Effects of capsicum oleoresin, garlic botanical, and turmeric oleoresin on gene expression profile of ileal mucosa in weaned pigs.

This study was conducted to characterize the effects of feeding 3 plant extracts on gene expression in ileal mucosa of weaned pigs. Weaned pigs (n = 32, 6.3 ± 0.2 kg BW, and 21 d old) were housed in individual pens for 9 d and fed 4 different diets: a nursery basal diet as control diet, basal diet supplemented with 10 mg/kg of capsicum oleoresin, garlic botanical, or turmeric oleoresin. Results reported elsewhere showed that the plant extracts reduced diarrhea and increased growth rate of weaning pigs. Total RNA (4 pigs/treatment) was extracted from ileal mucosa of pigs at d 9. Double-stranded cDNA was amplified, labeled, and further hybridized to the microarray. Microarray data were analyzed in R using packages from the Bioconductor project. Differential gene expression was tested by fitting a mixed linear model equivalent to ANOVA using the limma package. Bioinformatics analysis was conducted by DAVID Bioinformatics Resources. Three pairwise comparisons were used to compare each plant extract diet with the control diet. Quantitative real time PCR was applied to verify the mRNA expression detected by microarray. Compared with the control diet, feeding capsicum oleoresin altered (P < 0.05) the expression of 490 genes (280 up, 210 down), and feeding garlic botanical altered (P < 0.05) the expression of 64 genes (33 up, 31 down), while feeding turmeric oleoresin altered (P < 0.05) the expression of 327 genes (232 up, 95 down). Compared with the control diet, feeding capsicum oleoresin and turmeric oleoresin increased [Expression Analysis Systematic Explorer (EASE) < 0.05] the expression of genes related to integrity of membranes and tight junctions, indicating enhanced gut mucosa health, but decreased (EASE < 0.05) the cell cycle pathway. Feeding each of the 3 plant extracts enhanced (EASE < 0.05) the expression of genes associated with immune responses, indicating that feeding these plant extracts may stimulate the immune responses of pigs in the normal conditions. In conclusion, plant extracts regulated the expression of genes in ileal mucosa of pigs, perhaps providing benefits by enhancing the gut mucosa health and stimulating the immune system.

Dietary plant extracts modulate gene expression profiles in ileal mucosa of weaned pigs after an Escherichia coli infection.

This study was conducted to characterize the effects of infection with a pathogenic F-18 Escherichia coli and 3 different plant extracts on gene expression of ileal mucosa in weaned pigs. Weaned pigs (total = 64, 6.3 ± 0.2 kg BW, and 21-d old) were housed in individual pens for 15 d, 4 d before and 11 d after the first inoculation (d 0). Treatments were in a 2 × 4 factorial arrangement: with or without an F-18 E. coli challenge and 4 diets (a nursery basal, control diet [CON], 10 ppm of capsicum oleoresin [CAP], garlic botanical [GAR], or turmeric oleoresin [TUR]). Results reported elsewhere showed that the plant extracts reduced diarrhea in challenged pigs. Total RNA (4 pigs/treatment) was extracted from ileal mucosa of pigs at d 5 post inoculation. Double-stranded cDNA was amplified, labeled, and further hybridized to the microarray, and data were analyzed in R. Differential gene expression was tested by fitting a mixed linear model in a 2 × 4 factorial ANOVA. Bioinformatics analysis was conducted by DAVID Bioinformatics Resources 6.7 (DAVID; National Institute of Allergy and Infectious Diseases [NIAID, NIH], http://david.abcc.ncifcrf.gov). The E. coli infection altered (P < 0.05) the expression of 240 genes in pigs fed the CON (148 up- and 92 down-regulated). Compared with the infected CON, feeding CAP, GAR, or TUR altered (P < 0.05) the expression of 52 genes (18 up, 34 down), 117 genes (34 up- and 83 down-regulated), or 84 genes (16 up- and 68 down-regulated), respectively, often counteracting the effects of E. coli. The E. coli infection up-regulated (P < 0.05) the expression of genes related to the activation of immune response and complement and coagulation cascades, but down-regulated (P < 0.05) the expression of genes involved in protein synthesis and accumulation. Compared with the CON, feeding CAP and GAR increased (P < 0.05) the expression of genes related to integrity of membranes in infected pigs, indicating enhanced gut mucosa health. Moreover, feeding all 3 plant extracts reduced (P < 0.05) the expression of genes associated with antigen presentation or other biological processes of immune responses, indicating they attenuated overstimulation of immune responses caused by E. coli. These findings may explain why diarrhea was reduced and clinical immune responses were ameliorated in infected pigs fed plant extracts. In conclusion, plant extracts altered the expression of genes in ileal mucosa of E. coli-infected pigs, perhaps leading to the reduction in diarrhea reported previously.

Dietary plant extracts alleviate diarrhea and alter immune responses of weaned pigs experimentally infected with a pathogenic Escherichia coli.

A study was conducted to evaluate the effects of 3 different plant extracts on diarrhea, immune response, intestinal morphology, and growth performance of weaned pigs experimentally infected with a pathogenic F-18 Escherichia coli (E. coli). Sixty-four weaned pigs (6.3±0.2 kg BW, and 21 d old) were housed in individual pens in disease containment chambers for 15 d: 4 d before and 11 d after the first inoculation (d 0). Treatments were in a 2×4 factorial arrangement: with or without an F-18 E. coli challenge (toxins: heat-labile toxin, heat-stable toxin b, and Shiga-like toxin 2; 10(10) cfu/3 mL oral dose; daily for 3 d from d 0) and 4 diets [a nursery basal diet (CON) or 10 ppm of capsicum oleoresin, garlic botanical, or turmeric oleoresin]. The growth performance was measured on d 0 to 5, 5 to 11, and 0 to 11. Diarrhea score (1, normal, to 5, watery diarrhea) was recorded for each pig daily. Frequency of diarrhea was the percentage of pig days with a diarrhea score of 3 or greater. Blood was collected on d 0, 5, and 11 to measure total and differential white blood cell counts and serum tumor necrosis factor (TNF)-α, IL-10, transforming growth factor (TGF)-ß, C-reactive protein, and haptoglobin. On d 5 and 11, half of the pigs were euthanized to measure villi height and crypt depth of the small intestine and macrophage and neutrophil number in the ileum. The E. coli infection increased (P<0.05) diarrhea score, frequency of diarrhea, white blood cell counts, serum TNF-α and haptoglobin, and ileal macrophages and neutrophils but reduced (P<0.05) villi height and the ratio of villi height to crypt depth of the small intestine on d 5. In the challenged group, feeding plant extracts reduced (P<0.05) average diarrhea score from d 0 to 2 and d 6 to 11 and frequency of diarrhea and decreased (P<0.05) TNF-α and haptoglobin on d 5, white blood cell counts and neutrophils on d 11, and ileal macrophages and neutrophils on d 5. Feeding plant extracts increased (P<0.05) ileal villi height on d 5 but did not affect growth performance compared with the CON. In the sham group, feeding plant extract also reduced (P<0.05) diarrhea score, frequency of diarrhea, and ileal macrophages compared with the CON. In conclusion, the 3 plant extracts tested reduced diarrhea and inflammation caused by E. coli infection, which may be beneficial to pig health.

Zebrafish (Danio rerio) as a screen for attenuation of Lancefield group C streptococci and a model for streptococcal pathogenesis.

Group C streptococci are highly contagious pyogenic bacteria responsible for respiratory tract, lymph node, urogenital tract, and wound infections. Wild-type strains of Streptococcus equi ssp equi (S. equi) and Streptococcus equi ssp zooepidemicus (S. zoo) as well as a commercially available modified live vaccine strain of S. equi were evaluated for virulence in zebrafish. Survival times, histologic lesions, and relative gene expression were compared among groups. Based on the intramuscular route of infection, significantly shorter survival times were observed in fish infected with wild-type strain when compared to modified live vaccine and S. zoo strains. Histologically, S. zoo-infected fish demonstrated a marked increase in inflammatory infiltrates (predominantly macrophages) at the site of infection, as well as increased cellularity in the spleen and renal interstitium. In contrast, minimal cellular immune response was observed in S. equi-injected fish with local tissue necrosis and edema predominating. Based on whole comparative genomic hybridization, increased transcription of positive acute-phase proteins, coagulation factors, and antimicrobial peptides were observed in S. equi-injected fish relative to S. zoo-injected fish, while mediators of cellular inflammation, including CXC chemokines and granulin, were upregulated in S. zoo-injected fish relative to S. equi-injected fish. In a screen of 11 clinical isolates, S. equi strains with a single nucleotide deletion in the upstream region of szp, a known virulence factor of streptococci, were found to be significantly attenuated in zebrafish. These collective findings underscore the value of the zebrafish as a model of streptococcal pathogenesis.

Dietary clays alleviate diarrhea of weaned pigs.

Two experiments were conducted to determine whether 3 different clays in the nursery diet reduce diarrhea of weaned pigs experimentally infected with a pathogenic Escherichia coli. Weaned pigs (21 d old) were housed in individual pens of disease containment chambers for 16 d [4 d before and 12 d after the first challenge (d 0)]. The treatments were in a factorial arrangement: 1) with or without an E. coli challenge (F-18 E. coli strain; heat-labile, heat-stable, and Shiga-like toxins; 10(10) cfu/3 mL oral dose daily for 3 d from d 0) and 2) dietary treatments. The ADG, ADFI, and G:F were measured for each interval (d 0 to 6, 6 to 12, and 0 to 12). Diarrhea score (DS; 1 = normal; 5 = watery diarrhea) was recorded for each pig daily. Feces were collected on d 0, 3, 6, 9, and 12 and plated on blood agar to differentiate ß-hemolytic coliforms (HC) from total coliforms (TC) and on MacConkey agar to verify E. coli. Their populations on blood agar were assessed visually using a score (0 = no growth; 8 = very heavy bacterial growth) and expressed as a ratio of HC to TC scores (RHT). Blood was collected on d 0, 6, and 12 to measure total and differential white blood cell (WBC) counts, packed cell volume (PCV), and total protein (TP). In Exp. 1 (8 treatments; 6 replicates), 48 pigs (6.9 ± 1.0 kg of BW) and 4 diets [a nursery control diet (CON), CON + 0.3% smectite (SM), CON + 0.6% SM, and CON until d 0 and then CON + 0.3% SM] were used. The SM treatments did not affect growth rate of the pigs for the overall period. In the E. coli challenged group, the SM treatments reduced DS for the overall period (1.77 vs. 2.01; P < 0.05) and RHT on d 6 (0.60 vs. 0.87; P < 0.05) and d 9 (0.14 vs. 0.28; P = 0.083), and altered differential WBC on d 6 (neutrophils, 48 vs. 39%, P = 0.092; lymphocytes, 49 vs. 58%, P = 0.082) compared with the CON treatment. In Exp. 2 (16 treatments; 8 replicates), 128 pigs (6.7 ± 0.8 kg of BW) and 8 diets [CON and 7 clay treatments (CON + 0.3% SM, kaolinite, and zeolite individually and all possible combinations to total 0.3% of the diet)] were used. The clay treatments did not affect growth rate of the pigs. In the E. coli challenged group, the clay treatments reduced DS for the overall period (1.63 vs. 3.00; P < 0.05), RHT on d 9 (0.32 vs. 0.76; P < 0.05) and d 12 (0.13 vs. 0.39; P = 0.094), and total WBC on d 6 (15.2 vs. 17.7 × 10(3)/µL; P = 0.069) compared with the control treatment. In conclusion, dietary clays alleviated diarrhea of weaned pigs.

Oral lavage with polyethylene glycol reduces microbial colonization in the gastrointestinal tract of broilers.

Oral lavage of 7-wk-old broiler chickens with polyethylene glycol (PEG) was conducted to induce rapid evacuation of the gastro-intestinal tract (GIT) before processing. Three groups of birds were fed ad libitum (FF), subjected to feed withdrawal 12 h before processing (FW), or received 115 mL of PEG by oral lavage 3 h before processing (PEG(L)). The crop, a section of small intestine, and the right cecum were aseptically collected for determination of contents and Gram-negative bacilli (GNB) counts. The PEG(L) effectively cleansed the upper GIT and intestine of treated birds within 3 h before processing. However, the ceca were not completely empty for all the birds subjected to PEG(L). The PEG(L) group had a significantly lower crop and cecal GNB concentrations than the FW group. Birds on FW and PEG(L) treatments had significantly lower GNB concentrations in the small intestine than FF birds. Polyethylene glycol resulted in complete evacuation of the upper GIT, as well as the intestine, within 3 h of use, and significantly reduced GNB concentrations.

Identification of virulence attributes of gastrointestinal Escherichia coli isolates of veterinary significance.

The pathogenic strains of Escherichia coli recovered from the intestinal tract of animals fall into categories called enterotoxigenic, enteropathogenic, enterohemorrhagic and necrotoxigenic. The other two categories, enteroinvasive and enteroaggregative, have not been reported in animals. The pathogenicity of these strains is determined by the presence of certain genes that encode adhesins and toxins, are generally organized in large blocks in chromosomes, large plasmids or phages, and are often transmitted horizontally between strains. In this review, we summarize current knowledge of the virulence attributes that determine the pathogenic potential of E. coli strains and the methods available to assess the virulence of the strains. We also discuss the clinical symptoms, the gross and histological lesions, and the molecular diagnostic methods our laboratories have implemented for detecting pathogenic strains of E. coli that are isolated from the gastrointestinal tract of animals.

Cytotoxic activity of coagulase-negative staphylococci in bovine mastitis.

Secreted toxins play important roles in the pathogenesis of bacterial infections. In this study, we examined the presence of secreted cytotoxic factors of coagulase-negative staphylococci (CoNS) from bovine clinical and subclinical mastitis. A 34- to 36-kDa protein with cell-rounding cytotoxic activity was found in many CoNS strains, especially in Staphylococcus chromogenes strains. The protein caused cell detachment and cell rounding in several cell lines, including HEp-2, Int 407, CHO-K1, and Y-1 cells. Native protein recovered from nondenatured polyacrylamide gel electrophoresis showed both cytotoxic activity and casein hydrolysis activity. The purified protein had a pH optimal at 7.2 to 7.5 and a pI of 5.1 and was heat labile. The proteolytic activity could be inhibited by zinc and metal specific inhibitors such as 1, 10-phenanthroline and EDTA, indicating that it is a metalloprotease. Protein mass analysis and peptide sequencing indicated that the protein is a novel metalloprotease. Different bacterial strains expressed variable levels of 34- to 36-kDa protease, which may provide an indication of strain virulence.

Reservoir competence of Carcinops pumilio for Salmonella enteritidis (Eubacteriales: Enterobacteriaceae).

The histerid beetle Carcinops pumilio (Erichson) occurs naturally in poultry house manure and is an important predator of house fly eggs and larvae. Because efforts to commercially produce C. pumilio have been unsuccessful, one fly control strategy under consideration is the direct transport of adult C. pumilio between poultry houses to facilitate their establishment. However, we demonstrate that C. pumilio is a competent reservoir of Salmonella enteritidis (Gaertner). Adult C. pumilio exposed to S. enteritidis-inoculated house fly eggs harbored the bacterium externally and internally for up to 4 and 13 d, respectively, and feces were culture-positive for S. enteritidis for at least 14 d. This suggests that C. pumilio can be a reservoir of S. enteritidis; therefore its movement between poultry facilities should be carefully considered.

Unique Salmonella choleraesuis surface protein affecting invasiveness. Possible inv related sequence.

TnphoA mutagenesis of a Salmonella choleraesuis isolate recovered from septicemic infection of feeder pigs resulted in 56 PhoA+ KnR StrR mutants. Thirty-five mutants exhibited reduced levels of invasion in the Hep-2 cell model and were examined by SDS-PAGE Western Blot analysis using an anti-alkaline phosphatase antibody to visualize the insertion gene products. A mutant which produced a gene fusion product of 95 kDa and exhibited > 90% reduction in invasion was subcloned. A 10 Kb BamHI fragment of the chromosome containing the phoA insert was detected by hybridization and cloned into a pGEM vector. The resulting 1657 base sequence contained a 1104 bp ORF with two short regions of homology with S. typhimurium invF and invG. one region of homology with lcrD of Yersinia pseudotuberculosis but contained largely unique sequences not contained in Gene Bank. The full length sequence was not obtained as there was no stop codon detected. The % G+C was 44%, considerably lower than that of the Salmonella chromosome, but compatible with the proposed Yersinia origin of the inv genes. The NH2 387 a.a. sequence includes 5 transmembrane regions, resembling the model derived from the hydrophobicity plot of S. typhimurium InvA.

Epidemiologic evaluation of encephalitic listeriosis in goats.

OBJECTIVE: To evaluate host and environmental factors associated with the development of encephalitic listeriosis in goats. DESIGN: Retrospective analysis of diagnostic laboratory records and survey of veterinarians and goat producers. SAMPLE POPULATION: 355 goat herds accessible through laboratory records; 38 veterinarians who treated goats and 76 goat producers. PROCEDURE: Data regarding breed and use for goats affected with encephalitic listeriosis were obtained from surveys and case follow-up information. Listeria monocytogenes isolates from the brains of 7 affected goats were serotyped and subjected to DNA restriction analysis. RESULTS: Odds ratio for the development of encephalitis listeriosis in Angora (mohair-producing) goats was 22.9 by use of diagnostic laboratory records. Survey also revealed a high prevalence in herds of Angora and other breeds that subsisted on woody browse, although Angora goats feeding predominantly on hay or pasture were not affected. Listeria monocytogenes isolates from 4 Angora goats in 3 herds differed in DNA restriction patterns, although the pattern was identical in 3 other goats from another herd. CLINICAL IMPLICATIONS: Encephalitic listeriosis can be observed in all goat breeds, but a lifestyle of heavy browse consumption seems important to the development of disease in some herds.

Evaluation of laboratory tests for confirming the diagnosis of encephalitic listeriosis in ruminants.

Retrospective analysis of 93 bovine, ovine, and caprine cases diagnosed as listerial encephalitis revealed positive bacterial isolations in only 63% of 59 cases in which bacterial culture was attempted. Only 42% of 41 attempted bovine brain cultures were successful, compared with 67% from 6 sheep brains and 92% from 12 goat brains. Gram stains and Listeria-specific immunohistochemistry were evaluated as tools for verifying the presence of bacteria or listerial antigens in 38 animals. Sixteen of 17 animals in this group with positive bacterial isolations were immunochemically positive for listerial antigens (including 5/6 cattle), but Gram stains detected only 9/17 positive animals (including 1/6 cattle). Antigen was also detected in 15 of 21 animals (including 5/9 cattle) with unsuccessful or unattempted bacterial isolations. Of all 38 animals, the histologic diagnosis could be verified in 82% by immunohistochemistry, compared to 47% verified by Gram stains. Immunohistochemical testing was especially beneficial in locating antigen in lesions with few bacteria or bacterial antigens and is a rapid method of confirming the diagnosis of encephalitic listeriosis where inappropriate material is submitted for bacterial isolation or in culture-negative cases.

Use of a Salmonella typhimurium-derived virulence probe in the detection of Salmonella sp. and in the characterization of S. cholerae-suis virulence plasmids.

A genetic probe encoding a virulence gene from Salmonella typhimurium was useful in the detection of Salmonella from feces during an outbreak of salmonellosis at a local dairy. A 3.2-kb BamHI restriction endonuclease fragment of the S. typhimurium virulence plasmid, pStSR100, has been useful as a DNA probe for both detection of Salmonella sp. and characterization of virulence plasmids from numerous field isolates. This virA probe hybridizes to a highly conserved gene carried on the large virulence plasmids of invasive Salmonella isolates. Colony blots prepared from feces directly plated onto MaConkey's agar failed to detect low numbers of Salmonella sp. However, hybridization of the VirA probe to vacuum blots or colony blots prepared from feces in tetrathionate enrichment broth incubated for 16 hours at 37 C was effective for detecting Salmonella sp. and resulted in an 85.9% correlation with culture results. The probe also demonstrated the highly conserved nature (96%) of the virulence gene among S. cholerae-suis isolate plasmids detected using Southern blot analysis.

High technology diagnostics: detection of enterotoxigenic Escherichia coli, using DNA probes.

A relatively new technique termed colony blot hybridization appears to be a reliable replacement for the ligated porcine gut loop assay previously used to detect stable toxin-B producing Escherichia coli. The highly reproducible blot assay enables screening of large numbers of isolants for the presence of E coli stable-B toxin genes, thus avoiding variations in phenotypic expression and poor repeatability inherent in the in vivo assays. Availability of this diagnostic test can aid practitioners in identifying enterotoxigenic E coli related disease in swine and in determining the benefits of vaccination programs.