Potential non-invasive detection of lesions in broiler femur heads: application of the DXA imaging system.

Leg health is a significant economic and welfare concern for the poultry industry. Current methods of detection rely on visual assessment of the legs and gait scores and bone scoring during necropsy for full characterization. Additionally, the current scoring of femurs only examines the external surface of the femoral head. Through the use of the dual-energy X-ray absorptiometry (DXA) imaging system, we show the presence of a necrotic region in the femurs that would otherwise be considered healthy based on the current evaluation procedures. Importantly, these lesions were present in almost 60% (22 of 37) of femurs that scored normal for femoral head necrosis (FHN). Additionally, these femurs showed greater bone mineral content (BMC) relative to weight compared to their counterparts with no lucent lesions (6.95% ± 0.20% vs. 6.26% ± 0.25; p = 0.038). Identification of these lesions presents both a challenge and an opportunity. These subclinical lesions are likely to be missed in routine scoring procedures for FHN and can inadvertently impact the characterization of the disease and genetic selection programs. Furthermore, this imaging system can be used for in vivo, ex vivo, and embryonic (egg) studies and, therefore, constitutes a potential non-invasive method for early detection of bone lesions in chickens and other avian species.

Dietary inclusion of phytase and stimbiotic decreases mortality and lameness in a wire ramp challenge model in broilers.

RESEARCH HIGHLIGHTS: Wire ramp model reproducibly induced lameness/BCO in broilers.Treatments did not affect growth, but phytase with stimbiotic significantly reduced BCO.Phytase increased circulating inositol, and wire flooring decreased bone inositol.

Comparative- and network-based proteomic analysis of bacterial chondronecrosis with osteomyelitis lesions in broiler's proximal tibiae identifies new molecular signatures of lameness.

Bacterial Chondronecrosis with Osteomyelitis (BCO) is a specific cause of lameness in commercial fast-growing broiler (meat-type) chickens and represents significant economic, health, and wellbeing burdens. However, the molecular mechanisms underlying the pathogenesis remain poorly understood. This study represents the first comprehensive characterization of the proximal tibia proteome from healthy and BCO chickens. Among a total of 547 proteins identified, 222 were differentially expressed (DE) with 158 up- and 64 down-regulated proteins in tibia of BCO vs. normal chickens. Biological function analysis using Ingenuity Pathways showed that the DE proteins were associated with a variety of diseases including cell death, organismal injury, skeletal and muscular disorder, immunological and inflammatory diseases. Canonical pathway and protein-protein interaction network analysis indicated that these DE proteins were involved in stress response, unfolded protein response, ribosomal protein dysfunction, and actin cytoskeleton signaling. Further, we identified proteins involved in bone resorption (osteoclast-stimulating factor 1, OSFT1) and bone structural integrity (collagen alpha-2 (I) chain, COL2A1), as potential key proteins involved in bone attrition. These results provide new insights by identifying key protein candidates involved in BCO and will have significant impact in understanding BCO pathogenesis.

Role of autophagy machinery dysregulation in bacterial chondronecrosis with osteomyelitis.

Autophagy is a cell survival and homeostasis mechanism involving lysosomal degradation of cellular components and foreign bodies. It plays a role in bone homeostasis, skeletal diseases, and bacterial infections as both a cell-survival or cell-death pathway. This study sought to determine if autophagy played a role in bacterial chondronecrosis with osteomyelitis (BCO). BCO is a prominent cause of lameness in modern broilers and results from bacterial infection of mechanically stressed leg bone growth plates. The protein and gene expression of key autophagy machinery was analyzed in both normal and BCO-affected broilers using real-time qPCR and immunoblot, respectively. Gene expression showed a significant downregulation of key target signatures involved in every stage of autophagy in BCO-affected bone, such as ATG13, SQSTM1 (p62), ATG9B, ATG16L, ATG12, LC3C, and RAB7A. Additionally, protein expression for LC3 was also significantly lower in BCO. An in vitro study using human fetal osteoblast cells challenged with BCO isolate, Staphylococcus agnetis 908, showed a similar dysregulation of autophagy machinery along with a significant decrease in cell viability. When autophagy was inhibited via 3-methyladenine or chloroquine, comparable decreases in cell viability were seen along with dysregulation of autophagy machinery. Together, these results are the first to implicate autophagy machinery dysregulation in the pathology of BCO.

Local and Systemic Cytokine, Chemokine, and FGF Profile in Bacterial Chondronecrosis with Osteomyelitis (BCO)-Affected Broilers.

Complex disease states, like bacterial chondronecrosis with osteomyelitis (BCO), not only result in physiological symptoms, such as lameness, but also a complex systemic reaction involving immune and growth factor responses. For the modern broiler (meat-type) chickens, BCO is an animal welfare, production, and economic concern involving bacterial infection, inflammation, and bone attrition with a poorly defined etiology. It is, therefore, critical to define the key inflammatory and bone-related factors involved in BCO. In this study, the local bone and systemic blood profile of inflammatory modulators, cytokines, and chemokines was elucidated along with inflammasome and key FGF genes. BCO-affected bone showed increased expression of cytokines IL-1ß, while BCO-affected blood expressed upregulated TNFα and IL-12. The chemokine profile revealed increased IL-8 expression in both BCO-affected bone and blood in addition to inflammasome NLRC5 being upregulated in circulation. The key FGF receptor, FGFR1, was significantly downregulated in BCO-affected bone. The exposure of two different bone cell types, hFOB and chicken primary chondrocytes, to plasma from BCO-affected birds, as well as recombinant TNFα, resulted in significantly decreased cell viability. These results demonstrate an expression of proinflammatory and bone-resorptive factors and their potential contribution to BCO etiology through their impact on bone cell viability. This unique profile could be used for improved non-invasive detection of BCO and provides potential targets for treatments.

Impact of experimentally induced bacterial chondronecrosis with osteomyelitis (BCO) lameness on health, stress, and leg health parameters in broilers.

Stress and lameness negatively affect the health, production, and welfare of broilers. Bacterial chondronecrosis with osteomyelitis (BCO) is a leading cause of stress and lameness in commercial broilers. External changes in skin temperature related to changes in blood flow can be detected with infrared thermography (IRT), offering a noninvasive tool to assess the health of animals. This study compared physiological and noninvasive measures of stress and lameness in clinically healthy and lame male broiler chickens between 25 and 56 d. Birds were raised in pens within separate environmental chambers containing either litter flooring (sound) or wire flooring, with the latter established to induce BCO lameness (lame). Physiological and noninvasive measures of stress and lameness were collected: body weight, (BW), relative bursa weight, core body temperature, corticosterone (CORT) concentrations in serum and feathers, surface temperatures of the head (eye and beak) and leg (hock, shank, and foot) regions by infrared thermography (IRT), leg blood oxygen saturation (leg O2), and BCO lesion severity scores of tibial head necrosis (THN) and femoral head necrosis (FHN). Lame birds exhibited greater FHN and THN lesion severities, core body temperatures, and serum CORT (P < 0.05), but had lower BW, relative bursa weight, leg O2, and IRT surface temperatures of the beak, hock, shank, and foot compared with sound birds (P < 0.05). The difference in THN lesion severity between sound and lame birds decreased with age. Linear relationships between leg O2 with IRT leg surface temperatures were positive and negative between leg O2 with BCO lesion severity (P < 0.05). There were negative correlations between serum CORT with hock, shank and foot temperatures (P < 0.001), indicating that BCO is stressful. These results indicate that birds lame from BCO are stressed, have reduced oxygen saturation of blood in their legs, and that IRT surface temperatures can be used as noninvasive indicators of stress and lameness in broilers.

Evidence of Mitochondrial Dysfunction in Bacterial Chondronecrosis With Osteomyelitis-Affected Broilers.

A leading cause of lameness in modern broilers is bacterial chondronecrosis with osteomyelitis (BCO). While it is known that the components of BCO are bacterial infection, necrosis, and inflammation, the mechanism behind BCO etiology is not yet fully understood. In numerous species, including chicken, mitochondrial dysfunction has been shown to have a role in the pathogenicity of numerous diseases. The mitochondria is a known target for intracellular bacterial infections, similar to that of common causative agents in BCO, as well as a known regulator of cellular metabolism, stress response, and certain types of cell death. This study aimed to determine the expression profile of genes involved in mitochondrial biogenesis, dynamics, and function. RNA was isolated form the tibias from BCO-affected and healthy broilers and used to measure target gene expression via real-time qPCR. Mitochondrial biogenesis factors PGC-1α and PGC-1ß were both significantly upregulated in BCO along with mitochondrial fission factors OMA1, MTFR1, MTFP1, and MFF1 as well as cellular respiration-related genes FOXO3, FOXO4, and av-UCP. Conversely, genes involved in mitochondrial function, ANT, COXIV, and COX5A showed decreased mRNA levels in BCO-affected tibia. This study is the first to provide evidence of potential mitochondrial dysfunction in BCO bone and warrants further mechanistic investigation into how this dysfunction contributes to BCO etiology.

Microbiota Analysis of Chickens Raised Under Stressed Conditions.

A substantial progress has been made toward understanding stress-associated gut and extraintestinal microbiota. However, a comprehensive understanding of the extraintestinal microbiota of chickens raised under stressed conditions is lacking. In this study, chickens were raised on a wire-floor model to induce stress, and the microbiota in the gut (ceca) and extraintestinal sites (blood, femur, and tibia) were characterized at different ages (1, 17, and 56 days) using 16S rRNA gene microbiota profiling. Open reference OTU picking showed extraintestinal sites had a significantly higher number of unassigned OTUs compared to ceca across all ages of chickens. Extraintestinal sites of all ages, irrespective of body sites, as well as ceca of 1 day-old chickens had significantly lower alpha diversity than ceca of older chickens. Intriguingly, bacterial diversity (alpha and beta) and OTU interaction network analysis showed relatively stable bacterial composition within the extraintestinal sites of chickens regardless of age and sites compared to ceca. Furthermore, assessment using UniFrac distance suggested the gut as a possible source of extraintestinal bacteria. Lastly, LEfSe analysis showed that both commensal and pathogenic bacteria were translocated into the extraintestinal tissues and organs under the stress. Extraintestinal sites have highly abundant novel taxa that need to be further explored. In ovo microbiota colonization and/or translocation of circulating maternal blood microbiota into ovarian follicles might be the source of intestinal and extraintestinal microbiota in 1 day-old chickens. Our comprehensive microbiota data including extraintestinal sites in reference to gut provide unique insights into microbiota of chickens raised under stressed conditions, which may be relevant in other animal species as well.

Chondronecrosis with osteomyelitis in broilers: further defining a bacterial challenge model using standard litter flooring and protection with probiotics.

This report demonstrates that high levels of lameness can be induced by a limited bacterial challenge in drinking water for birds raised on litter flooring, comparable with lameness induced by the gold standard for inducing lameness, growth on suspended wire flooring. The bacterium used in the challenge was cultured from lesions in birds induced for bacterial chondronecrosis with osteomyelitis (BCO) in the wire-flooring model so the epidemiology appears similar. The litter-flooring model could better approximate broiler operations. Furthermore, the work demonstrates that 2 commercial probiotics (GalliProTect and GalliProMax) can reduce lameness in the bacterial challenge litter-flooring model. Lameness attributable to BCO is one of the most significant animal welfare issues for broiler production. The wire-flooring and litter-flooring models afford alternatives for understanding the etiology, and epidemiology of BCO, and development of management strategies to reduce lameness. Probiotics afford a promising management strategy. The results suggest that the probiotic protection may extend beyond just intestinal health and intestinal barrier function.