We used an embryo lethality assay (ELA) to assess virulence for different isolates from cases of bacterial chondronecrosis with osteomyelitis (BCO) in broilers. Lameness is among the most significant ...animal welfare issues in the poultry industry. Bacterial infections are a major cause of lameness and different bacterial species have been obtained from lame broilers. Reliable lab-based assays are required to assess relative virulence of bacteria obtained from lame broilers. ELA has been used to assess lethal dosage of Enterococcus faecalis and Enterococcus cecorum. We hypothesized that ELA could substitute for more laborious and costly assessments of BCO isolate pathogenicity using live birds. We evaluated 2 different levels of bacteria injected into eggs from layer and commercial broiler embryos. Significant findings include 1) Escherichia coli from neighboring farms operated by the same integrator had very different embryo lethality, 2) isolate Staphylococcus agnetis 908 had low virulence in ELA, even though this isolate can induce more than 50% BCO lameness, 3) Enterococcus cecorum 1415 also had low pathogenicity; even though it was recovered from severe bilateral tibial dyschondroplasia, 4) human and chicken BCO isolates of S. aureus had significant pathogenicity, 5) virulence for some isolates was highly variable possibly corresponding with quality of the embryos/fertile eggs used, and 6) ELA pathogenicity was much lower for our BCO isolates than previous reports which may reflect maternal environment. Overall, ELA virulence and BCO virulence are not always concordant indicating that ELA may not be an effective measure for assessing virulence with respect to BCO.
DNA extraction for downstream molecular diagnostic applications can be an expensive, time-consuming process. We devised a method to quickly extract total bacterial DNA from environmental samples ...based on the sodium hydroxide lysis of cells with or without capture by magnetic beads for subsequent PCR or quantitative PCR. The final DNA extraction method using NaOH is extremely low-cost and can be completed in as little as 10 min at room temperature with dilution, or the DNA can be further purified using silica-coated paramagnetic beads. NaOH extraction was effective for Gram-negative and Gram-positive bacteria in samples from air, soil, sewage, food, laboratory surfaces, and chicken cloacal swabs. The NaOH extraction method was comparable to commercial kits for extraction of DNA from pig fecal samples for 16S amplicon sequencing analyses. We demonstrated that an impinger and portable pump can efficiently capture bacteria from the air in poultry facilities for rapid NaOH extraction to quantify total bacteria and for detection of specific species using qPCR. The air sampling and NaOH extraction procedures are well suited for routine, high-throughput screening and for metagenomic analyses for specific pathogens, even in resource-limited situations.
Our prior research demonstrated a 20% to 25% reduction in bacterial chondronecrosis with osteomyelitis (BCO) lameness in broilers with organic Zn, Mn, and Cu (Availa® ZMC) supplementation. Expanding ...on this, we investigated the optimal timing for Availa® ZMC feeding to mitigate BCO lameness and reduce feed additive costs in the poultry industry. In this study, we compared the application of 0.15% Availa® ZMC for 56 days, the first 28 days, and the last 28 days. The experimental design was a randomized block design involving 1560 one-day-old chicks distributed across two wire-floor pens as BCO source infection and four treatment groups with six replicates. The source of BCO infection exhibited a cumulative lameness incidence of 83%, whereas the negative control group showed a 77% cumulative incidence of lameness (p = 0.125). Administering 0.15% of Availa® ZMC during the initial 28 d resulted in a 41.3% reduction in BCO incidence, significantly different from the supplementation during the last 28 d (p < 0.05). However, this reduction did not differ substantially (p > 0.05) from the 56d application period. Hence, administering 0.15% Availa® ZMC during the first four weeks emerges as the optimal timing protocol, providing a defense against lameness comparable to the continuous supplementation throughout the complete production duration. Implementing this feeding approach reduces the cost of feed additive, promotes the health of skeletal bones, and effectively protects against BCO lameness in broilers, offering a valuable consideration for producers seeking optimal outcomes in the poultry industry.
has been previously associated with subclinical or clinically mild cases of mastitis in dairy cattle and is one of several Staphylococcal species that have been isolated from the bone and blood of ...lame broilers. We reported that
could be obtained frequently from bacterial chondronecrosis with osteomyelitis (BCO) lesions of lame broilers Al-Rubaye
, PLoS One 10:e0143336, 2015, https://doi.org/10.1371/journal.pone.0143336. A particular isolate,
908, can induce lameness in over 50% of exposed chickens, exceeding normal BCO incidences in broiler operations. We reported the assembly and annotation of the genome of isolate 908. To better understand the relationship between dairy cattle and broiler isolates, we assembled 11 additional genomes for
isolates, an additional chicken BCO strain, and ten isolates from cattle milk, mammary gland secretions or udder skin, from the collection at the University of Missouri. To trace phylogenetic relationships, we constructed phylogenetic trees based on multi-locus sequence typing, and Genome-to-Genome Distance Comparisons. Chicken isolate 908 clustered with two of the cattle isolates along with three isolates from chickens in Denmark and an isolate of
we isolated from a BCO lesion on a commercial broiler farm in Arkansas. We used a number of BLAST tools to compare the chicken isolates to those from cattle and identified 98 coding sequences distinguishing isolate 908 from the cattle isolates. None of the identified genes explain the differences in host or tissue tropism. These analyses are critical to understanding how Staphylococci colonize and infect different hosts and potentially how they can transition to alternative niches (bone vs dermis).
has been recently recognized as associated with disease in dairy cattle and meat type chickens. The infections appear to be limited in cattle and systemic in broilers. This report details the molecular relationships between cattle and chicken isolates in order to understand how this recently recognized species infects different hosts with different disease manifestations. The data show the chicken and cattle isolates are very closely related but the chicken isolates all cluster together suggesting a single jump from cattle to chickens.
Lameness is a major issue in animal welfare and the broiler industry. Bacterial chondronecrosis with osteomyelitis (BCO) is one of the main causes of lameness. Many staphylococcal species, including ...Staphylococcus agnetis isolate 908, have been isolated from the bones and blood of lame broilers at the University of Arkansas. Staphylococcus agnetis is a coagulase-variable, Gram-positive bacterial species that has been previously associated with subclinical or mild clinical cases of mastitis in dairy cattle. The annotated complete genome of hypervirulent strain 908 was published at NCBI. In this study, it has been compared to nine genomes we assembled for hypervirulent isolates in dairy cattle. Phylogenomic analyses of chicken and cattle isolates of S. agnetis and Staphylococcus hyicus suggest a very close relationship between the cattle and chicken isolates. The hypervirulent chicken isolate, 908, clustered with two of the cattle isolates, including strain 1379. A catalogue of gene differences between the cattle and chicken isolates was constructed using reciprocal blast analyses at the nucleotide and polypeptide level. More than 40 genes and 3 plasmids from strain 908 are absent or poorly conserved in any of the cattle S. agnetis isolates. No transformation protocol has been described for S. agnetis. Subsequently, an electroporation procedure has been optimized for DNA transformation of Staphylococcus agnetis. Therefore, we have optimized an electroporation method for DNA transformation so that we regularly obtain 10 to 20 transformants per ng using a Gram+/Gram- shuttle vector. Moreover, among the BCO pathogens isolated from the lame broilers, there are a number of Staphylococcus species, such as, S. agnetis, S. hyicus, S. chromogenes, S. aureus, S. cohnii, S. saprophyticus, S. epidermidis, and S. capitis, which are hard to accurately identify based just on genes like 16S rDNA. Therefore, using pfbA gene, a novel PCR assay was optimized for Staphylococcus species discrimination and strain typing. Moreover, extraction of bacterial DNA for subsequent molecular diagnostic applications remains a costly and time-consuming operation. We developed a technique for rapidly extracting genomic DNA from the BCO pathogens and other environmental bacteria based on sodium hydroxide cell lysis with or without magnetic bead capture. Finally, the BCO pathogens are transmitted via air. Our efficient air sampling system was designed for the quick screening of these airborne BCO pathogens and is transferable to monitor agriculturally important pathogenic bacteria.
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