A. S. Kiess

Phenotypic and genotypic methods for typing Campylobacter jejuni and Campylobacter coli in poultry

Human campylobacteriosis, an infection caused by the bacterium Campylobacter, is a major issue in the United States food system, especially for poultry products. According to the Center for Disease Control, campylobacterosis is estimated to affect over 2.4 million people annually. Campylobacter jejuni and Campylobacter coli are 2 species responsible for the majority of campylobacterosis infections. Phenotypic and genotypic typing methods are often used to discriminate between bacteria at the species and subspecies level and are often used to identify pathogenic organisms, such as C. jejuni and C. coli. This review describes the design as well as advantages and disadvantages for 3 current phenotypic techniques (biotyping, serotyping, and multilocus enzyme electrophoresis) and 6 genotypic techniques (multilocus sequence typing, PCR, pulse-field gel electrophoresis, ribotyping, flagellin typing, and amplified fragment length polymorphisms) for typing pathogenic Campylobacter spp.

The combined efficacy of carvacrol and modified atmosphere packaging on the survival of Salmonella, Campylobacter jejuni and lactic acid bacteria on Turkey breast cutlets

The primary objective of this study was to determine the efficacy of carvacrol in combination with modified atmosphere packaging (MAP) in reducing Salmonella on turkey breast cutlets stored at 4 °C. In experiment I, carvacrol (0.5, 1, and 2% v/v) was applied as surface treatment and samples were stored under aerobic condition or as surface and dip treatments followed by storage in an environment of 100% carbon dioxide. The findings of the experiment I revealed the synergistic activity of carvacrol with carbon dioxide in reducing Salmonella when used as dip treatment compared to the surface treatment. In experiment II, turkey breast cutlets were dip treated with carvacrol (0.25, 0.5, and 1% v/v) for 30 s and stored under MAP (95% carbon dioxide and 5% oxygen) to evaluate the efficacy against Salmonella, Campylobacter jejuni and lactic acid bacteria on turkey breast cutlets. In experiment II, the combined application of carvacrol and MAP resulted in 1.0-2.0 log CFU/g reduction (P ≤ 0.05) of both Salmonella and Campylobacter on turkey breast cutlets for 7 d storage at 4 °C. MAP alone and in combination with carvacrol reduced lactic acid bacteria (P ≤ 0.05) on cutlets stored at 4 °C for 21 d period. There was no difference (P ≤ 0.05) in meat color among treatments and controls except for an increased paleness of meat (P ≤ 0.05) observed for the 1% carvacrol treated cutlets stored under MAP after 21 d of storage. The high concentration of carbon dioxide and carvacrol treatments did not cause any alteration in meat pH (P ≤ 0.05). In conclusion, carvacrol was effective at a low concentration of 0.25% (v/v) in reducing Salmonella and C. jejuni by ∼1.0 log CFU/g when stored under MAP.

The effect of cage and house design on egg production and egg weight of White Leghorn hens: An epidemiological study

Hen performance can be affected by many interacting variables related to cage design, such as floor area, height, tier arrangement, and feeder and drinker type and placement within the cage. Likewise, features of house design such as waste management and lighting can also affect hen productivity. The influence of these design aspects on hen performance has not been fully assessed. Determining the effects of numerous, interacting variables is impractical in a traditional experiment; therefore, an epidemiological approach, using variability in cage and house design among and within commercial producers, was employed to identify features that affect egg production and egg weight. A universal cage measurement system was created to calculate cage design variables. A database for recording information on cage design, resource location, waste management, environmental conditions, and hen productivity was developed. Production outcomes were assessed from placement to 60 wk of age in White Leghorns (n = 165-168 houses). Using GLM, a statistical model was identified that best described the variance in egg traits. Eggs/hen-housed increased with greater feeder space allocation (P = 0.031); taller cages (P = 0.029); rear (vs. front) drinker location in vertical cages (P = 0.026); and regular removal of manure from the house (P = 0.005). Case weight of eggs was greater in A-frame houses where manure was removed regularly instead of being left in the house (P < 0.001); with increasing cage floor slope (P = 0.001); in cages where drinkers were placed more toward the front or back of the cage as compared with the middle of the cage (P < 0.001); with more space/hen (P = 0.024); and with higher caloric intake (P < 0.001). Perhaps because of its negative correlation with egg production, case weight of eggs increased with less feeder space allocation (P = 0.004) and shorter cage heights (P < 0.001). These results reveal important effects of feeder space, floor space, cage height, drinker position, and waste management on hen productivity.

Evaluation of different selective media and culturing techniques for the quantification of Campylobacter ssp. from broiler litter

Poultry is a major reservoir for Campylobacter, the leading cause of foodborne illness in the United States, but how broilers become initially colonized is still under debate. Broiler litter is a potential source, but the best technique for quantifying Campylobacter from litter is still unknown. Therefore, our objectives were to determine if certain media are more selective for quantifying Campylobacter and if enrichment allows for the detection of stressed or viable but nonculturable cells from broiler litter samples. In this trial, 5 media and 2 culturing techniques were used to enumerate Campylobacter from broiler litter. The media used were campy-Line agar (CLA), campy-cefex agar (CCA), modified CCA, Campylobacter agar plates (CAP), and modified charcoal cefoperazone deoxycholate agar. Litter samples were obtained from a commercial broiler house. Each sample was equally divided and diluted 10-fold into peptone, for direct plating, or 4-fold into Campylobacter enrichment broth. Samples diluted in peptone were direct-plated onto each media and incubated under microaerophilic conditions for 48 h at 42 degrees C. Samples diluted in enrichment broth were incubated under the same conditions for 24 h, then further diluted to 10-fold before plating. Plates from enriched samples were incubated for an additional 24 h after plating. After incubation, all plates (direct and enriched) were counted and presumptive positive colonies were confirmed using a Campylobacter latex agglutination kit. Results indicated that there was no difference in the ability of any of the selective media tested to grow Campylobacter. Direct-plated samples had a higher Campylobacter isolation rate compared with enriched samples. The CLA and CAP were able to suppress total bacterial growth better than modified charcoal cefoperazone deoxycholate, modified CCA, and CCA. The CLA and CAP were the only media able to detect total bacterial population shifts over time. In conclusion, it is important before making a final decision on a selective medium to consider the mediums ability to suppress total bacterial growth as well as isolate Campylobacter.

Reduction of Salmonella on chicken meat and chicken skin by combined or sequential application of lytic bacteriophage with chemical antimicrobials.

The effectiveness of recently approved Salmonella lytic bacteriophage preparation (SalmoFresh™) in reducing Salmonella in vitro and on chicken breast fillets was examined in combination with lauric arginate (LAE) or cetylpyridinium chloride (CPC). In another experiment, a sequential spray application of this bacteriophage (phage) solution on Salmonella inoculated chicken skin after a 20s dip in chemical antimicrobials (LAE, CPC, peracetic acid, or chlorine) was also examined in reducing Salmonella counts on chicken skin. The application of phage in combination with CPC or LAE reduced S. Typhimurium, S. Heidelberg, and S. Enteritidis up to 5 log units in vitro at 4 °C. On chicken breast fillets, phage in combination with CPC or LAE resulted in significant (p<0.05) reductions of Salmonella ranging from 0.5 to 1.3 log CFU/g as compared to control up to 7 days of refrigerated storage. When phage was applied sequentially with chemical antimicrobials, all the treatments resulted in significant reductions of Salmonella. The application of chlorine (30 ppm) and PAA (400 ppm) followed by phage spray (10(9)PFU/ml) resulted in highest Salmonella reductions of 1.6-1.7 and 2.2-2.5l og CFU/cm(2), respectively. In conclusion, the surface applications of phage in combination with LAE or CPC significantly reduced Salmonella counts on chicken breast fillets. However, higher reductions in Salmonella counts were achieved on chicken skin by the sequential application of chemical antimicrobials followed by phage spray. The sequential application of chlorine, PAA, and phage can provide additional hurdles to reduce Salmonella on fresh poultry carcasses or cut up parts.

Reduction of Salmonella in skinless chicken breast fillets by lauric arginate surface application

Lauric arginate (LAE) has been found to be effective against various foodborne pathogens. In this study, the antimicrobial efficacy of LAE against Salmonella and mesophilic organisms was evaluated in fresh, skinless, boneless, uncooked chicken breast fillets. The effect of LAE treatments on pH and color of breast fillets was also assessed. Chicken breast fillets were inoculated with a 4-strain Salmonella cocktail (Salmonella Enteritidis ATCC 4931, Salmonella Heidelberg ATCC 8326, Salmonella Kentucky ATCC 9263, and Salmonella Typhimurium ATCC 14028) and then treated with sterile dionized water (positive control) and 200 ppm and 400 ppm of LAE. The chicken breast fillets were stored at 4 ± 1°C and analyzed on d 0, 1, 3, 5, and 7 for Salmonella, total aerobes, color, and pH. The fillets destined for color analysis were not inoculated with Salmonella cocktail and stored under conditions simulating the retail display. The fillets treated with 400 ppm LAE had lower (P < 0.05) Salmonella counts compared with the positive control from d 0 through d 7 of storage except on d 3, when no effect of LAE was observed. Treating fillets with 200 ppm of LAE caused a significant reduction in Salmonella counts (P < 0.1) on d 0, 1, and 7. Reductions in Salmonella spp. were 0.7 log cfu/g and 0.7 to 1.0 log cfu/g for 200 and 400 ppm treatments, respectively. Lauric arginate did not exhibit any treatment effect on the growth of mesophilic microorganisms, pH, and color of chicken breast fillets (P > 0.05) when applied at 200 and 400 ppm concentrations. These results indicate that surface application of LAE in chicken breast fillets significantly reduces Salmonella during refrigerated aerobic storage without negatively affecting the color of chicken breast fillets.

Impact of 6 different intestinal bacteria on broiler breeder sperm motility in vitro

Male fertility is often evaluated by measuring sperm parameters, including concentration, viability, and motility. This is important because after copulation occurs, sperm must overcome many barriers in the female reproductive tract to fertilize the ovum. In mammalian species, sperm have been shown to have reduced motility when bacteria are present. In male broiler breeders, bacteria have been associated with spermatozoa, but their effect on motility has not been investigated. The sperm quality index is a modern rapid method of evaluating avian sperm motility. Therefore, the objective of this study was to use the sperm quality index to determine if broiler breeder sperm motility is reduced when semen is exposed to various bacteria. In this experiment, semen was collected from 20 broiler breeders to obtain a pooled neat semen sample. Six different intestinal bacteria, Salmonella enterica, Escherichia coli, Campylobacter jejuni, Clostridium bifermentans, Lactobacillus acidophilus, and Bifidobacterium animalis were cultured overnight. For each bacterium, 50 µL of semen was diluted in 450 µL of saline, sterile broth, or the overnight culture, creating 3 treatments. The experiment was repeated twice. In each treatment, 3 replicates were evaluated at 0 and 10 min postinoculation, creating a completely randomized design with a split plot over time. Also, the pH was measured for each treatment at 0 and 10 min. The results indicated that all broths containing bacteria immediately reduced broiler breeder sperm motility when compared with the controls (P < 0.0001), but broths containing Bifidobacterium or Lactobacillus virtually made sperm immotile. Although broth containing Salmonella, Campylobacter, and Bifidobacterium immediately reduced sperm motility, the reduction did not change over time. Broths containing E. coli, Clostridium, and Lactobacillus reduced sperm motility immediately, but over time motility continued to decrease. However, pH was increased when semen was exposed to the E. coli and Campylobacter treatment, but when semen was exposed to Bifidobacterium and Lactobacillus treatments, pH was reduced. In conclusion, the results indicate that bacteria can reduce broiler breeder sperm motility upon exposure.

Genetic selection increases parthenogenesis in Chinese painted quail (Coturnix chinensis)

Parthenogenesis, embryonic development of an unfertilized egg, occurs naturally in turkey, chicken, and quail species. In fact, parthenogenesis in turkeys and chickens can be increased by genetic selection. However, it is unknown if genetic selection for parthenogenesis is effective in quail or if selection for parthenogenesis affects egg production. Therefore, the objectives of this study were to determine if the incidence of parthenogenesis in quail could be increased by genetic selection and if selection for this trait affects egg production. To prevent fertilization, 1,090 females were caged separately from males at 4 wk of age and then caged individually at 6 wk of age to monitor egg production. Eggs were collected daily, labeled, and stored for 0 to 3 d. After 10 d of incubation, 20 unfertilized eggs from each hen were examined for the occurrence of parthenogenesis and embryonic growth. In the parent (P) generation and subsequent generations (1 to 4), hens laying eggs containing parthenogenetic development and males whose sisters or mothers exhibited parthenogenesis were used for breeding. There was a linear increase in the percentage of hens exhibiting parthenogenesis as generation of selection increased. With each successive generation, there was a quadratic response in the percentage of eggs positive for parthenogenesis. When compared with the P generation, parthenogenesis was almost 3 times greater for eggs laid by the fourth generation (4.6 to 12.5%, respectively). Even when only hens exhibiting parthenogenesis were examined, the percentage of eggs demonstrating embryonic development responded quadratically with generation of selection. The embryonic size at 10 d of incubation was greater for each subsequent generation when compared with the P generation. There was a linear decrease in both egg production and the average position of an egg in a clutch as generation of selection increased. In conclusion, genetic selection for parthenogenesis increased the incidence of parthenogenesis and embryonic size but decreased egg production and average position of an egg in a clutch as generations of selection increased.

Effects of prebiotics, probiotics, and their combination on growth performance, small intestine morphology, and resident Lactobacillus of male broilers.

Effects of commercial antimicrobials and the individual and combinational use of commercial prebiotics and probiotics in feed from d zero to 41 on the growth performance, small intestine size, jejunal morphology, and ileal resident bacteria population of broiler chickens were determined. A total of 1,040 one-day-old male Ross × Ross 708 broilers were randomly distributed to 80 floor pens (5 treatments, 16 replications per treatment, 13 chicks per pen). Five dietary treatments were employed: 1) a corn soybean-meal basal diet (served as a negative control diet, NC); 2) a basal diet supplemented with a commercial prebiotic product (Pre); 3) a basal diet supplemented with a probiotic product containing Bacillus subtilis spores (Pro); 4) a basal diet supplemented with both prebiotic and probiotic products (Pre + Pro); and 5) a basal diet supplemented with commercial antimicrobials (served as a positive control diet, PC). At d 14, Pre diets improved the relative level of Lactobacillus in ileal mucosa as compared to NC, Pro, or PC diets (P = 0.045) without improving broiler BW. Broilers fed PC diets exhibited the highest BW gain from d 15 to 27, the lowest duodenum, jejunum, and ileum relative weights as percentage of BW at d 27, and the highest breast weight at d 42 (P = 0.026, 0.035, 0.002, 0.025, and 0.035, respectively). Broilers fed Pro or Pre + Pro diets exhibited higher BW gain from d 28 to 41 (P = 0.005) and higher overall BW gain from d zero to 41 (P = 0.039) than those fed other diets. Dietary treatments did not affect jejunal morphology or ileal resident Escherichia coli level at any age. From our results, including spores of Bacillus subtilis in feed may stimulate growth at a later age and may facilitate broilers in reaching their target weight sooner. Therefore, probiotics are recommended as potential alternatives to antimicrobials in chicken diets, especially in grower and finisher feed.

The relationship of parthenogenesis in virgin Chinese Painted quail (Coturnix chinensis) hens with embryonic mortality and hatchability following mating1

Unfertilized chicken, turkey, and quail eggs are capable of developing embryos by parthenogenesis. However, it is unknown if the physiological mechanisms regulating parthenogenesis in virgin hens may actually work against fertilization, embryonic development, and hatchability of eggs from these same hens following mating. Additionally, because most parthenogenic development closely resembles early embryonic mortality in fertilized eggs during the first 2 to 3 d of incubation, it is possible that many unhatched eggs classified as containing early embryonic mortality may actually be unfertilized eggs that contain parthenogens. Therefore, the objective of this study was to examine the relationship of parthenogenesis before mating with embryonic development and hatchability characteristics after mating. Based upon their ability to produce unfertilized eggs that contain parthenogens, 372 virgin Chinese Painted quail hens were divided into 7 groups, according to their incidence of parthenogenesis: 0, 10, 20, 30, 40, 50, and greater than 50% parthenogenesis. Males were then placed with these hens so that fertility, embryonic mortality, and hatchability could be evaluated for each hen. Hatchability of eggs set, hatchability of fertile eggs, and late embryonic mortality declined dramatically as the incidence of parthenogenesis increased. On the other hand, early embryonic mortality increased as parthenogenesis increased. Fertility was not different across the 7 parthenogenesis hen groups, perhaps because unfertilized eggs that exhibited parthenogenesis resembled and were therefore classified as early embryonic mortality. In conclusion, virgin quail hens that exhibit parthenogenesis appear to have impaired embryonic development and hatchability following mating. Additional sperm-egg interaction and embryonic research is needed to determine if a large portion of the early embryonic mortality experienced by mated hens that exhibit parthenogenesis as virgin hens is in fact embryonic development in unfertilized eggs.