Ambient ammonia does not appear to inhibit the immune response to infectious bronchitis virus vaccination and protection from homologous challenge in broiler chickens.

Commercial broilers are commonly exposed to gaseous ammonia (NH3) originating from degradation of nitrogen-containing excreta in the litter during the grow-out period. Ammonia concentrations in the air are higher in poorly ventilated houses and appear to coincide with the elevated incidence of respiratory disease occurring during the winter months. This study examined the effect of NH3 on the immune response to infectious bronchitis virus (IBV) vaccination and protection against homologous serotype challenge in commercial broiler chickens. One-day-old chicks were administered IBV vaccine and exposed to 30-60 ppm of NH3. At 28 DOA, birds were challenged oculonasally with a pathogenic homologous IBV, and protection was measured by viral detection, clinical signs, ciliostasis, and presence of airsacculitis. IBV-specific serum IgG and lacrimal fluid IgA titers, as well as Harderian gland (HG) immune cell phenotypes, were evaluated. Ammonia exposure was associated with an increased incidence of airsacculitis among non-vaccinated, challenged birds. Vaccinated, NH3-exposed birds were completely protected from IBV challenge. Ammonia had subtle effects on cilia morphology and function but did not affect vaccine or challenge virus replication and clearance, clinical signs, ciliostasis, tracheal histopathology scores, or immune responses. In the HG of vaccinated birds, the percent of leukocytes, MHC I+/MHC IIhi expression, IgM+ expression, and CD8+ expression was increased, while mucosal IgA and serum IgG titers were nominal. Non-vaccinated, IBV-challenged birds exhibited an increased percent of leukocytes, MHC I+/MHC IIhi expression, and IgM+ expression in the HG at 5 dpc, followed by increased mucosal IgA and serum IgG titers and CD8+ expression at 10-14 dpc. In contrast, vaccinated, IBV-challenged birds had a minimal increase in MHC I+/MHC IIhi expression, and serum IgG antibody titers in vaccinated birds increased rapidly. The results indicate that commercial broilers exposed to moderate levels of ambient NH3 are equally protected against IBV challenge if appropriately vaccinated, and the absence of robust immune activation in vaccinated, challenged birds suggests that the challenge virus was efficiently neutralized before establishing infection. In contrast, ambient NH3 exposure was associated with a higher incidence of airsacculitis in non-vaccinated, challenged birds, despite the apparent lack of differences in the immune response between birds in the NH3-exposed and NH3 control groups.

Impact of litter Salmonella status during feed withdrawal on Salmonella recovery from the broiler crop and ceca.

Research was conducted to evaluate the impact of litter Salmonella status during feed withdrawal on Salmonella recovery from the crop and ceca following feed withdrawal. In 4 experiments, pens of broilers in separate rooms were challenged with marker strains of either Salmonella Montevideo or Salmonella Heidelberg. Three d post challenge, a 12-hour feed withdrawal was initiated, and one pen of broilers was switched between rooms for each Salmonella serotype. In experiments 3 and 4, non-challenged broilers also were added to the Salmonella challenge pens. The litter of each pen was sampled before and after the feed withdrawal period, the broilers euthanized, and the crop and ceca aseptically removed for Salmonella isolation. Results showed that only the challenge Salmonella serotype was recovered from the litter in challenge pens where broilers were not moved, while both Salmonella serotypes were recovered from the litter of the switched pens. Salmonella was recovered from 56/80 crops and from 66/80 ceca of challenged broilers that remained in the challenge pens. The challenge Salmonella serotype was recovered from 50/80 crops and from 60/80 ceca, and the switched pens' litter Salmonella serotype was recovered from 19/80 crops but not from the ceca in broilers challenged with Salmonella and then switched between pens. For experiments 3 and 4, Salmonella was recovered from 19/40 crops and from only 2/40 ceca from the non-challenged broilers placed into the Salmonella challenge pens. The results from broilers that were switched between Salmonella challenge pens indicate that the recovery of Salmonella from the crop of broilers following feed withdrawal (on Salmonella-contaminated litter) appears to depend mainly on the initial challenge Salmonella (62%) and less on the litter Salmonella (24%) status during the feed withdrawal period. In contrast, only the initial challenge Salmonella was recovered from the ceca (79%) from broilers that remained in challenge pens or were switched between Salmonella challenge pens. However, when non-challenged broilers were placed into the Salmonella challenge pens and commingled during the 12-hour feed and water withdrawal period, it was possible to recover the pen litter Salmonella from the ceca at a low level of 5% (2/40).

Zona pellucida protein B2 messenger ribonucleic acid expression varies with follicular development and granulosa cell location.

The freshly ovulated ovum in avian species is surrounded by a protein layer called the inner perivitelline layer (IPVL). The IPVL contains zona pellucida proteins and 6 distinct zona pellucida genes have been identified (ZPA, ZPB1, ZPB2, ZPC, ZPD and ZPX1) in the chicken. In the present research, the expression of the mRNA for ZPA, ZPB2, and ZPX1 was investigated in 2 lines of turkey hens selected for either increased egg production (E line) or increased body weight (F line). Theca and granulosa cell expression of the mRNA for ZPA and ZPB2 was also investigated in hierarchical and prehierarchical follicles from broiler breeder hens. Granulosa tissue was collected from F1 through F4 and F1 through F10 follicles in E line and F line hens, respectively. A one cm2 section of the granulosa layer around the germinal disc (GD) and an equivalent sized nongerminal disc (NGD) area was also collected from the F1 and F2 follicles from other hens from each genetic line. Granulosa and theca tissue was collected from hierarchical and prehierarchical follicles of broiler breeder hens. Total RNA was extracted from the samples. Minor groove-binding probes and primers for detecting ZPA, ZPB2, and ZPX1, were made for real-time PCR analyses. Expression of ZPA, ZPB2, and ZPX1 was detected in all follicle sizes from both genetic lines of hens. No significant differences in ZPA and ZPX1 mRNA expression were detected between the GD and NGD granulosa cells. However, the expression of the mRNA for ZPB2 was significantly greater in the GD granulosa cells when compared to the NGD granulosa cells in F1 and F2 follicles from E line and F line hens. In broiler breeder hens, the mRNA expression of ZPA and ZPB2 was greatest in the smallest prehierarchical follicles. The results suggest that higher expression of ZPB2 in the germinal disc area may be important for the preferential binding of sperm to this region of the IPVL.

Expression of genes for interleukins, neuropeptides, growth hormone receptor, and leptin receptor in adipose tissue from growing broiler chickens.

In this study, total RNA was collected from abdominal adipose tissue samples obtained from 10 broiler chickens at 3, 4, 5, and 6 wk of age and prepared for quantitative real-time PCR analysis. Quantitative real-time PCR analysis was used to examine the influence of age on the expression of the adipose tissue genes for IL-1ß, -6, -10, -15, -18; brain-derived neurotropic factor; ciliary neurotropic factor; interferon γ, neuropeptide Y receptor Y1; neuropeptide Y; nucleobindin 2; growth hormone receptor; leptin receptor; and visfatin. Between 3 and 6 wk of age, leptin receptor expression decreased (P=0.013) with age, whereas expression of IL-15 (P=0.015) and growth hormone receptor (P=0.002) increased. Furthermore, IL-18 (P<0.001) and visfatin (P=0.007) expression increased between 4 and 6 wk of age. This is a unique exhibition of age-related changes in cytokine gene expression in chicken adipose tissue. Future studies are needed to elucidate the role of adipose tissue cytokines in growth and, possibly, disease resistance.

Footpad dermatitis in poultry.

Footpad dermatitis (FPD) is a condition that causes necrotic lesions on the plantar surface of the footpads in growing broilers and turkeys. This condition not only causes downgrades and condemnations of saleable chicken paws, the portion of the leg below the spur, but is also an animal welfare concern in both the United States and in Europe.. Revenue from chicken paws in 2008 alone was worth $280 million. Harvesting large, unblemished paws has become a priority to poultry companies all over the world. Research on this subject has been ongoing since the 1940s and has looked into many different areas including nutrition, environment, and genetics. Early research looked at nutritional deficiencies such as riboflavin and biotin mainly in turkey poults. This early research was most likely looking at a separate form of dermatitis than what is being investigated now. Recent findings have suggested that there is a myriad of interacting factors that lead to FPD. Litter moisture appears to be the most likely culprit in the onset of this condition. Research has also shown a possible genetic link in the susceptibility to development of FPD lesions. Current chicken paw prices have skyrocketed due to a large export market in Asia. To produce unblemished paws for both increased profit and comply with current animal welfare recommendations, further research is needed to understand how the condition develops and what strategies can be used to prevent it.

Transmission of Salmonella to broilers by contaminated larval and adult lesser mealworms, Alphitobius diaperinus (Coleoptera: Tenebrionidae).

The ability of the lesser mealworm, Alphitobius diaperinus (Panzer), commonly known as the darkling beetle, to transmit marker Salmonella Typhimurium to day-of-hatch broiler chicks was evaluated, as well as the spread to nonchallenged pen mates. In trial 1, day-of-hatch chicks were orally gavaged with 4 larval or 4 adult beetles that had been exposed to marker Salmonella-inoculated feed for 72 h. In addition, chicks were gavaged with the marker Salmonella in saline solution. These chicks were then placed into pens to serve as challenged broilers. In trial 2, all pens received 2 challenged chicks that were gavaged with larvae or beetles that had been exposed to marker Salmonella-inoculated feed for 24 h and then removed from the inoculated feed for a period of 7 d. At 3 wk of age, cecal samples from the marker Salmonella-challenged broilers and from 5 pen mates in trial 1, or 10 pen mates in trial 2, were evaluated for the presence of the marker Salmonella in their ceca, and at 6 wk of age, all remaining pen mates were sampled. To monitor the presence of the marker Salmonella within pens, stepped-on drag swab litter samples were taken weekly. For the Salmonella-saline pens, 29 to 33% of the broilers that had been challenged and 10 to 55% of the pen mates were positive at 3 wk of age, and only 2 to 6% had positive ceca at 6 wk. For the pens challenged with adult beetles, 0 to 57% of the challenged broilers and 20 to 40% of the pen mates had positive ceca at 3 wk, and 4 to 7% were positive at 6 wk. The pens challenged with larvae had the greatest percentage of marker Salmonella-positive broilers; 25 to 33% of the challenged broilers and 45 to 58% of pen mates were positive at 3 wk, and 11 to 27% were positive at 6 wk. These results demonstrated that ingestion of larval or adult beetles contaminated with a marker Salmonella could be a significant vector for transmission to broilers.

The mRNA for zona pellucida proteins B1, C and D in two genetic lines of turkey hens that differ in fertility.

The avian inner perivitelline layer (IPVL) contains zona pellucida protein-B1 (ZPB1), zona pellucida protein-C (ZPC) and zona pellucida protein-D (ZPD). These three proteins may be involved in sperm binding to the IPVL. ZPB1 is produced by the liver and transported to the developing preovulatory follicle, while ZPC and ZPD are synthesized and secreted by the granulosa cells of the preovulatory follicle. The mRNA of ZPB1, ZPC, and ZPD was investigated in two lines of turkey hens selected for over 40 generations for either increased egg production (E line) or increased body weight (F line). Total RNA was extracted from the liver and from 1cm(2) sections of the granulosa layer around the germinal disc and a nongerminal disc area of the F(1) and F(2) follicles of hens from each genetic line. Northern analysis was performed using chicken cDNA probes for all three ZP proteins. Hepatic mRNA for ZPB1 was greater (P<0.05) in turkey hens from the E line than the F line. Although, there was no difference in ZPC mRNA between the germinal disc and nongerminal disc region of the two largest follicles in E line hens, ZPC mRNA was greater in the nongerminal disc region compared to the germinal disc region in the two largest follicles obtained from the F line hens. There were no differences in ZPD mRNA between the germinal disc and nongerminal disc regions of the F(1) and F(2) follicles for either genetic line. The results suggest that the greater rates of fertility previously observed in eggs from the E line hens compared with the F line of hens may be related to differential amounts of the potential sperm binding proteins ZPB1 and ZPC.

Follicular development and expression of the messenger ribonucleic acid for the inhibin/activin subunits in two genetic lines of turkey hens that differ in total egg production.

The characterization of the follicular hierarchy and the expression of the mRNA for the inhibin/activin subunits was investigated in the follicles of 2 lines of turkey hens selected for over 40 generations for increased egg production (Egg line) or increased body weight (Growth line). The follicular hierarchies of 6 hens from the Egg and Growth lines were characterized in middle (45 wk of age) and late production (58 wk of age). Relative follicular weights for individual hierarchical follicles (>12 mm), pooled small yellow follicles (5 to 12 mm), and large white follicles (2 to 5 mm) were calculated. Total RNA was extracted for Northern blot analysis from individual granulosa cell layers of the F1 through F4 follicles, and from the combined granulosa and theca layers of small yellow follicles and large white follicles from an additional 6 hens from each genetic line. Egg line hens displayed a more distinct follicular size hierarchy than Growth line hens at 45 and 58 wk. Although total follicular weight relative to body size was greater at 45 and 58 wk of age for the Egg line hens than the Growth line hens, the total number of hierarchical follicles was greater in the Growth line hens at 45 and 58 wk of age. Expression of follistatin and the inhibin beta(B)-subunit was highest in nonhierarchical follicles, whereas the expression of the inhibin alpha- and beta(A)-subunits was highest in the hierarchical follicles. The inhibin alpha- and beta(A)-subunit mRNA expression pattern in the 4 largest follicles of the Growth line hens was not similar to the Egg line hens or characteristic of laying hens that have a high rate of egg production. The unusual inhibin subunit mRNA expression in the largest hierarchical follicles of the Growth line hens may account for their development of an abnormal follicular size hierarchy and for their poor egg production.

Comparison of four sampling methods for the detection of Salmonella in broiler litter.

Experiments were conducted to compare litter sampling methods for the detection of Salmonella. In experiment 1, chicks were challenged orally with a suspension of naladixic acid-resistant Salmonella and wing banded, and additional nonchallenged chicks were placed into each of 2 challenge pens. Nonchallenged chicks were placed into each nonchallenge pen located adjacent to the challenge pens. At 7, 8, 10, and 11 wk of age the litter was sampled using 4 methods: fecal droppings, litter grab, drag swab, and sock. For the challenge pens, Salmonella-positive samples were detected in 3 of 16 fecal samples, 6 of 16 litter grab samples, 7 of 16 drag swabs samples, and 7 of 16 sock samples. Samples from the nonchallenge pens were Salmonella positive in 2 of 16 litter grab samples, 9 of 16 drag swab samples, and 9 of 16 sock samples. In experiment 2, chicks were challenged with Salmonella, and the litter in the challenge and adjacent nonchallenge pens were sampled at 4, 6, and 8 wk of age with broilers remaining in all pens. For the challenge pens, Salmonella was detected in 10 of 36 fecal samples, 20 of 36 litter grab samples, 14 of 36 drag swab samples, and 26 of 36 sock samples. Samples from the adjacent nonchallenge pens were positive for Salmonella in 6 of 36 fecal droppings samples, 4 of 36 litter grab samples, 7 of 36 drag swab samples, and 19 of 36 sock samples. Sock samples had the highest rates of Salmonella detection. In experiment 3, the litter from a Salmonella-challenged flock was sampled at 7, 8, and 9 wk by socks and drag swabs. In addition, comparisons with drag swabs that were stepped on during sampling were made. Both socks (24 of 36, 67%) and drag swabs that were stepped on (25 of 36, 69%) showed significantly more Salmonella-positive samples than the traditional drag swab method (16 of 36, 44%). Drag swabs that were stepped on had comparable Salmonella detection level to that for socks. Litter sampling methods that incorporate stepping on the sample material while in contact with the litter appear to detect Salmonella in greater incidence than traditional sampling methods of dragging swabs over the litter surface.

Presence of inoculated Campylobacter and Salmonella in unabsorbed yolks of male breeders raised as broilers.

Day-old male broiler breeder chicks were obtained from a commercial hatchery and raised as broilers. For Experiment 1, at 5 wk of age, the broilers were orally inoculated with a 10(6) cfu/ml of a characterized strain of Campylobacter jejuni and a cocktail (three naladixic acid-resistant strains) of Salmonella serovars. One week after inoculation, the birds were euthanatized and defeathered. The abdominal cavity was examined and any unabsorbed yolk material (and remaining yolk stalk) and ceca were aseptically removed for microbiological analyses. For each pooled sample (two birds per pool), an aerobic plate count (APC), an Enterobacteriaceae (ENT) count, and a test for the presence of Campylobacter and Salmonella was performed. For Experiment 2, at 5 wk of age, the broilers were orally inoculated with 10(5) cfu/ml of a characterized strain of Campylobacter jejuni. One week after inoculation, the birds (n = 20) were killed, defeathered, and the yolk stalk, attached yolk, or free-floating yolk and ceca were individually analyzed for presence of Campylobacter. For Experiment 1, the Salmonella-inoculated birds had 2/12 ceca and 0/12 unabsorbed yolk samples positive for Salmonella. The average yolk APC was log10 3.4 cfu/g and the average ENT was log10 1.9 cfu/g. For the Campylobacter-inoculated birds, 12/12 ceca and 9/12 unabsorbed yolk samples were positive for Campylobacter. The average yolk APC was log10 3.5 cfu/g and the average ENT was log10 3.1 cfu/g. For Experiment 2, the inoculated Campylobacter birds had 19/20 ceca, 5/20 free floating yolks, and 19/20 yolk stalks positive. In Experiment 1, the inoculated Campylobacter colonized the ceca in every instance and were present in 75% of the unabsorbed yolks. Alternatively, the inoculated Salmonella were not found in any of the unabsorbed yolks and only rarely in the ceca. In Experiment 2, the inoculated Campylobacter was found in very high numbers in the yolk and internal body samples. Determining to what extent these internal bodies and unabsorbed yolks play in bacterial colonization and contamination of the birds at processing has not been determined. The next step will be to determine the incidence of unabsorbed yolks and presence of Campylobacter and Salmonella in these bodies of commercial broilers at processing.

Incidence of unabsorbed yolk sacs in broilers, broiler breeder roosters, white Leghorn hens, and Athens-Canadian randombred control broilers.

Unabsorbed yolk sacs are being investigated as a possible reservoir for internal Campylobacter and salmonellae contamination of processed poultry carcasses. However, it is unknown at what frequency that unabsorbed yolk sacs persist at the time of processing of broilers and spent breeders. Seven sets of 100 broiler carcasses (at 6 or 8 wk of age) were obtained from commercial processing plants. In addition, 100 52-wk-old broiler breeder males, 100 102-wk-old Leghorn hens, and 300 8-wk-old Athens-Canadian randombred control (ACRBC) broilers were euthanized, and their abdominal cavities were opened for determination of the presence of unabsorbed yolk sacs. Carcasses with obliterated yolk stalks or stalks with no detectable yolk material were categorized as normal. Those with unabsorbed yolk sacs were further separated into 2 groups: 1) attached by the yolk stalk to the small intestine or 2) unattached within the abdominal cavity. Yolk sacs were further classified by size: 1) small was <2 mm in diameter, 2) medium was 2 to 10 mm, and 3) large was >10 mm. From the 300 commercial broiler carcasses that were 6 wk old, 54% were categorized as normal with no detectable yolk sac, 35% had an unabsorbed yolk sac attached to the yolk stalk, and 12% had unattached yolk sacs. From the 400 commercial broiler carcasses that were 8 wk old, 49% of the carcasses were normal, 31% had attached unabsorbed yolk sacs, and 20% had unattached yolk sacs. From the 100 rooster carcasses sampled, 73% were normal, 8% had attached unabsorbed yolk sacs, and 19% had unattached yolk sacs. From the 100 White Leghorn hen carcasses sampled, 88% were normal, 8% had attached unabsorbed yolk sacs, and 4% were unattached yolk sacs. From the 300 ACRBC carcasses sampled, 76% were normal, 4% had attached unabsorbed yolk sacs, and 20% were unattached yolk sacs. The incidence of unabsorbed yolk sacs in present day commercial broilers appears twice as high as for mature roosters, hens, or ACRBC broilers.

A novel delivery of oxytetracycline in turkey breeder hens.

A novel product (SQ12) for subcutaneous (SQ) injectable delivery of oxytetracycline (OTC) has been developed for use in livestock. SQ12 employs microfluidic spheres encasing OTC crystals, which allows for longer release of the OTC compared with other injectable antibiotics. The objectives of the study were to determine serum and tissue levels of SQ12 in turkey breeder hens to 14 days postinjection and to evaluate effects of SQ12 on reproductive status. Thirty photostimulated hens were housed in litter floor pens and provided with 14.5 hr of light per day in a curtain-sided facility. Six hens served as untreated controls. Twelve hens per treatment group received SQ injections in the neck with SQ12 at 11.4 (L dose group) or 22.7 mg/kg (H dose group) to assess low and high doses, respectively. Serum samples were obtained from each hen at predose and 6, 12, 24, 48, 72, 96, 168, 240, and 336 hr postinjection. All hens were euthanatized at 14 and 15 days postinjection. One-half of the hens in each treatment group were sampled (liver, lung, kidneys, and breast muscle) for tissue residue levels of OTC. The control group had no detectable OTC in serum or tissues at any sample collection time. There were no detectable serum levels of OTC in either treatment group prior to injection. The average serum concentrations of the L and H dose groups showed similar depletion curves although the H dose group was 42% higher at maximum concentration than the L group. Average tissue concentration of OTC for all tissues sampled from the H dose group was twice that of the L dose group. All tissue levels were below the OTC residue tolerance limit. SQ12 provided an extended source of OTC in serum of turkey breeder hens with no effect on reproductive status. SQ12 may provide for a novel treatment of bacterial infection in turkey breeder hens with longer lasting serum levels compared with other single injectable OTC products.

Photostimulation of turkey eggs accelerates hatching times without affecting hatchability, liver or heart growth, or glycogen content.

The objective of the current study was to determine the effects of incubating turkey eggs in the presence of incandescent light on hatching times, as well as liver and heart growth and function of the hatched poult. In each of two independent trials, 180 commercial turkey eggs were incubated either in a 12-h incandescent light:dark schedule or continuous darkness throughout the incubation period (n = 360). Hatching time was observed at 8-h intervals following 25 d of incubation. Hatchability was calculated as a percentage of total eggs set, and was also calculated as a percentage of fertilized eggs. Poult weights, blood glucose, liver weights, and heart weights were measured at hatch. Liver and heart glycogen concentrations were analyzed, and comparisons were made of light-treated hatchlings and controls exposed to continuous darkness. The photostimulation of eggs accelerated hatching times (P < or = 0.01) without affecting hatchability or poult weight at hatching. Neither organ weights nor organ glycogen contents of photostimulated poults differed significantly from controls incubated in the dark. Results of this experiment indicate that the incubation length of turkey eggs may be shortened by photostimulation of eggs during the incubation period without significantly affecting embryonic survival, liver or heart growth, or glycogen content.