dogs in Spain a Decade after the Avoparcin Ban in Europe

One hundred and 26 faecal samples from healthy dogs (2009) and 157 faecal samples from healthy humans (2007) from La Rioja region (Spain) were tested to know the carriage of vancomycin‐resistant enterococci (VRE). VRE with intrinsic resistance (vanC) were found in 12% of healthy dogs and humans (29 Enterococcus gallinarum and four Enterococcus casseliflavus). Nevertheless, VRE with acquired mechanism of resistance were not detected among these samples. Four Enterococcus faecalis isolates with vancomycin MIC of 8‐16 mg L^?1 were recovered in human samples, but no single organism with known mechanism of acquired resistance could be identified. These 37 VRE isolates (33 E. gallinarum/E. casseliflavus and four E. faecalis) of dog and human origin were further characterized (PCR detection of antibiotic resistance, virulence and bacteriocin genes). High prevalence of tetracycline resistance was identified (70%), especially among dog isolates harbouring tet(M) ± tet(L) genes; erythromycin resistance was also higher among isolates from dogs and they harboured the erm(B) gene, associated with erm(A) gene in one case. Virulence genes were only identified among E. faecalis isolates of human origin (agg, cpd and/or gelE) and never among E. gallinarum/E. casseliflavus of human or dog origin. Five E. gallinarum isolates of dog and three E. faecalis of human origin expressed bacteriocin activity; among them, only one E. faecalis presented activity against Listeria monocytogenes. The bacteriocin structural gene ef1097 was identified in 3 bacteriocin‐producing E. faecalis isolates, associated with ent1071 in one of them.     

In Vitro Comparison of a Novel External Fixator and Traditional Full‐Limb Transfixation Pin Cast in Horses

Objective: To compare the mechanical properties and failure modes of a standardized short oblique distal radial metaphyseal osteotomy stabilized using either a transfixation pin cast (TPC), a modular‐sidebar external skeletal fixator (ESF), or a solid‐sidebar ESF (modular‐ or solid‐ESF, respectively) using static or cyclic axial loading to failure. Study Design: In vitro study. Animals: Equine cadaver forelimbs. Methods: A 30° oblique distal radial osteotomy was created and stabilized using 1 of the 3 fixation methods: (1) TPC, (2) modular‐ESF, or (3) solid‐ESF. Limbs were tested using static (TPC, modular‐ESF, and solid‐ESF) or cyclic (TPC and solid‐ESF) axial loading to failure. The stiffness, yield load, yield displacement, failure load, and failure displacement for static loading and the cycles to failure for cyclic loading at 75% failure load were obtained. Data were analyzed using a Kruskal–Wallis test. Level of significance was P<.05. Results: The solid‐ESF had a greater stiffness, higher yield and failure load and a lower yield and failure displacement than the TPC (P=.01) and the modular‐ESF (P=.02). TPC had a higher yield load, failure load, and yield displacement than the modular‐ESF (P=.01). Mean cycles to failure for TPC was 2996±657 at a load of 16,000 N and for solid‐ESF 6560±90 cycles at a load of 25,000 N. Conclusions: The solid‐ESF was stiffer and stronger than the TPC and modular‐ESF and failed at a greater number of cycles in axial loading compared with the TPC. Clinical Relevance: This study is an initial step in evaluating the solid‐ESF. Further testing needs to be performed, but this fixation may offer a viable alternative to the traditional TPC for stabilization of long bone fractures in adult horses.     

Gene constitution of South-East Asian native chickens, commercial chickens and jungle fowl using polymorphisms of four calpain genes

The gene constitution of polymorphisms of the four calpain genes (µ‐calpain, m‐calpain, p94, and µ/m‐calpain) were analyzed in South‐East Asian native chickens, White Leghorn and Broiler commercial chickens, and Red and Green jungle fowl. Polymorphisms were detected at all loci in chickens and Red jungle fowl, but only for CAPN1 (µ‐calpain gene) in Green jungle fowl. CAPN2 and CAPN1.5 are linked on chicken chromosome 3, and the genotype for these loci were treated as haplotype. Some combinations of calpain loci were tested using principal component analysis, and the best combination (CAPN1, CAPN3, and CAPN1.5) was determined. The proportion of polymorphic loci (P_poly) and heterozygosity (H?) were 1.00 and 0.316–0.465 in domestic chickens and red jungle fowl, and 0.33 and 0.137 in Green jungle fowl, respectively. G_ST values suggested that the degree of subdivision among native chickens was relatively low except for Thailand, which was highest. Pair‐wise F_ST testing, dendrogram and principal component analysis from the results of calpain loci showed that the four South‐East Asian native and commercial chicken populations were close genetically.     

Presence and Distribution of Urocortin and its Receptors in the Epididymis of Alpaca (Vicugna pacos)

Urocortin 1 (UCN) is a 40‐amino acid peptide belonging to the corticotrophin‐releasing hormone (CRH) family. The biological effects of this peptide are modulated by binding two G‐coupled receptors named CRH receptor 1 (CRHR1) and CRH receptor 2 (CRHR2). CRHR2 has high affinity for UCN. The aim of the present study was to investigate the presence and distribution of UCN, CRHR1 and CRHR2 in the epididymis of the South America camelid Alpaca (Vicugna pacos) by Western blotting analysis and immunohistochemistry. Tissue extracts of the organ reacted with the anti‐UCN, anti‐CRHR1 and anti‐CRHR2 antibodies, recognizing in all the cases a single specific protein band. UCN‐ and CRHR2‐immunoreactivities (IRs) were found in the cytoplasm of the principal cells (PCs) of the caput epididymis. A prevalent supranuclear localization of granular‐shaped positive material was observed. CRHR1‐IR was observed in the fibromuscular stromal cells encircling the tubules and in the smooth musculature of the blood vessels throughout the three epididymal segments. In addition, in the cauda, CRHR1‐IR was observed in some apical epithelial cells (ACs) which were morphologically similar to apical mitochondria‐rich cells (AMRCs). These results suggest that UCN, CRHR1 and CRHR2 are expressed in the alpaca epididymis and that CRH‐related peptides might play multiple roles in maturation and storage of spermatozoa.     

Short‐term ingestion of deoxynivalenol in naturally contaminated feed alters piglet performance and gut hormone secretion

The mycotoxin deoxynivalenol (DON) generally exists in cereals and affects human and animal health. The aim of this study is to analyze the impacts of DON in naturally contaminated feed on piglet growth performance and intestinal hormone secretion in the short term. We randomly divided 5‐week‐old piglets into four groups: Control, DON 1,000, DON 2,000 and DON 3,000 groups. Piglets received a feed naturally contaminated with DON (approximately 400, 1,000, 2,000 or 3,000 μg/kg) for 21 days. Body weight showed no significant difference following exposure to DON. The balance of anti‐oxidation and oxidation was disrupted by DON after 21 days. The concentration of tumor necrosis factor‐alpha (TNF‐α) and cyclooxgenase‐2 (COX‐2) significantly increased (p < .001) in all DON‐treated groups. Gut anorexigenic hormone secretion of peptide YY (PYY) and cholecystokinin (CCK) had a time‐ and dose‐dependent relationship with DON exposure; however, there was no effect on orexigenic hormone ghrelin secretion. Changes of histomorphology in the jejunum were observed in DON‐treated groups, including villi flattening and fusion, and apical necrosis of villi. These results indicated that DON could suppress piglet growth performance and alter gut hormone secretion in the short term.     

The In Vitro Secretion of Acetylcholinesterase by Adult Stages of Heligmosomoides polygyrus: the Effects of Broad‐Spectrum Anthelmintics

The secretion of acetylcholinesterase (AChE) by female and male Heligmosomoides polygyrus was studied in different in vitro culture media. AChE secretion was increased in the presence of fetal calf serum or bovine serum albumin (BSA). In the absence of crowding effects, specific AChE activity in excretion/secretion products was higher for male (2.41 ± 0.07 µmol min^?1 l^?1 mg^?1) than for female (0.56 ± 0.04 µmol min^?1 mg^?1) worms but on a per nematode basis both sexes showed comparable rates of secretion. Acetylthiocholine iodide was the favoured substrate of the enzyme. When the nematodes were incubated in vitro with albendazole (ABZ), ricobendazole (RBZ), mebendazole (MBZ), levamisole (LVM), morantel (MRT) or ivermectin (IVM), at concentrations from 1 mM to 10 nM, in RPMI medium for 2 or 6 h and then transferred to a drug‐free medium (RPMI medium supplemented with 0.5 % BSA) for 24 h or continuously exposed to the drugs in supplement‐free medium (24 h), the concentration‐ and time‐dependent inhibitory effects on AChE secretion were observed. The continued exposure to the drugs for all incubation periods (with a single exception for LVM 1 mM) produced the highest levels of inhibition. Under these conditions, the concentrations inhibiting the secretion of AChE by 50 % (IC₅₀) relative to drug‐free controls were estimated. The IC₅₀ values ranged from 0.012 µM (IVM) to 2.96 µM (MRT). The potential of this bioassay for the selective primary evaluation of new compounds with broad‐spectrum anti‐nematodal activity is discussed.