Comparative efficacy of postbiotic, probiotic, and antibiotic against necrotic enteritis in broiler chickens.

Prevention of necrotic enteritis (NE), caused by Clostridium perfringens (C. perfringens), is one of the most important goals to improve the profitability of broiler chickens. This work aimed to compare the efficacy of 2 antibiotic alternatives including a postbiotic (dry feed additive and aqueous nonviable Lactobacillus (L.) species fermentation) and a probiotic (dry feed additive and aqueous Bacillus (B.) subtilis and B. lischeniformis mixture) with an antibiotic (amoxicillin in water) against NE. Four hundred, day-old broiler chicks were divided into 8 equal groups (Gs), n = 50 each (5 replicates; 10 each). Chickens of G1 (postbiotic dry-feed additive), G2 (postbiotic and antibiotic in drinking water), G3 (postbiotic dry and aqueous), G4 (probiotic dry-feed additive), G5 (probiotic and antibiotic in drinking water), G6 (probiotic dry and aqueous), and G7 (nontreated) were orally inoculated with a toxigenic C. perfringens type A on the d 19 to 21 of age and predisposed with 3X coccidial vaccine for induction of NE. However, chickens of G8 were kept nontreated or challenged. The severity of NE signs was markedly decreased in G3 in comparison with other challenged treatment groups, and the mortality rates were 22%, 10%, 16%, 22%, 12%, 20%, and 36% in Gs 1, 2, 3, 4, 5, 6, and 7, respectively. The best significant (P ≤ 0.05) feed conversion ratio was detected in G3 (1.51), G6 (1.54), and G2 and G8 (1.61). In addition, the European production efficiency factor was significantly (P ≤ 0.05) improved in G3 (279.33) and G2 (266.67), but it was decreased in G7 (177.33) when compared with G8 (339.33). An improvement in intestinal and hepatic pathology and liver function tests, as well as a significant (P ≤ 0.05) decrease in bacterial counts were observed in Gs 2, 5, 3, 6, 1, and 4, respectively in comparison with G7. Immunologically, the highest significant (P ≤ 0.05) hemagglutination inhibition antibody titers for Newcastle disease virus vaccine were in Gs 1 and 3 (6.4 log2). In conclusion, the combined feed and water postbiotic treatment demonstrated promising results in ameliorating the severity of NE and improving the hepatic and the immune status of broiler chickens when compared with the commonly used probiotic and antibiotic.

Altered fecal microbiota, IgA, and fermentative end-products in adult dogs fed prebiotics and a nonviable Lactobacillus acidophilus.

A study investigating the use of a nonviable Lactobacillus acidophilus (NVL: Culbac; TransAgra, Storm Lake, IA) and a mixed prebiotic (MP) blend (beet pulp, fructooligosaccharide (FOS), mannanoligosaccharide (MOS), inulin, and kelp) was done to evaluate changes in fecal microbiota, fermentative end products, and gut immune health in healthy female and male adult Beagle dogs (n = 24; 5.74 ± 2.18 yr; 9.30 ± 1.32 kg). The study protocol was first approved by the facility's Institutional Animal Care and Use Committee (Summit Ridge Farms; Susquehanna, PA) and followed throughout. Each of four test diets (control, NVL, MP, and MP + NVL [formulated to crude protein 25%, crude fat 14%, crude fiber 10% as-fed]) was fed once daily to maintain body weight for 21 d in a randomized-crossover design (four treatment periods and four washout periods). Fecal samples were collected on days 0 and 21 only for immunoglobulin A (IgA) and microbiota evaluation (16S rRNA V4 region and qPCR for Escherichia coli and Bifidobacterium), and fecal fermentative end-products and fecal pH were assessed only on day 21. Over the test periods, apparent total tract nutrient digestibility and stool quality were assessed. Data were analyzed by ANOVA (SAS v9.4, Cary, NC) or Kruskal-Wallis for between-diet effects, and paired t-test or Wilcoxon for time effects. Statistical significance was set at P ≤ 0.05. Apparent total tract nutrient digestibility revealed feeding MP-containing diets resulted in lower (P < 0.05) crude protein and fat digestibility vs. control and NVL diets. When dogs were fed MP, they had lower (P < 0.05) fecal pH compared with control and NVL diets, whereas fecal pH was lower in (P < 0.05) MP + NVL- vs. NVL-fed dogs. Fecal E. coli was (P < 0.05) lower at day 21 vs. day 0 when dogs were fed MP. Fecal Fusobacterium spp. was lower (P < 0.05) in both MP diets vs. control. Fecal Lactobacillus spp. increased (P < 0.05) from baseline with MP. Both diets with MP elicited greater (P < 0.05) fecal acetate and propionate concentration vs. control diet. At day 21, fecal IgA was greater (P < 0.05) in MP and MP + NVL compared with NVL diet. Only when dogs were fed MP did they have increased (P < 0.05) fecal IgA from day 21 vs. day 0. The MP + NVL diet decreased (P < 0.05) fecal isovalerate, isobutyrate, phenol, and indole vs. control. Overall, the MP elicited the most changes on microbiota, fermentative end-products, and IgA. Further investigation into NVL's gut health benefits is warranted.

In vitro activity and rodent efficacy of clinafloxacin for bovine and swine respiratory disease.

Clinafloxacin is a broad-spectrum fluoroquinolone that was originally developed and subsequently abandoned in the late 1990s as a human health antibiotic for respiratory diseases. The purpose of this study was to investigate the activity of clinafloxacin as a possible treatment for respiratory disease in cattle and pigs. Minimum inhibitory concentration (MIC) values were determined using Clinical and Laboratory Standards Institute recommended procedures with recent strains from the Zoetis culture collection. Rodent efficacy was determined in CD-1 mice infected systemically or intranasally with bovine Mannheimia haemolytica or Pasteurella multocida, or swine Actinobacillus pleuropneumoniae, and administered clinafloxacin for determination of ED50 (efficacious dose-50%) values. The MIC90 values for clinafloxacin against bovine P. multocida, M. haemolytica, Histophilus somni, and M. bovis were 0.125, 0.5, 0.125, and 1 µg/ml, respectively, and the MIC90 values against swine P. multocida, A. pleuropneumoniae, S. suis, and M. hyopneumoniae were í0.03, í0.03, 0.125, and í0.008 µg/ml, respectively. Efficacy in mouse models showed average ED50 values of 0.019 mg/kg/dose in the bovine M. haemolytica systemic infection model, 0.55 mg/kg in the bovine P. multocida intranasal lung challenge model, 0.08 mg/kg/dose in the bovine P. multocida systemic infection model, and 0.7 mg/kg/dose in the swine A. pleuropneumoniae systemic infection model. Clinafloxacin shows good in vitro activity and efficacy in mouse models and may be a novel treatment alternative for the treatment of respiratory disease in cattle and pigs.

Glucocorticoids stimulate ENaC upregulation in bovine mammary epithelium.

Mammary epithelia produce an isotonic, low-Na(+) fluid that is rich in nutrients. Mechanisms that account for the low electrolyte concentration have not been elucidated, although amiloride-sensitive ion transport has been reported in some situations. We hypothesized that corticosteroid exposure modulates epithelial Na(+) channel (ENaC) expression and/or activity in bovine mammary epithelial cells. BME-UV cells were grown to confluent monolayers on permeable supports with a standard basolateral medium and apical medium of low-electrolyte, high-lactose composition that resembles the ionic composition of milk. Ion transport was assessed in modified Ussing flux chambers. Exposure to glucocorticoids (dexamethasone, cortisol, or prednisolone), but not aldosterone, increased short-circuit current (I(sc)), a sensitive measure of net ion transport, whereas apical exposure to amiloride or benzamil reduced corticosteroid-induced I(sc) close to basal levels. Quantitative RT-PCR indicated a glucocorticoid-induced increase in mRNA for beta- and gamma-ENaC, whereas alpha-ENaC mRNA expression was only mildly affected. Exposure to mifepristone (a glucocorticoid receptor antagonist), but not spironolactone (a mineralocorticoid receptor antagonist), precluded both the corticosteroid-induced elevation in amiloride-sensitive I(sc) and the induced changes in beta- and gamma-ENaC mRNA. We conclude that Na(+) movement across mammary epithelia is modulated by corticosteroids via a glucocorticoid receptor-mediated mechanism that regulates the expression of the beta- and gamma-subunits of ENaC. ENaC expression and activity could account for the low Na(+) concentration that is typical of milk.

Apical electrolyte concentration modulates barrier function and tight junction protein localization in bovine mammary epithelium.

In vitro mammary epithelial cell models typically fail to form a consistently tight barrier that can effectively separate blood from milk. Our hypothesis was that mammary epithelial barrier function would be affected by changes in luminal ion concentration and inflammatory cytokines. Bovine mammary epithelial (BME-UV cell line) cells were grown to confluence on permeable supports with a standard basolateral medium and either high-electrolyte (H-elec) or low-electrolyte (L-elec) apical medium for 14 days. Apical media were changed to/from H-elec medium at predetermined times prior to assay. Transepithelial electrical resistance (R(te)) was highest in monolayers continuously exposed to apical L-elec. A time-dependent decline in R(te) began within 24 h of H-elec medium exposure. Change from H-elec medium to L-elec medium time-dependently increased R(te). Permeation by FITC-conjugated dextran was elevated across monolayers exposed to H-elec, suggesting compromise of a paracellular pathway. Significant alteration in occludin distribution was evident, concomitant with the changes in R(te), although total occludin was unchanged. Neither substitution of Na(+) with N-methyl-d-glucosamine (NMDG(+)) nor pharmacological inhibition of transcellular Na(+) transport pathways abrogated the effects of apical H-elec medium on R(te). Tumor necrosis factor alpha, but not interleukin-1beta nor interleukin-6, in the apical compartment caused a significant decrease in R(te) within 8 h. These results indicate that mammary epithelium is a dynamic barrier whose cell-cell contacts are acutely modulated by cytokines and luminal electrolyte environment. Results not only demonstrate that BME-UV cells are a model system representative of mammary epithelium but also provide critical information that can be applied to other mammary model systems to improve their physiological relevance.

PVD9902, a porcine vas deferens epithelial cell line that exhibits neurotransmitter-stimulated anion secretion and expresses numerous HCO3(-) transporters.

Epithelial ion transport disorders, including cystic fibrosis, adversely affect male reproductive function by nonobstructive mechanisms and by obstruction of the distal duct. Continuous cell lines that could be used to define ion transport mechanisms in this tissue are not readily available. In the present study, porcine vas deferens epithelial cells were isolated by standard techniques, and the cells spontaneously immortalized to form a porcine vas deferens epithelial cell line that we have titled PVD9902. Cells were maintained in continuous culture for >4 yr and 200 passages in a typical growth medium. Frozen stocks were generated, and thawed cells exhibited growth characteristics indistinguishable from their nonfrozen counterparts. Molecular and immunocytochemical studies confirmed the origin and epithelial nature of these cells. When seeded on permeable supports, PVD9902 cells grew as electrically tight (>6,000 ohms x cm2), confluent monolayers that responded to forskolin with an increase in short-circuit current (I(sc); 8 +/- 1 microA/cm2) that required Cl-, HCO3(-), and Na+, and was partially sensitive to bumetanide. mRNA was expressed for a number of anion transporters, including CFTR, electrogenic Na+-HCO3(-) cotransporter 1b (NBCe1b), downregulated in adenoma, pendrin, and Cl-/formate exchanger. Both forskolin and isoproterenol caused an increase in cellular cAMP levels. In addition, PVD9902 cell monolayers responded to physiological (i.e., adenosine, norepinephrine) and pharmacological [i.e., 5'-(N-ethylcarboxamido)adenosine, isoproterenol] agonists with increases in I(sc). Unlike their freshly isolated counterparts, however, PVD9902 cells did not respond to glucocorticoid exposure with an increase in amiloride-sensitive I(sc). RT-PCR analysis revealed the presence of both glucocorticoid and mineralocorticoid receptor mRNA as well as mRNA for the alpha- and gamma-subunits of the epithelia Na+ channels (alpha- and gamma-ENaC), but not beta-ENaC. Nonetheless, PVD9902 cells recapitulated most observations in freshly isolated cells and thus represent a powerful new tool to characterize mechanisms that contribute to male reproductive function.

Functional and molecular evidence for Na(+)-HCO cotransporter in porcine vas deferens epithelia.

This study focused on the role of sodium-bicarbonate cotransporter (NBC1) in cAMP-stimulated ion transport in porcine vas deferens epithelium. Ion substitution experiments in modified Ussing chambers revealed that cAMP-mediated stimulation was dependent on the presence of Na(+), HCO, and Cl(-) for a full response. HCO-dependent current was unaffected by acetazolamide, bumetanide, or amiloride but was inhibited by basolateral 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Na(+)-driven, HCO-dependent, stilbene-inhibitable anion flux was observed across the basolateral membrane of selectively permeabilized monolayers. Results of radiotracer flux studies suggest a 4,4'-dinitrostilbene-2,2'-disulfonate-sensitive stoichiometry of 2 base equivalents per Na(+). Antibodies raised against rat kidney NBC epitopes (rkNBC; amino acids 338-391 and 928-1035) identified a single band of ~145 kDa. RT-PCR detected NBC1 message in porcine vas deferens epithelia. These results demonstrate that vas deferens epithelial cells possess the proteins necessary for the vectoral transport of HCO and that these mechanisms are maintained in primary culture. Taken together, the results indicate that vas deferens epithelia play an active role in male fertility and have implications for our understanding of the relationship between cystic fibrosis and congenital bilateral absence of the vas deferens.