Evaluating the impact of gypsum as a novel bedding material on broiler performance, foot pad health, and fear response.

Flue Gas Desulfurization (FGD) gypsum is a byproduct of the coal-fired power plant process commonly used to remove sulfur dioxide emissions from the flue gas. FGD gypsum has numerous industrial, agricultural, and environmental applications. This study aimed to explore a novel approach involving the use of FGD gypsum combined with different litter treatments as bedding for broiler production. It focused on performance metrics, including adjusted feed conversion ratio (AFCR) and average body weight (BW), foot pad dermatitis (FPD), and fear response over 5 consecutive flocks. A total of 1,800 one-day-old Ross 708 chicks were randomly assigned to 24 pens (75 birds/pen), divided into 6 treatment groups (4 pens/treatment), with 5 replications and raised until 42 d old (d). Treatments were gypsum that was decaked (D), rotovated (E), and rotovated then windrowed (F) between flocks. Control treatments using pine shavings were decaked (A), rotovated (B), and windrowed postrotovating (C). AFCR, average BW, and mortality were used as a measure of production. Foot pad dermatitis scores were taken on d42 using a scale of 0 (absence), 1 (mild), and 2 (severe). Response to observer and human approach test were used to measure fear response. Data were analyzed as a 2-way ANOVA (Proc Glimmix) for the main effects of bedding type and litter treatment. Means were identified using Tukey's HSD. No effect of bedding type or litter treatment was found for AFCR, BW, or mortality. FPD scores 2 and 1, were higher with pine shavings than gypsum (P = 0.01 and P = 0.01, respectively). While FPD scores 0 were higher for gypsum than the pine shaving (P = 0.01). No difference in fear response was found among birds raised on any of the gypsum litter treatments and any of the pine shaving litter treatments. Overall, the use of gypsum as bedding results in equivalent production and fear response to pine shavings, while increasing FPD quality when compared to pine shaving.

Vaccination Against Poultry Parasites.

The complexity of parasites and their life cycles makes vaccination against parasitic diseases challenging. This review highlights this by discussing vaccination against four relevant parasites of poultry. Coccidia, i.e., Eimeria spp., are the most important parasites in poultry production, causing multiple billions of dollars of damage worldwide. Due to the trend of antibiotic-free broiler production, use of anticoccidia vaccines in broilers is becoming much more important. As of now, only live vaccines are on the market, almost all of which must be produced in birds. In addition, these live vaccines require extra care in the management of flocks to provide adequate protection and prevent the vaccines from causing damage. Considerable efforts to develop recombinant vaccines and related work to understand the immune response against coccidia have not yet resulted in an alternative. Leucozytozoon caulleryi is a blood parasite that is prevalent in East and South Asia. It is the only poultry parasite for which a recombinant vaccine has been developed and brought to market. Histomonas meleagridis causes typhlohepatitis in chickens and turkeys. The systemic immune response after intramuscular vaccination with inactivated parasites is not protective. The parasite can be grown and attenuated in vitro, but only together with bacteria. This and the necessary intracloacal application make the use of live vaccines difficult. So far, there have been no attempts to develop a recombinant vaccine against H. meleagridis. Inactivated vaccines inducing antibodies against the poultry red mite Dermanyssus gallinae have the potential to control infestations with this parasite. Potential antigens for recombinant vaccines have been identified, but the use of whole-mite extracts yields superior results. In conclusion, while every parasite is unique, development of vaccines against them shares common problems, namely the difficulties of propagating them in vitro and the identification of protective antigens that might be used in recombinant vaccines.

Estudio recapitulativo- Vacunación contra los parásitos de las aves de corral. La complejidad de los parásitos y sus ciclos de vida hace que la vacunación contra las enfermedades parasitarias sea un desafío. Esta revisión destaca este concepto al discutir la vacunación contra cuatro parásitos relevantes en la avicultura. Las coccidias, como, Eimeria spp., son los parásitos más importantes en la producción avícola y causan daños por miles de millones de dólares en todo el mundo. Debido a la tendencia de la producción de pollos de engorde sin antibióticos, el uso de vacunas anticoccidianas en pollos de engorde se está volviendo mucho más importante. Por el momento, sólo hay en el mercado vacunas vivas y casi todas ellas deben producirse en aves. Además, estas vacunas vivas requieren un cuidado especial en el manejo de las parvadas para brindar una protección adecuada y evitar que las vacunas causen daños. Los esfuerzos considerables para desarrollar vacunas recombinantes y los trabajos relacionados para comprender la respuesta inmune contra coccidias aún no han dado como resultado una alternativa. Leucozytozoon caulleryi es un parásito sanguíneo que prevalece en el este y el sur de Asia. Es el único parásito de las aves de corral para el que se ha desarrollado y comercializado una vacuna recombinante. El parásito Histomonas meleagridis causa tiflohepatitis en pollos y pavos. La respuesta inmune sistémica después de la vacunación intramuscular con parásitos inactivados no es protectora. El parásito se puede cultivar y atenuar in vitro, pero sólo junto con bacterias. Esto y la necesaria aplicación intracloacal dificultan el uso de vacunas vivas. Hasta el momento no ha habido intentos de desarrollar una vacuna recombinante contra H. meleagridis. Las vacunas inactivadas que inducen anticuerpos contra el ácaro rojo de las aves Dermanyssus gallinae tienen el potencial de controlar las infestaciones por este parásito. Se han identificado antígenos potenciales para vacunas recombinantes, pero el uso de extractos completos de ácaros produce resultados superiores. En conclusión, si bien cada parásito es único, el desarrollo de vacunas contra ellos comparte problemas comunes, por ejemplo, las dificultades de propagarlos in vitro y la identificación de antígenos protectores que podrían usarse en vacunas recombinantes.

Quantification of Salmonella Infantis transfer from transport drawer flooring to broiler chickens during holding.

Transportation is a potential point of cross-contamination before broiler chickens arrive at the processing plant for slaughter. Previous studies have associated the use of uncleaned transport containers with the introduction of pathogenic bacteria onto uncontaminated broilers. The objective of this study was to quantify the transfer of Salmonella from transport drawer perforated flooring to broiler chickens during different holding times. For traceability, the flooring of each drawer was inoculated with fecal content slurry containing a marker strain of Salmonella Infantis. Three drawers per treatment were used, and each drawer was subjected to one of the following treatments: pressure wash, disinfectant, and pressure wash (A), pressurized steam followed by forced hot air (B), or no cleaning (C). Drawers were classified as top, middle, or bottom based on their relative position with each other. After treatment, broilers were introduced to each drawer and held for 2, 4, or 6 h. At each timepoint, broilers were removed from drawers, euthanized, and carcasses rinsed to obtain Salmonella counts. Samples under the limit of direct plating detection were enriched, plated, and later confirmed positive or negative. Differences were observed per treatment, holding time, and drawer relative position (P < 0.0001). Broilers placed in transport containers that underwent a cleaning procedure (A or B) had lower levels of Salmonella when compared to broilers placed in noncleaned containers (C). However, most of the samples below the limit of detection were positive after enrichment, indicating that both procedures evaluated need improvement for efficient pathogen inactivation. A decrease in Salmonella transfer was observed after 6 h in rinsates obtained from broilers placed in noncleaned containers (C). Rinsates obtained from top drawers had less Salmonella than the middle or bottom drawers when broilers were placed in transport containers that underwent a cleaning procedure (A and B). The application of pressurized steam and forced hot air was comparable to the use of water washes and disinfectant indicating a potential role in cleaning poultry transport containers.

Application of pressurized steam and forced hot air for cleaning broiler transport container flooring.

In the United States, cleaning poultry transport containers prior to arrival at the broiler grow-out farm is not currently a widely adopted practice in the industry. However, previous studies have shown that transport containers have an important role in cross-contamination before the broilers arrive at the processing plant. The objective of this study was to evaluate the efficacy of pressurized steam followed by forced hot air to clean transport container flooring and compare it to conventional cleaning procedures. Fiberglass and plastic flooring were cut into even pieces and inoculated with chicken intestinal contents containing Salmonella Infantis or Campylobacter jejuni. The cleaning treatments were pressurized steam, forced hot air, pressurized steam followed by forced hot air, water pressure washing, water pressure washing before and after disinfectant, and no cleaning. Counts for Salmonella, Campylobacter, Escherichia coli, coliforms, and aerobic bacteria were assessed. All reductions were made in comparison to noncleaned samples. Forced hot air applied by itself was not efficient in reducing Campylobacter, coliforms, and E. coli; and limited reductions (less than 1 log10 CFU/cm2) were observed for Salmonella and aerobic bacteria. Then, for all bacteria types evaluated, pressurized steam by itself showed reductions of 2.4 to 3.5 log10 CFU/cm2. Samples that were cleaned with a single-pressure water wash showed reductions of 4.0 to 4.6 log10 CFU/cm2 for all bacteria types. For Salmonella, Campylobacter, and E. coli, the greatest reductions were observed when samples were cleaned with pressurized steam followed by forced hot air (4.3-6.1 log10 CFU/cm2) or water washed before and after disinfectant (4.5-6.2 log10 CFU/cm2), and these treatments did not differ from each other. Pressurized steam followed by forced hot air was shown to be an efficient cleaning procedure to reduce poultry-associated pathogens on transport cage flooring, with the benefit of using less water than conventional water cleaning. Processors may be able to adapt this process to reduce potential cross-contamination and lessen the level of pathogens entering the processing plant.

Buffering Capacity Comparison of Tris Phosphate Carbonate and Buffered Peptone Water Salmonella Pre-Enrichments for Manufactured Feed and Feed Ingredients.

Various culture-based methods to detect Salmonella in animal feed have been developed due to the impact of this bacterium on public and animal health. For this project, tris phosphate carbonate (TPC) and buffered peptone water (BPW) buffering capacities were compared as pre-enrichment mediums for the detection of Salmonella in feed ingredients. A total of 269 samples were collected from 6 feed mills and mixed with the pre-enrichments; pH was measured before and after a 24 h incubation. Differences were observed when comparing pH values by sample type; DDGS and poultry by-product meal presented lower initial pH values for TPC and BPW compared to the other samples. For both TPC and BPW, meat and bone meal presented higher final pH values, while soybean meal and peanut meal had lower final pH values. Furthermore, for BPW, post cooling, pellet loadout, and wheat middlings reported lower final pH values. Additionally, most feed ingredients presented significant differences in pH change after 24 h of incubation, except DDGS. From meat and bone meal samples, four Salmonella isolates were recovered and identified: three using BPW and one using TPC. TPC provided greater buffer capacity towards neutral pH compared to BPW, but BPW was more effective at recovering Salmonella.

In vitro Effect of Photoactive Compounds Curcumin and Chlorophyllin Against Single Strains of Salmonella and Campylobacter.

Salmonella and Campylobacter are two of the most common foodborne pathogens associated with poultry meat. Regulatory restrictions and consumer concerns have increased the interest for plant-derived antimicrobials and emerging novel technologies. The objective of this study was to determine the antimicrobial activity of photoactive compounds curcumin (CUR) and chlorophyllin (CH) followed by activating light exposure for the reduction of Salmonella and Campylobacter. Peroxyacetic acid (PAA) was also evaluated as a poultry industry standard antimicrobial processing aid. CUR and CH were evaluated in 96-well plates at concentrations of 100, 500, and 1,000 ppm, along with PAA at 100, 200, and 300 ppm, or distilled water (DW). Each well was inoculated with 105 CFU/mL of Salmonella Typhimurium or Campylobacter jejuni, and plates were exposed to activating light (430 nm) for 0 or 5 min. No detectable reductions were observed for Salmonella or Campylobacter when treated with CUR, CH, or 100 ppm PAA. However, when Salmonella was treated with 200 ppm PAA, counts were reduced from 4.57 to 2.52 log10 CFU/mL. When Salmonella was treated with 300 ppm PAA, counts were reduced to below detectable levels (5 CFU/mL). Campylobacter was reduced from 4.67 to 2.82 log10 CFU/mL when treated with 200 ppm PAA. However, no further reductions were observed when Campylobacter was treated with 300 ppm PAA (2.50 log10 CFU/mL). These results indicate that CUR and CH were not effective as antimicrobials under the evaluated conditions, particularly in comparison to the commonly used antimicrobial, PAA.

Mapping the poultry insectome in and around broiler breeder pullet farms identifies new potential Dipteran vectors of Histomonas meleagridis.

BACKGROUND: Histomonas meleagridis can infect chickens and turkeys. It uses the eggs of the cecal worm Heterakis gallinarum as a vector and reservoir. Litter beetles (Alphitobius diaperinus) and other arthropod species have been implicated as potential vectors, but little information about other arthropod species as potential vectors is known. METHODS: Four broiler breeder pullet farms were sampled every 4 months. On each farm, three types of traps were set inside and outside two houses. Trapped arthropod specimens were morphologically identified at order level and grouped into families/types when possible. Selected specimens from abundant types found both inside and outside barns were screened for H. meleagridis and H. gallinarum by qPCR. RESULTS: A total of 4743 arthropod specimens were trapped. The three most frequently encountered orders were Diptera (38%), Coleoptera (17%), and Hymenoptera (7%). Three hundred seventeen discrete types were differentiated. More arthropods were trapped outside than inside. Alpha diversity was greater outside than inside but not significantly influenced by season. The composition of the arthropod populations, including the insectome, varied significantly between trap location and seasons. Up to 50% of litter beetles tested positive for H. meleagridis DNA 4 months after an observed histomonosis outbreak. Sporadically litter beetles were positive for H. gallinarum DNA. Thirteen further arthropod types were tested, and specimens of four Dipteran families tested positive for either one or both parasites. CONCLUSIONS: This study describes the insectome in and around broiler breeder pullet farms and identifies new potential vectors of H. meleagridis through qPCR. The results show a limited but present potential of arthropods, especially flies, to transmit histomonosis between farms.

Improvement in the Immunity- and Vitamin D3-Activity-Related Gene Expression of Coccidiosis-Challenged Ross 708 Broilers in Response to the In Ovo Injection of 25-Hydroxyvitamin D3

Effects of the in ovo administration of two vitamin D3 sources (vitamin D3 (D3) and 25-hydroxyvitamin D3 (25OHD3)) on the expression of D3 activity- and immunity-related genes in broilers subjected to a coccidiosis infection were investigated. At 18 d of incubation (doi), five in ovo injection treatments were administrated to live embryonated Ross 708 broiler hatching eggs: non-injected (1) and diluent-injected (2) controls, or diluent injection containing 2.4 µg of D3 (3) or 2.4 µg of 25OHD3 (4), or their combination (5). Birds in the in ovo-injected treatments were challenged at 14 d of age (doa) with a 20× dosage of a live coccidial vaccine. At 14 and 28 doa, the expression of eight immunity-related genes (IL-2, IL-6, IL-10, TLR-4, TLR-15, MyD88, TGF-ß4, and IFN-γ) and four D3 activity-related genes (1α-hydroxylase, 25-hydroxylase, 24-hydroxylase, and VDR) in the jejunum of one bird in each treatment-replicate group were evaluated. No significant treatment effects were observed for any of the genes before challenge. However, at 2 weeks post-challenge, the expression of 1α-hydroxylase, TGF-ß4, and IL-10 increased in birds that received 25OHD3 alone in comparison to all the other in ovo-injected treatment groups. Additionally, the expression of 24-hydroxylase and IL-6 decreased in birds that received 25OHD3 in comparison to those injected with diluent or D3 alone. It was concluded that the in ovo injection of 2.4 µg of 25OHD3 may improve the intestinal immunity as well as the activity of D3 in Ross 708 broilers subjected to a coccidiosis challenge.

Effects of Feed-Through Sulfur on Growth Performance, Atmospheric Ammonia Levels, and Footpad Lesions in Broilers Raised Beginning with Built-Up Litter.

To the poultry industry, ammonia accumulation within poultry houses can be a costly issue, as this can lead to problems with bird performance, damage to economically important parts such as paws, and customer disapproval due to animal welfare concerns. Common management practices for ammonia control can be quite effective; however, these methods are used variably from farm to farm, which necessitates ammonia control measures that poultry companies can more uniformly implement across all contract growers. One possible measure is ammonia control through feed additives, which would allow poultry companies more direct control over the treatment. This project explored the efficacy of elemental sulfur added directly to the feed (feed-through sulfur) in controlling litter ammonia levels, live performance, and paw quality of broilers raised on built-up litter over three successive flocks. Feed-through sulfur on its own showed inconsistent effects on performance or footpad lesions after 38 days of production compared to sodium bisulfate or control treatments. However, combination of feed-through sulfur and sodium bisulfate showed a potential synergistic effect on ammonia levels and litter pH, although there were few differences between treatments and controls; therefore, additional research must be explored to confirm these observations.

Effects of Common Litter Management Practices on the Prevalence of Campylobacter jejuni in Broilers.

Campylobacter is an important foodborne pathogen and is naturally found in chickens. During broiler production, litter can become contaminated with Campylobacter when birds defecate, and this litter, in some countries, is typically reused for the next flock, potentially causing cross-contamination. The goal of this experiment was to observe if reusing contaminated litter could spread Campylobacter between flocks and to observe if common litter treatments could prevent this cross-contamination. To determine this, a flock of birds was inoculated with Campylobacter jejuni and allowed to naturally contaminate the litter for 42 days. After grow-out, birds were terminated, and litter was given five treatments: uninoculated fresh litter, untreated re-used litter, composted re-used litter, re-used litter treated with sodium bisulfate (45 kg/305 m2), and re-used litter composted and treated with sodium bisulfate (45 kg/305 m2). A second flock was placed on the litter, grown for 42 days, and tested for C. jejuni prevalence. Following inoculation of the first flock, high prevalence of C. jejuni was observed; however, after a 19-day down-time between flocks, no C. jejuni was detected in any samples from the second flock. These results indicate that re-used litter was not a significant reservoir for cross-contamination of broilers when provided a significant down-time between flocks.

Research Note: Bacterial composition of settled dust during growout of broiler chickens.

Dust present in poultry houses can disseminate bacteria in air and deposit them on surfaces. This study evaluated bacteria in settled dust during growout of broilers from 2 flocks (Flocks A and B). Dust samples for bacteria analyses were obtained during 6 wk of growout (Flocks A and B) and 1 wk after flock termination (Flock B) by environmental swabbing and collecting dust in petri dishes from multiple locations inside the poultry house. For weekly swabbing, dust deposited during each wk of the sampling period (noncumulatively, n = 12/wk) and cumulatively (n = 12/wk) throughout the sampling period was collected. Swabbed dust samples were analyzed for counts (log10 CFU/28 cm2) of aerobic bacteria, E. coli, coliforms, and Salmonella recovery. For petri dish dust collection, dust was collected in weekly and bi-weekly time spans during the sampling period and then analyzed for Salmonella recovery. Data were analyzed by one-way ANOVA and Fisher's Exact Test and means were separated using LSD. Only aerobic plate counts changed over time in dust during growout (Flocks A and B; P < 0.0001). In noncumulatively settled dust, aerobic bacteria (Flocks A and B; P < 0.0001), E. coli (Flock A; P = 0.0432), and coliforms (Flock B; P = 0.0303) varied during growout with peak counts on wk 5 or wk 6, wk 4, and wk 4, respectively, after bird placement. Salmonella recovery did not vary in cumulatively (3/72, 10/84) and noncumulatively (0/12, 10/84) settled dust during growout in both flocks. In dust sampled by bi-weekly collection in petri dishes, Salmonella recovery was highest (5/6) between wk 2 to wk 4 for Flock B (P = 0.0118). Overall, this study displayed that settled dust bacteria levels can fluctuate during broiler growout, and dust can contain Salmonella.

Investigation of the potential of aerosolized Salmonella Enteritidis on colonization and persistence in broilers from day 3 to 21.

The presence of Salmonella in air of poultry houses has been previously confirmed. Therefore, it is important to investigate the entry of Salmonella into broilers through air. The present study aimed to evaluate different levels of Salmonella Enteritidis aerosol inoculations in broiler chicks for colonization of ceca, trachea, and liver/spleen and persistence over time. In 3 independent trials, 112 one-day-old birds were randomly divided into 4 groups (n = 28/group). On d 1 of age, one group was exposed to an aerosol of sterile saline and the remaining three groups were exposed to an aerosol generated from one of 3 doses (103, 106, or 109 CFU/mL) of S. Enteritidis inoculum. Aerosol exposure time was 30 min/group and was performed using a nebulizer. On d 3, 7, 14, and 21 of age, ceca, trachea, and liver/spleen were aseptically removed. Ceca were cultured for Salmonella counts (log10 CFU/g) and all tissues were cultured for Salmonella prevalence. All tissues from the control group were Salmonella negative for all sampling days. On sampling d 3 and 7, ceca Salmonella counts were highest (5.14 and 5.11, respectively) when challenged with 109Salmonella (P ≤ 0.0281). Ceca Salmonella counts increased from d 3 (2.43) to d 7 (4.43), then remained constant when challenged at 103Salmonella, and counts decreased over time for all other groups. Tissue Salmonella prevalence increased with increasing challenge levels at all sampling timepoints (P ≤ 0.0213). Salmonella prevalence was low (0/18 to 4/18) and did not change over time following 103Salmonella challenge (P ≥ 0.2394). Prevalence decreased over time in ceca and trachea following 106 and 109Salmonella challenge (P ≤ 0.0483). Liver/spleen Salmonella prevalence increased from d 3 (13/18) to d 14 (18/18) and then decreased at d 21 (10/18) in birds exposed to an aerosol of 109Salmonella but remained constant over time for rest of the Salmonella inoculated groups. Overall, this study demonstrated the Salmonella colonization and persistence in different tissues in broilers following exposure to aerosolized Salmonella.

Heterakis gallinarum and Histomonas meleagridis DNA persists in chicken houses years after depopulation.

The poultry pathogen Histomonas meleagridis is transmitted by chicken cecal worms (Heterakis gallinarum) and is potentially transmitted by second order insect vectors and paratenic hosts. Darkling beetles (Alphitobius diaperinus) are poultry farm pests that infest barns. An outstanding question is the degree to which darkling beetles transmit both Heterakis and Histomonas. In this study we monitored populations of darkling beetles and assessed their positivity for both Heterakis and Histomonas by PCR. Uniquely, this study was conducted during the scheduled deconstruction of Auburn University's Poultry Research Farm. Therefore, we were able to monitor beetle and litter infection status months and years after bird depopulation. The duration of our monitoring continued through three seasons. We show that environmental DNA from both Heterakis and Histomonas persist in the environment long after prior infections, even in the absence of living Heterakis and its hosts. Finally, in an intensive search for live Heterakis, we discovered reniform nematodes (plant parasitic nematodes) residing in the soil floor of poultry farms.

A Comparison of Colonizing Ability Between Salmonella Enteritidis and Salmonella Heidelberg in Broiler Chickens Challenged Through Feed Administration.

With over 1 million estimated cases per year in the United States, foodborne salmonellosis is an important public health issue. Chicken products are frequent sources of foodborne Salmonella infection. These bacteria readily colonize the gastrointestinal tract of broiler chickens, and feed is a known vector. Past research has demonstrated that the survivability of Salmonella in feed is dependent on the serovar and strain. Therefore, the objective of this research was to compare colonization incidence of these two serovars in broiler chicken tissues by administration of feed contaminated with Salmonella enterica serovar Enteritidis (SE) or Salmonella enterica serovar Heidelberg (SH). A comparison was made with equal conditions so that there was no influence of other factors. Birds were inoculated by addition of Salmonella to the feed (1 × 104 colony-forming unit [CFU]/g of feed) at 14 days of age, and the following tissue samples were collected from each bird after grow-out (days 34-41 depending on the trial): abdominal cavity swab, bone marrow swab, cloaca swab, lung swab, breast, bursa and thymus, ceca, crop, kidney, liver and spleen, skin, spinal cord, thigh, and trachea. A higher percentage of birds inoculated with SE were positive in at least one tissue compared with SH (68% and 9%, respectively), and the SE inoculated birds also showed a higher number of positive tissue samples than SH (13.1% and 0.7%, respectively). Recovery of SH was low for all tissue samples. However, recovery of SE was variable between samples, with ceca showing the highest percentage (50%). These results indicate that challenge at day 14 through feed administration results in greater colonization by SE compared with SH, suggesting that monitoring and control methods for Salmonella in feed should focus on SE to have the greatest positive effect.

Impact of poultry litter Salmonella levels and moisture on transfer of Salmonella through associated in vitro generated dust.

Dust present in poultry houses can contain high concentrations of microorganisms and has the potential to include pathogens from the litter. The objective of this study was to examine in vitro the potential for litter to dust transfer of aerobic bacteria, Salmonella, E. coli, and coliforms, and the role of the litter moisture on this process. Poultry litter was inoculated with 102 to 109 CFU/mL of Salmonella Typhimurium to evaluate litter to dust transfer of bacteria (Experiment 1). To evaluate the effect of litter moisture on litter to dust microbial transfer (Experiment 2), litter was inoculated with 109S. Typhimurium with increasing amounts of sterilized water added for moisture adjustment. Dust was generated by blowing air in a direct stream onto inoculated litter while simultaneously collecting dust through impingement. Following litter and dust sample collection, microbial analyses for aerobic plate counts (APC),Salmonella, E. coli, and coliforms were conducted. Both experiments were repeated 5 times and their data analyzed by one-way ANOVA and simple logistic regression. In Experiment 1, APC of litter (log10 CFU/g) and dust samples (log10 CFU/L) were 10.55 and 4.92, respectively. Salmonella ranged from 1.70 to 6.16 log10 CFU/g in litter and only one dust sample had 1.10 log10 CFU/L of Salmonella. As Salmonella levels in litter increased, the probability of obtaining a dust Salmonella positive result also increased. In Experiment 2, attained moisture percentages were 13.0, 18.2, 23.0, 28.2, and 33.3%. Litter recovery for APC, Salmonella, E. coli, and coliforms counts did not differ (P > 0.05) with increasing moisture levels. Dust sample bacterial counts significantly decreased with increasing moisture levels (P < 0.0001). Results from this in vitro study indicate that there is potential for Salmonella to be present in generated dust and the higher levels of Salmonella in litter increase the likelihood of detecting Salmonella in dust. Additionally, with higher litter moisture percentage, prevalence of Salmonella in generated dust was decreased.

Prevalence of Select Intestinal Parasites in Alabama Backyard Poultry Flocks.

Keeping chickens as backyard pets has become increasingly popular in the United States in recent years. However, biosecurity is generally low in backyard flocks. As a consequence, they can serve as reservoirs for various pathogens that pose a risk for commercial poultry or human health. Eighty-four fecal samples, 82 from chickens and two from turkeys, from 64 backyard flocks throughout the state of Alabama were collected in the summers of 2017 and 2018. Coccidia oocysts were seen in 64.1% of flocks with oocyst counts in most samples below 10,000 oocysts per gram. Eggs of Ascaridia spp. or Heterakis gallinarum were observed in 20.3% of the flocks, and eggs of Capillaria spp. in 26.6% of the flocks. Egg counts were low, rarely exceeding 1000 eggs per gram. DNA extracted directly from fecal samples was investigated by PCR for other relevant parasites. The results showed that 4.7% of flocks were positive for Histomonas meleagridis, 18.8% of flocks for Tetratrichomonas gallinarum, 18.8% of flocks for Cryptosporidium spp. and 87.5% of flocks for Blastocystis spp. The results will help to provide information that can be used to design outreach programs to improve health and wellbeing of birds in backyard flocks.

Inoculation Route and Serotype Alter Salmonella Recovery in the Broiler's Digestive Tract.

Salmonella enterica serotypes Enteritidis (SE) and Heidelberg (SH) are consistently linked to poultry-related foodborne outbreaks and can be isolated from broiler parts in processing facilities. In order to control this pathogen's establishment in the broiler, entryways at the farm that lead to colonization must be considered. The objective of these trials was to determine if the inoculation route of either SE or SH altered its recovery in a market-age broiler's digestive tract if chicks were dosed on day of hatch. Chicks were given a 104 colony-forming units inoculation of SE or SH on day 0 via one of five inoculation routes (oral, intratracheal, subcutaneous, ocular, or cloacal) and then placed in pens (60-100 chicks/treatment). Broilers were reared for 32-36 days, then euthanatized, and samples of trachea, crop, liver and spleen (pooled), cecum, and a cloacal swab were collected. Samples were enriched and then analyzed on yes/no criteria based on Salmonella growth. Data were analyzed in JMP Pro 14.1 using the GLM procedure with the Student t-test to separate serotype means and a Tukey honestly significant difference test to separate inoculation means (P ≤ 0.05). All samples collected and all inoculation routes resulted in recovery of either serotype. The intratracheal inoculation, mimicking inhaled fomites, resulted in significantly higher recovery of Salmonella serotypes than did the other inoculation routes (P < 0.0001), indicating the importance of controlling respiratory contamination. When comparing serotypes, there was a significantly greater recovery of SH compared to SE based on samples collected (P = 0.001). SH also had significantly greater recovery from the cecum (P < 0.001) and the cloacal swab (P = 0.02). These trials indicate the need for further investigation of the intratracheal route, as well as reinforcing that the potential of systemic infection through grow out with either serotype is highly probable preharvest.

Influence of Eimeria spp. Infection on Chicken Jejunal Microbiota and the Efficacy of Two Alternative Products Against the Infection.

Eimeria spp. are important intestinal pathogens of chickens (Gallus gallus domesticus). Anticoccidial feed additives, chemicals, and ionophores have traditionally been used to control Eimeria infections in broiler production. Thus, the trend toward antibiotic-free and organic production requires new approaches to coccidiosis prevention. Two not mutually exclusive methods are the use of plant extracts with antiparasitic activity and manipulation of the intestinal microbiota by pre- and probiotics. In the present study, birds were inoculated with a combination of Eimeria acervulina, Eimeria maxima, and Eimeria tenella. We profiled the jejunal microbiome at multiple time points postinfection to investigate the changes in jejunum microbiota and to identify the time point of the maximum difference between infected and noninfected birds. Additionally, we assessed the anticoccidial effects of two anecdotal treatment methods, green tea and apple cider vinegar, as well as amprolium. Green tea and apple cider vinegar had no effect on oocyst shedding, but green tea reduced the mild unspecific lesions in coccidia-infected birds; there was no influence on unspecific lesions in uninfected controls. Jejunal contents were collected on the day of the infection and 1, 2, 4, 6, 10, and 14 days postinfection (dpi) for investigation of the intestinal microbiota by 16S ribosomal (r)RNA gene sequencing. Comparison of the untreated-uninfected and the untreated-infected groups showed a maximum community dissimilarity of 10 dpi. From 4 days after infection, Clostridiales were significantly enriched at the expense of Lactobacillales in infected compared with uninfected birds. Interestingly, treatment with green tea prevented proliferation of Clostridiales induced by the coccidia and increased the relative abundance of Melainabacteria.

Influencia de la infección de Eimeria spp. en el microbiota yeyunal del pollo y en la eficacia de dos productos alternativos contra la infección. Los protozoarios del género Eimeria spp. son importantes patógenos intestinales de los pollos (Gallus gallus domesticus). Los aditivos anticoccidiales en el alimento, los productos químicos y los ionóforos se han utilizado tradicionalmente para controlar las infecciones por Eimeria en la producción de pollos de engorde. Por lo tanto, la tendencia hacia la producción orgánica y libre de antibióticos requiere nuevos enfoques para la prevención de la coccidiosis. Dos métodos no mutuamente excluyentes son el uso de extractos de plantas con actividad antiparasitaria y la manipulación de la microbiota intestinal por prebióticos y probióticos. En el presente estudio, las aves fueron inoculadas con una combinación de Eimeria acervulina, Eimeria maxima y Eimeria tenella. Se determinó el perfil del microbioma yeyunal en múltiples puntos de tiempo después de la infección, para investigar los cambios en la microbiota del yeyuno e identificar el momento donde se observa la diferencia máxima entre las aves infectadas y no infectadas. Además, se evaluaron los efectos anticoccidiales de dos métodos de tratamiento basados por datos anecdóticos, el té verde y el vinagre de sidra de manzana, así como el amprolium. El té verde y el vinagre de sidra de manzana no tuvieron ningún efecto sobre el desprendimiento de oocistos, pero el té verde redujo las lesiones leves inespecíficas en aves infectadas con coccidia; no hubo influencia en lesiones inespecíficas en controles no infectados. Los contenidos yeyunales se recogieron el día de la infección y a los días 1, 2, 4, 6, 10 y 14 después de la infección (dpi) para la investigación de la microbiota intestinal mediante secuenciación del gene del ARN ribosómico 16S. La comparación de los grupos no tratados-no infectados y no tratados-infectados mostró una diferencia máxima en la comunidad a los diez días después de la inoculación. A partir de los cuatro días después de la infección, los agentes Clostridiales se enriquecieron significativamente a expensas de los Lactobacillales en aves infectadas en comparación con las aves no infectadas. Curiosamente, el tratamiento con té verde evitó la proliferación de Clostridiales inducida por los coccidios y aumentó la abundancia relativa de melainabacterias.

Evaluation of Next-Generation Amplicon Sequencing to Identify Eimeria spp. of Chickens.

Identifying Eimeria spp. circulating in a poultry flock assists in designing vaccine preventive programs, as different species do not cross-protect. Because species differ in anticoccidial drug susceptibility, species identification can also be used to optimize anticoccidial medication. In the present study, we designed pan-Eimeria-specific primers for the 18S rDNA and the cytochrome oxidase I (COI) genes, and tested whether next-generation sequencing of their amplicons allowed reliable identification of Eimeria spp. in samples of isolated oocysts. For each gene, two sets of primers to be used in a nested PCR (nPCR) system were designed. In silico evaluation of the primers using published sequences showed that nucleotide sequence identities of the nested amplicons were less than 97% between most species, while only identities of 18S rDNA genes of Eimeria necatrix and Eimeria tenella and between the COI genes of Eimeria mitis and Eimeria mivati were higher than 97%. Three vaccines and five Eimeria samples from chickens in backyard flocks were investigated by nPCRs and by direct PCRs (dPCR) using the nested (inner) primers with genomic DNA as the template. Seventeen further Eimeria samples from chickens in backyard flocks and three Eimeria samples from commercial broiler flocks were investigated only by nPCR. Sequencing nPCR products tended to detect more species than sequencing dPCR products and sequencing 18S rDNA products tended to detect more species than sequencing COI products. Regarding the detected species, there was a clear difference between the commercial broiler flocks and the backyard flocks. Eimeria acervulina, Eimeria maxima, and E. tenella/E. necatrix were the only species detected in broiler flocks, while the population in the backyard flocks was more varied, with Eimeria brunetti and E. mitis/E. mivati and the previously described operational taxonomic unit Y being more prevalent. Several sequences having less than 97% identity with one of the sequences used for clustering were detected in samples from backyard flocks. In conclusion, next-generation amplicon sequencing can be a useful tool to determine which Eimeria spp. are circulating in chicken flocks.

Evaluación de la secuenciación de nueva generación de amplicones para identificar Eimeria spp. de pollos. La identificación de Eimeria spp.que está circulando en una parvada avícola ayuda a diseñar programas preventivos de vacunas, ya que entre las diferentes especies no existe protección cruzada. Debido a que las especies difieren en la susceptibilidad a los medicamentos anticoccidiales, la identificación de especies también se puede utilizar para optimizar la medicación anticoccidial. En el presente estudio, se diseñaron iniciadores específicos genéricos de Eimeria para los genes de ADNr 18S y de citocromo oxidasa I (COI) y se evaluó si la secuenciación de nueva generación de los amplicones permitía la identificación confiable de Eimeria spp. en muestras de ooquistes aislados. Para cada gene, se diseñaron dos conjuntos de iniciadores que se utilizaron en un sistema de PCR anidado (nPCR). La evaluación in silico de los iniciadores usando secuencias publicadas mostró que las identidades de la secuencia de nucleótidos de los amplicones anidados eran inferiores al 97% entre la mayoría de las especies, mientras que solo las identidades de los genes de ADNr 18S de Eimeria necatrix y Eimeria tenella y entre los genes de citocromo oxidasa I de Eimeria mitis y Eimeria mivati fueron superiores al 97%. Se analizaron tres vacunas y cinco muestras de Eimeria de parvadas de pollos de traspatio con el sistema de PCR anidado y con PCR directa (dPCR) utilizando los iniciadores anidados (internos) con ADN genómico como modelo. Diecisiete muestras adicionales de Eimeria de parvadas de pollos de traspatio y tres muestras de Eimeria de parvadas de pollos de engorde comerciales fueron analizadas solo por PCR anidada. La secuenciación de productos PCR anidada tendió a detectar más especies que la secuenciación de productos por el método directo de PCR y la secuenciación de productos de ADNr 18S pareció detectar más especies que la secuenciación de productos de citocromo oxidasa I. Con respecto a las especies detectadas, hubo una clara diferencia entre las parvadas comerciales de engorde y las parvadas de traspatio. Eimeria acervulina, Eimeria maxima y E. tenella/E. Necatrix fueron las únicas especies detectadas en las parvadas de engorde, mientras que la población en las parvadas de traspatio fue más variada, con Eimeria brunetti y E. mitis/E. mivati, también la unidad taxonómica operativa Y descrita anteriormente fue más prevalente. Varias secuencias con menos del 97% de identidad con una de las secuencias utilizadas para la agrupación fueron detectadas en muestras de parvadas de traspatio. En conclusión, la secuenciación de nueva generación de amplicones puede ser una herramienta útil para determinar que especies de Eimeria spp. están circulando en parvadas de pollos.

Use of a Multiplex PCR for the Detection of Toxin-Encoding Genes netB and tpeL in Strains of Clostridium perfringens.

Some studies have shown that the NetB toxin may be an important virulence factor of Clostridium perfringens associated necrotic enteritis in poultry. Additionally, research has shown that strains of C. perfringens positive for both the netB gene and a second toxin-encoding gene, tpeL, appear to be more virulent than strains with only netB. In the past, detection of these genes has been performed relatively inefficiently using two single locus PCRs. This report describes a novel multiplex PCR developed to detect netB and tpeL simultaneously in C. perfringens strains isolated from cases of necrotic enteritis in broilers, providing a more efficient diagnostic tool in the screening of strains for these genes.