Collagen adhesin protein and necrotic enteritis B-like toxin as biomarkers for early diagnosis of necrotic enteritis in commercial broiler chickens.

Mouse monoclonal antibodies (mAbs) reactive with Clostridium perfringens collagen adhesin protein (CNA) and necrotic enteritis B-like toxin (NetB) were developed. The best capture/detection mAb pairs for CNA and NetB were selected based on their affinity and specificity to develop sandwich enzyme-linked immunosorbent assays (ELISAs) to detect CNA and NetB proteins, respectively, in jejunal digesta samples from commercial broiler farms in the United States. Prior to the analysis of samples from commercial broiler flocks, the specificity and sensitivity of the CNA and NetB ELISAs were validated using sera, jejunal digesta, and fecal samples from chickens coinfected with Eimeria maxima and CNA+/NetB+C. perfringens in an animal model of necrotic enteritis (NE). Subsequently, a total of 251 field samples were collected from 74 commercial poultry farms. Among these, 18 samples were from 6 broiler farms that used certified organics (CO), and 155 samples were from 42 farms with nonantibiotics (NA). In jejunal digesta samples, CNA levels ranged from 0.02 to 0.59 ng/mL and NetB levels ranged from 0.09 to 1.91 ng/mL. CNA and NetB levels showed a positive correlation with each other (Pearson correlation coefficient r = 0.772, P < 0.001). CNA and NetB levels in jejunal digesta were significantly decreased in CO farms compared with those from NA farms (P < 0.001). In conclusion, these new C. perfringens antigen-specific sandwich ELISAs offer a sensitive and specific means to detect C. perfringens CNA and NetB proteins as biomarkers of early NE occurrence in field samples from commercial broiler chickens.

Bacteria and fungi in day-old turkeys vary among companies, collection periods, and breeder flocks.

Microbial colonization of the intestinal tract of commercial poultry is highly variable, likely due to the fact that poults and chicks are hatched and raised without exposure to adult birds and their microbiota. In industrial poultry production, it is hypothesized that most of the microbiota is obtained through horizontal transmission from the environment and very little by maternal transmission. The initial gut microbiota will therefore differ between flocks and companies based on environmental conditions at the hatchery. Day-old poults were collected from the hatchery of 2 companies at 3 different time points to monitor the initial colonizing microbiota by sequencing amplicons of marker genes for bacteria, lactic acid bacteria (LAB), fungi, and archaea. Bacterial colonizers were distinct by company (pseudo-F 38.7, P ≤ 0.05) with the predominant bacteria at Company A being clostridia, specifically Clostridium celatum group, C. paraputrificum, and C. tertium. Predominant bacteria at Company B were Enterobacteriaceae, belonging to 2 different groups, one that included Escherichia; Shigella and Salmonella and the other Klebsiella; Enterobacter; and others. The predominant LAB at both companies were Enterococcus faecalis and E. gallinarum, confirmed by sequencing the 16S ribosomal RNA (rRNA) gene of colonies picked from lactobacilli agar plate counts. The predominant fungi were Aspergillus niger and Saccharomyces cerevisiae, with Candida sake or Alterneria sp. in some samples of Company A. Archaeal sequences were detected only in a single poult from Company B. The initial gastrointestinal colonizers of poults vary across company and time, signifying a strong environmental effect on microbiota acquisition. There was an indication of maternal effects in certain breeder flocks from Company B. Further work is necessary to determine how this variability affects microbiota succession and impacts growth and production of the birds.

Short communication: Determination of Salmonella clustered regularly interspaced short palindromic repeats (CRISPR) diversity on dairy farms in Wisconsin and Minnesota.

Salmonella enterica ssp. enterica is a foodborne pathogen able to cause disease in both humans and animals. Diverse serovars of this pathogen exist, some of which are host specific, causing a range of clinical symptoms from asymptomatic infection through morbidity and mortality. According to a 2007 survey by the USDA National Animal Health Monitoring System, fecal shedding of Salmonella from healthy cows occurs on 39.7% of dairy farms in the United States. Certain serovars are frequently isolated from dairy farms and the majority of isolates from the National Animal Health Monitoring System study were represented by 5 serovars; however, genotypic diversity was not examined. The objective of this study was to determine the diversity of clustered regularly interspaced short palindromic repeats (CRISPR) loci in Salmonella collected from 8 dairy farms with a previous history of salmonellosis. None of the cows or calves sampled on 2 of the 8 dairy farms were shedding Salmonella, although Salmonella was detected in a cow bedding sample on 1 of these farms. Salmonella populations were discrete on each farm, according to CRISPR typing, with the exception of an Anatum var. 15+ type on farms 5 and 6 and the Montevideo type on farms 1 and 2. One to 4 distinct CRISPR genotypes were identified per farm. The CRISPR typing differed within serovars, as Montevideo, Anatum var. 15+, and Muenster serovars had no overlap of spacer content, even on the same farm, reflecting between- and within-serovar genetic diversity. The dynamic nature of Salmonella populations was shown in a farm that was sampled longitudinally over 13.5 mo. Changes in serovar from 3,19:-:z27 to Montevideo was observed between the first sampling time and 8 mo later, with concomitant change in CRISPR alleles. The results indicate that Salmonella strains present in smaller dairy herds (<500 head) are specific to that farm and new Salmonella strains may emerge over time.

Effect of supplementation with an electrolyte containing a Bacillus-based direct-fed microbial on immune development in dairy calves.

Immune characteristics in 65 calves were evaluated in response to a Bacillus-based direct-fed microbial (DFM) provided in electrolyte scour treatment. Blood samples were analyzed for cell surface markers and α(1)-acid glycoprotein (AGP) concentration. AGP increased in scouring calves given electrolyte containing Bacillus at day 7 post-placement compared to scouring calves administered electrolyte alone and non-scouring calves, enhancing the inflammatory response for pathogen clearance. The Bacillus promotes T cell subsets including greater proportions of activated, mature cells (CD8(-)CD25(+), CD8(-)CD45RO(+), CD8(-)TCR1(+)) in calves given electrolyte containing Bacillus than scouring calves administered electrolyte alone and non-scouring calves. Also, the Bacillus may be alleviating inflammation at day 3 post-placement as the proportion of monocytes and granulocytes lacking L-selectin (CD172a(+)CD62L(-)) was greater in scouring calves given electrolyte compared to the other groups. Electrolyte containing Bacillus administered at the onset of scours influences components of innate and adaptive immune development during and following the scouring event.

Multilocus sequence typing subtypes of poultry Clostridium perfringens isolates demonstrate disease niche partitioning.

Clostridium perfringens is a ubiquitous and versatile pathogenic bacterium and is implicated in the etiology of the poultry diseases necrotic enteritis (NE) and poultry gangrene (PG). In this study, multilocus sequence typing was used to investigate genotypic relationships among 139 C. perfringens isolates from 74 flocks. These isolates had multiple disease, host, and environmental origins. The results indicated a polymorphic yet highly clonal population, with 79.6% of all isolates partitioning into one of six clonal complexes or two dominant sequence types, ST-9 and ST-31. The most prolific clonal complex, CC-1, contained 27.3% of all isolates and was not clearly associated with one particular disease. The subtypes CC-4 and ST-31 were highly associated with NE and represented 9.4% and 7.2% of the total isolates, respectively. No PG-associated and NE-associated C. perfringens isolates shared the same sequence type or clonal complex. NE-associated subtypes were more clonal and appeared more evolutionarily divergent than PG-associated subtypes, which tended to cluster in the more ancestral lineages alongside isolates from asymptomatic chickens and turkeys. Toxin gene screening identified cpb2 throughout these isolates and correlated the presence of netB with NE pathology. Previous investigations into the genetic basis of C. perfringens pathogenicity have focused on toxins and other variable genetic elements. This study presents the first sequence-based comparison of C. perfringens isolates recovered in clinical cases of PG and NE and demonstrates that niche specialization is observable in the core genomes of poultry-associated C. perfringens isolates, a concept with both epidemiological and evolutionary significance.

Quantitative real-time PCR assay for Clostridium septicum in poultry gangrenous dermatitis associated samples.

Clostridium septicum is a spore-forming anaerobe frequently implicated in cases of gangrenous dermatitis (GD) and other spontaneously occurring myonecrotic infections of poultry. Although C. septicum is readily cultured from diseased tissues it can be difficult to enumerate due to its tendency to swarm over the surface of agar plates. In this study a quantitative real-time PCR assay was developed in order to more accurately measure the levels of C. septicum in healthy as well as GD associated poultry samples. The assay was specifically designed to target the C. septicum alpha toxin gene, csa, which is, to our knowledge, carried by all strains of C. septicum and has been shown to be essential for virulence. Genomic DNAs from a diverse collection of bacterial species, including closely related Clostridium chauvoei, Clostridium carnis, Clostridium tertium as well as several strains of Clostridium perfringens, all failed to produce a positive reaction. An approximate reproducible limit of detection in spiked extracts of at least 10(3) cfu/g of C. septicum was observed for a variety of different sample types. C. septicum levels in broiler chicken field samples estimated from the results of qPCR were statistically correlated to culture based enumerations obtained from those same tissues.

Effects of direct-fed microbials on growth performance, gut morphometry, and immune characteristics in broiler chickens.

This study was conducted to compare growth performance, gut morphometry, and parameters of local and systemic immunity in broiler chickens fed for 22 consecutive days with a diet supplemented with Bacillus spp. as direct-fed microbials (DFM), a commercial product incorporating 3 DFM, or a nonsupplemented diet. Direct-fed microbials did not significantly modify BW gain and most failed to affect serum antibody levels in response to immunization with a recombinant Eimeria protein. However, altered intestinal morphometric measurements were readily apparent in DFM-fed chickens as revealed by increased villus height and crypt depth compared with non-DFM-fed controls. In addition, serum levels of alpha-1-acid glycoprotein as an inflammatory marker were reduced in DFM-fed birds, whereas splenic lymphocyte proliferation, intestine intraepithelial lymphocyte subpopulations, and cytokine mRNA levels in intraepithelial lymphocytes were increased, decreased, or unchanged compared with controls depending on the DFM used. These results provide a rational scientific basis for future studies to investigate DFM as immunomodulating agents to enhance host protective immunity against enteric pathogens in broiler chickens.

Benefits of Supplementation of an Electrolyte Scour Treatment with a Bacillus-Based Direct-Fed Microbial for Calves.

A Bacillus-based direct-fed microbial (DFM), Omni-BosCB™, added to an electrolyte was evaluated as a therapy for scours. Fecal shedding of presumptive Clostridium perfringens at day 7 was reduced in scouring calves treated with electrolyte plus DFM compared to scouring calves treated with electrolyte alone. Total therapeutic treatment costs during the first 2 weeks were significantly reduced by supplementing the electrolyte with the DFM: $18.69 and $21.57 for electrolyte plus DFM and electrolyte treated calves, respectively. Electrolyte treated calves experienced more severe scours than electrolyte plus DFM treated calves as additional therapy with Lactated Ringer's Solution was only necessary for electrolyte treated calves. The DFM may have other ancillary benefits after supplementation has ended, as evidenced by decreased recurrence of a second scouring event. This is the first report demonstrating efficacy of a DFM used therapeutically for mitigating calf scours. These findings have implications as alternatives to chemical interventions for disease control.

Effects of feeding yeast and propionibacteria to dairy cows on milk yield and components, and reproduction*.

To determine the effect of supplemental feeding of Diamond V-XP yeast (XPY) alone or in combination with propionibacteria strain P169 on milk production, milk components, body weight, days to first and second ovulation, plasma insulin, and plasma and milk glucose, 31 primiparous and multiparous (MP) Holstein cows were fed one of three dietary treatments between 2 weeks prepartum to 30 weeks postpartum: (i) control (n = 10), fed a corn silage-based total mixed ration (TMR); (ii) XPY (n = 11), fed control TMR plus XPY (at 56 g/head/day); and (iii) P169+XPY (n = 10), received control TMR plus XPY plus P169 (at 6 x 10(11) cfu/head/day). After parturition, daily milk weights were recorded, and milk samples were collected twice weekly for milk component analyses. Daily uncorrected milk, solids-corrected milk, and 4% fat-corrected milk production for MP cows fed P169+XPY was 9-16% greater than control MP cows, but these increases were only evident during mid lactation (9-30 weeks). The percentage of milk fat was 8-18% greater in control than XPY and P169+XPY groups. Milk lactose percentage in MP cows fed P169+XPY was 3-5% greater than in control and XPY MP cows. Primiparous and MP cows fed P169+XPY had 28-32% greater milk glucose levels than control and XPY-fed cows. Diurnal plasma glucose concentration was not affected by diet in MP cows. Plasma insulin levels in MP cows fed P169+XPY were 30-34% greater than in other groups of MP cows. Milk glucose and plasma insulin responses to P169+XPY feeding suggest that P169+XPY might have enhanced gluconeogenesis and increased glucose uptake by the mammary gland in Holstein cows. Thus, a combined feed supplement of P169 and XPY may hold potential as a natural feed alternative to hormones and antibiotics to enhance lactational performance.

Effects of propionibacteria and yeast culture fed to steers on nutrient intake and site and extent of digestion.

The effects of feeding Propionibacterium strain P169 (P169), yeast culture (XPY), and their combination on nutrient intake, site and extent of digestion, and ruminal kinetics were evaluated in a completely randomized experimental design. Ruminally and duodenally cannulated Angus x Hereford steers (n = 12) were assigned to 1 of 4 treatments in each of 2 periods: 1) control, fed a sorghum silage-based total mixed ration; 2) P169, fed the control plus P169 (6 x 10(11) cfu/steer per d); 3) XPY, fed the control plus XPY (56 g/steer per d); and 4) P169 + XPY, fed the control plus P169 and XPY (at 6 x 10(11) cfu/steer per d and 56 g/steer per d, respectively). Each period lasted 21 d; d 1 to 15 were used for diet adaptation and d 16 to 21 were used for fecal, duodenal, ruminal, and blood sample collection. Steers were individually housed and fed. Feeding XPY tended to decrease intake of organic matter, acid detergent fiber, and N, and decreased intake of neutral detergent fiber. However, feeding XPY alone tended to increase total tract digestibility of organic matter, N, neutral detergent fiber, and acid detergent fiber. Ruminal digestibility, duodenal flow, microbial N synthesis, microbial efficiency, and fluid and particulate passage rates were not affected by dietary treatments. Feeding P169 tended to decrease molar proportion of acetate, increased molar proportion of propionate (by 9.7%), and tended to decrease acetate:propionate ratio compared with control steers. No other effects of XPY or P169 on ruminal fermentation were observed. Plasma glucose and insulin concentrations were not affected by dietary treatment. Our results suggest that feeding P169 alters ruminal metabolism toward increased propionate without affecting feed intake or ruminal kinetics, whereas feeding XPY alone tended to increase total tract digestibilities of nutrients.

Effects of feeding propionibacteria to dairy cows on milk yield, milk components, and reproduction.

Two weeks before parturition, 38 Holstein primiparous and multiparous cows were assigned to 1 of 3 treatment groups: control animals (n = 13) received regular total mixed rations (TMR), the low-dose group (n = 14) received the control TMR plus 6 x 10(10) cfu/cow of Propionibacterium strain P169 (P169), and the high-dose group (n = 11) received the control TMR plus 6 x 10(11) cfu/cow of P169 from -2 to 30 wk postpartum. Weekly milk samples were analyzed for percentage of milk fat, protein, lactose, and SNF, milk urea nitrogen, and somatic cell counts. Daily milk production expressed as 4% fat-corrected milk was affected by treatment and week x parity. High-dose and low-dose P169-treated cows exhibited 7.1 and 8.5% increases above controls in daily 4% fat-corrected milk, respectively. Treatment x parity and week significantly influenced percentage of milk fat, lactose, and protein, whereas treatment x parity and treatment x week influenced SNF. Ruminal propionate levels were influenced by treatment such that high-dose P169 cows had greater molar percentage of propionate than did low-dose P169 and control cows. Change in body weight postpartum was influenced by week x parity and treatment x parity such that high-dose and low-dose P169 multiparous cows exhibited a more rapid recovery of wk-1 body weight than did control multiparous cows. There was no treatment, parity, or interaction on days to first postpartum ovulation or on estrous behavior at 45 and 90 d postpartum. We concluded that P169 might have potential as an effective direct-fed microorganism to increase milk production in dairy cows.

Characterization of Propionibacterium plasmids.

Plasmid DNAs from 15 Propionibacterium strains were characterized by using restriction endonuclease analyses, DNA-DNA hybridizations, and curing experiments. Restriction endonuclease analysis identified seven distinct plasmids (pRGO1 through pRGO7). Detailed restriction maps were constructed for four of these plasmids. DNA-DNA hybridization analysis revealed that plasmids pRGO1 and pRGO2 had extensive sequence homology and that both were homologous to pRGO7 and to similar sequences of pRGO5. Plasmids pRGO4 and pRGO6 did not have any significant sequence homology with any of the other plasmids. Plasmid pRGO3 had partial sequence homology only with pRGO7. Curing of plasmids pRGO1, pRGO2, and pRGO5 was achieved by treatment with acriflavin, but we failed to identify any plasmid-encoded bacteriocin production, carbohydrate fermentation, or antibiotic resistance. However, physical evidence was obtained that tentatively linked the clumping phenotype of Propionibacterium jensenii P38 with plasmid pRGO5.

Microbiological Quality of Commercial Tofu.

A study was done to investigate the microbiological quality of commercial tofu available in local retail outlets. A sampling method was first developed to obtain accurate and representative microbial counts of individual pieces of tofu. Plate count determination of total aerobic organisms, psychrotrophs, coliforms, sporeformers, yeasts and molds, and staphylococci were made on 60 tofu samples (representing three lots each of four different brands) obtained within 24 h after delivery to the retail store. In addition, for two brands that provided manufacturer's pull dates, the same microbial counts were obtained for samples stored in the laboratory at 10°C until the pull date. Of the tofu sampled immediately after purchase, 83% of the lots tested had total counts greater than 106 colony-forming units (CFU)/g and psychrotrophic counts greater than 104 CFU/g. In addition, 67% of the lots tested had confirmed coliform counts greater than 103 CFU/g. Very low levels (less than 10 CFU/g) of all other microbial groups tested for were found in the majority of lots. Samples held until the manufacturer's pull date contained higher total and psychrotrophic counts but lower or stable counts of other organisms compared with samples tested immediately after purchase. To improve the microbiological quality of tofu, processors need to reduce initial loads by improving sanitation and processing techniques, and retailers should provide more consistent and colder refrigerated storage.