[Show abstract][Hide abstract] ABSTRACT: Background
The Smyth line (SL) chicken is the only animal model for autoimmune vitiligo that spontaneously displays all clinical and biological manifestations of the human disorder. To understand the genetic components underlying the susceptibility to develop SL vitiligo (SLV), whole genome resequencing analysis was performed in SLV chickens compared with non-vitiliginous parental Brown line (BL) chickens, which maintain a very low incidence rate of vitiligo.
Illumina sequencing technology and reference based assembly on Red Jungle Fowl genome sequences were used. Results of genome resequencing of pooled DNA of each 10 BL and SL chickens reached 5.1x and 7.0x coverage, respectively. The total number of SNPs was 4.8 and 5.5 million in BL and SL genome, respectively. Through a series of filtering processes, a total of ~1 million unique SNPs were found in the SL alone. Eventually of the 156 reliable marker SNPs, which can induce non-synonymous-, frameshift-, nonsense-, and no-start mutations in amino acid sequences in proteins, 139 genes were chosen for further analysis. Of these, 14 randomly chosen SNPs were examined for SNP verification by PCR and Sanger sequencing to detect SNP positions in 20 BL and 70 SL chickens. The results of the analysis of the 14 SNPs clearly showed differential frequencies of nucleotide bases in the SNP positions between BL and SL chickens. Bioinformatic analysis showed that the 156 most reliable marker SNPs included genes involved in dermatological diseases/conditions such as ADAMTS13, ASPM, ATP6V0A2, BRCA2, COL12A1, GRM5, LRP2, OBSCN, PLAU, RNF168, STAB2, and XIRP1. Intermolecular gene network analysis revealed that candidate genes identified in SLV play a role in networks centered on protein kinases (MAPK, ERK1/2, PKC, PRKDC), phosphatase (PPP1CA), ubiquitinylation (UBC) and amyloid production (APP).
Various potential genetic markers showing amino acid changes and potential roles in vitiligo development were identified in the SLV chicken through genome resequencing. The genetic markers and bioinformatic interpretations of amino acid mutations found in SLV chickens may provide insight into the genetic component responsible for the onset and the progression of autoimmune vitiligo and serve as valuable markers to develop diagnostic tools to detect vitiligo susceptibility.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-707) contains supplementary material, which is available to authorized users.
[Show abstract][Hide abstract] ABSTRACT: A genome-wide SNP survey was used to identify chromosomal regions that showed linkage disequilibrium with respect to ascites susceptibility and ventricular hypertrophy in an F2 cross between previously described ascites-resistant and -susceptible lines. Variable number tandem repeats were used to obtain genotype data to further characterize these regions. A region on chromosome 9 (12 to 13 Mbp in 2011 assembly) shows association with ascites in the ascites lines and in several commercial broiler breeder lines with a significant sex effect. There are 2 candidate genes, AGTR1 (an angiotensin II type 1 receptor) and UTS2D (urotensin 2 domain containing), in this region that have been associated with hypertension and hypoxic response in mammals.
[Show abstract][Hide abstract] ABSTRACT: Newly hatched turkey poults were infected with 10(2) oocysts of Eimeria adenoeides and subsequently reinfected with 10(3) and 10(4) oocysts at 6 and 12 d of age, respectively. Three peaks in oocyst production were observed in the feces of poults following this series of infections. A second group of poults given the same dosing regimen was challenged with 5 × 10(4) oocysts/poult at different times to evaluate the acquisition of immunity. Judging by weight gain and mortality, no protection had been acquired at 6 d of age, but partial protection was observed by 12 and 18 d of age. A third group of poults were also infected with 10(2) oocysts and subsequently reinfected with 10(3) and 10(4) oocysts at 6 and 12 d of age to evaluate cellular immune responses to infection. Sections of ceca from infected poults showed a significantly higher leukocyte infiltration on d 6, 10, 12, 16, and 18 after infection than uninfected controls. The percent area occupied by CD4+ and CD8+ lymphocytes in the ceca, as assessed by immunohistochemistry, was significantly elevated in infected poults on d 12, 16, and 18. The relative expression of chemokine CXCLi2, and cytokines IL1β, IFNγ, IL10, IL13, IL2, IL12b, and IL18 was measured by real-time reverse-transcription PCR. The expression of CXCLi2 and IL10 was found to be elevated on d 12, and IFNγ on d 10, 12, and 16. Expression of IL13 and IL18 was increased on d 10 and IL2 on d 10 and 16, and that of IL12b on d 16 in infected poults. Increase in the infiltration of leukocytes, percent area occupied by CD4+ and CD8+ lymphocytes, and changes in the relative expression of cytokines in the ceca characterize the dynamics of immune responses in turkey poults infected with E. adenoeides early in life.
[Show abstract][Hide abstract] ABSTRACT: Pulmonary arterial hypertension (PAH) syndrome in broilers (also known as ascites syndrome and pulmonary hypertension syndrome) can be attributed to imbalances between cardiac output and the anatomical capacity of the pulmonary vasculature to accommodate ever-increasing rates of blood flow, as well as to an inappropriately elevated tone (degree of constriction) maintained by the pulmonary arterioles. Comparisons of PAH-susceptible and PAH-resistant broilers do not consistently reveal differences in cardiac output, but PAH-susceptible broilers consistently have higher pulmonary arterial pressures and pulmonary vascular resistances compared with PAH-resistant broilers. Efforts clarify the causes of excessive pulmonary vascular resistance have focused on evaluating the roles of chemical mediators of vasoconstriction and vasodilation, as well as on pathological (structural) changes occurring within the pulmonary arterioles (e.g., vascular remodeling and pathology) during the pathogenesis of PAH. The objectives of this review are to (1) summarize the pathophysiological progression initiated by the onset of pulmonary hypertension and culminating in terminal ascites; (2) review recent information regarding the factors contributing to excessively elevated resistance to blood flow through the lungs; (3) assess the role of the immune system during the pathogenesis of PAH; and (4) present new insights into the genetic basis of PAH. The cumulative evidence attributes the elevated pulmonary vascular resistance in PAH-susceptible broilers to an anatomically inadequate pulmonary vascular capacity, to excessive vascular tone reflecting the dominance of pulmonary vasoconstrictors over vasodilators, and to vascular pathology elicited by excessive hemodynamic stress. Emerging evidence also demonstrates that the pathogenesis of PAH includes characteristics of an inflammatory/autoimmune disease involving multifactorial genetic, environmental, and immune system components. Pulmonary arterial hypertension susceptibility appears to be multigenic and may be manifested in aberrant stress sensitivity, function, and regulation of pulmonary vascular tissue components, as well as aberrant activities of innate and adaptive immune system components. Major genetic influences and high heritabilities for PAH susceptibility have been demonstrated by numerous investigators. Selection pressures rigorously focused to challenge the pulmonary vascular capacity readily expose the genetic basis for spontaneous PAH in broilers. Chromosomal mapping continues to identify regions associated with ascites susceptibility, and candidate genes have been identified. Ongoing immunological and genomic investigations are likely to continue generating important new knowledge regarding the fundamental biological bases for the PAH/ascites syndrome.
[Show abstract][Hide abstract] ABSTRACT: Listeria monocytogenes continues to be a major foodborne pathogen that causes food poisoning, and sometimes death, among immunosuppressed people and abortion among pregnant women. In this study, magnetic nanoparticles with a diameter of 30 nm were functionalized with anti-L. monocytogenes antibodies via biotin-streptavidin bonds to become immunomagnetic nanoparticles (IMNPs) to capture L. monocytogenes in a sample during a 2-h immunoreaction. A magnetic separator was used to collect and hold the IMNPs-L. monocytogenes complex while the supernatants were removed. After the washing step, the nanoparticle-L. monocytogenes complex was separated from the sample and injected into a microfluidic chip. The impedance change caused by L. monocytogenes was measured by an impedance analyzer through the interdigitated microelectrode in the microfluidic chip. For L. monocytogenes in phosphate-buffered saline solution, up to 75% of the cells in the sample could be separated, and as few as three to five cells in the microfluidic chip could be detected, which is equivalent to 10(3) CFU/ml of cells in the original sample. The detection of L. monocytogenes was not interfered with by other major foodborne bacteria, including E. coli O157:H7, E. coli K-12, L. innocua, Salmonella Typhimurium, and Staphylococcus aureus. A linear correlation (R(2) = 0.86) was found between the impedance change and the number of L. monocytogenes in a range of 10(3) to 10(7) CFU/ml. Equivalent circuit analysis indicated that the impedance change was mainly due to the decrease in medium resistance when the IMNPs-L. monocytogenes complexes existed in mannitol solution. Finally, the immunosensor was evaluated with food sample tests; the results showed that, without preenrichment and labeling, 10(4) and 10(5) CFU/ml L. monocytogenes in lettuce, milk, and ground beef samples could be detected in 3 h.
[Show abstract][Hide abstract] ABSTRACT: White striping is a condition associated with heavier broiler breast fillets and is observed grossly as white striations seen parallel to the direction of the muscle fibers. The present study was intended to assess the consumer acceptance of broiler fillets with different degrees of white striping condition. High resolution digital images of fillets, representative of varying degrees of white striping, were shown to 75 consumers in a blind study. Individual images were presented using a completely randomized design. There were 4 replicates of individual fillets within each white striping category (normal = NORM, moderate = MOD, and severe = SEV) and one picture of tray pack (3 fillets) for each category. The consumers were asked to express their overall liking for appearance with a 9-point hedonic scale (9 = like extremely; 1 = dislike extremely) and purchase intent using a 5-point scale (5 = definitely would buy; 1 = definitely would not buy). An open-ended comments section was also included. The results showed that NORM fillets had a significantly higher hedonic score (6.9) than the MOD fillets (6.1), which was also significantly higher than the SEV fillets (4.5), indicating that as severity of white striping increased, the consumer acceptance decreased. From the distribution of the responses, 10.7, 22.4, and 56.7% of the consumers disliked the NORM, MOD, and SEV fillets, respectively. Furthermore, the average purchase intent score for the NORM fillets (3.6) was significantly higher than those with 2 degrees of white striping (2.4 and 2.5, respectively), suggesting that the consumers were more likely to buy NORM fillets. Over 50% of the consumers indicated that they would probably not or definitely not buy MOD or SEV fillets. The correspondence analysis of open-ended comments revealed the major reasons for the dislike of the white-striped meat was that the fillets had a more fatty or marbled appearance. The results of the study suggest that the white striping does affect the consumer acceptance based on the appearance of the fillets.
[Show abstract][Hide abstract] ABSTRACT: The Smyth line (SL) of chicken is an excellent avian model for human autoimmune vitiligo. The etiology of vitiligo is complicated and far from clear. In order to better understand critical components leading to vitiligo development, cDNA microarray technology was used to compare gene expression profiles in the target tissue (the growing feather) of SL chickens at different vitiligo (SLV) states.
Compared to the reference sample, which was from Brown line chickens (the parental control), 395, 522, 524 and 526 out of the 44 k genes were differentially expressed (DE) (P ≤ 0.05) in feather samples collected from SL chickens that never developed SLV (NV), from SLV chickens prior to SLV onset (EV), during active loss of pigmentation (AV), and after complete loss of melanocytes (CV). Comparisons of gene expression levels within SL samples (NV, EV, AV and CV) revealed 206 DE genes, which could be categorized into immune system-, melanocyte-, stress-, and apoptosis-related genes based on the biological functions of their corresponding proteins. The autoimmune nature of SLV was supported by predominant presence of immune system related DE genes and their remarkably elevated expression in AV samples compared to NV, EV and/or CV samples. Melanocyte loss was confirmed by decreased expression of genes for melanocyte related proteins in AV and CV samples compared to NV and EV samples. In addition, SLV development was also accompanied by altered expression of genes associated with disturbed redox status and apoptosis. Ingenuity Pathway Analysis of DE genes provided functional interpretations involving but not limited to innate and adaptive immune response, oxidative stress and cell death.
The microarray results provided comprehensive information at the transcriptome level supporting the multifactorial etiology of vitiligo, where together with apparent inflammatory/innate immune activity and oxidative stress, the adaptive immune response plays a predominant role in melanocyte loss.
[Show abstract][Hide abstract] ABSTRACT: Idiopathic pulmonary arterial hypertension (IPAH) is a disease of unknown cause that is characterized by elevated pulmonary arterial pressure and pulmonary vascular resistance, and by extensive vascular remodelling. In human IPAH patients, remodelling of the pulmonary vasculature results in the formation of plexiform lesions in the terminal pulmonary arterioles. Various molecules are expressed in the human plexiform lesions, including alpha smooth muscle actin, von Willebrand factor, vascular endothelial growth factor, vascular endothelial growth factor receptor type 2, hypoxia inducible factor-1α, survivin, tenascin, collagen, fibronectin, and various immune/inflammatory cells such as, cytotoxic lymphocytes, B lymphocytes, MHC class II cells, and monocytes/macrophages are also present. Plexiform lesions rarely develop in the lungs of laboratory animals, but plexiform-like complex vascular lesions (CVL) do develop spontaneously in the lungs of broiler chickens from an IPAH-susceptible line. To examine angioproliferative and immune-system-related activities associated with CVL in broiler lungs, paraformaldehyde-fixed, paraffin-embedded lung sections from 8-week-old to 24-week-old broiler chickens were stained immunohistochemically using monoclonal or polyclonal antibodies specific for angioproliferative molecules and immune/inflammatory cells. The CVL in the lungs of broiler chickens exhibited positive staining for both angioproliferative molecules and immune/inflammatory cells. These observations combined with the close histological resemblance of broiler CVL to the plexiform lesions of human IPAH patients further validates chickens from our IPAH-susceptible line as an excellent animal model of spontaneous plexogenic arteriopathy.
[Show abstract][Hide abstract] ABSTRACT: Differences in serum protein profiles were analyzed to identify possible biomarkers associated with a poultry
leg problem named tibial dyschondroplasia (TD) that can lead to lameness. A bead-based affinity matrix (ProteoMinerTM)
containing a combinatorial library of hexapeptides was used to deplete high abundant proteins and enrich the less
abundant ones to compare between the sera of six-week old normal and TD affected chickens. Equal amounts of proteins
in ProteoMiner depleted serum from control and TD-affected birds were subject to 2D gel electrophoresis, image analysis,
and compared to identify the differentially expressed protein spots. The protein spots were characterized using in-gel
trypsin digestion followed by mass spectrometry (MS). Of 46 matched protein spots in the gels, 33 were identified by
peptide mass finger printing (PMF) and tandem mass spectrometry (MS/MS). Eight spots corresponding to
immunoglobulins (Ig) were up-regulated in birds with TD and two spots down regulated. The up-regulated Ig proteins
belonged to IgM and IgY(IgG) classes indicated by the identification of ‘mu’ chain and Fc fragment associated peptides
respectively. Enzyme linked immunosorbent assay corroborated an increase in serum IgM levels but not IgG. Although
the significance of the increase in IgM proteins in the serum of TD-affected chickens is not understood, it is likely that
IgM plays some role in the removal of apoptotic chondrocytes which abound within TD lesions.
The Open Proteomics Journal 02/2012; 5(1875-0397):1-7.
[Show abstract][Hide abstract] ABSTRACT: The Smyth line (SL) of chicken is an excellent animal model for human autoimmune vitiligo. In SL vitiligo (SLV), postnatal loss of melanocytes in feathers appears to be due to cell-mediated immunity. In this study, leukocyte infiltration and associated expression (RNA) of immune function-related cytokines in growing feathers were investigated throughout SLV development and progression. Both leukocyte infiltration and cytokine expression levels started to increase near visible SLV onset (early SLV), reached peak levels during active SLV, and decreased to near pre-vitiligo levels after complete loss of melanocytes. Specifically, significant increases were noticed in relative proportions of T cells, B cells, and major histocompatibility complex (MHC) II-expressing cells during active SLV. Levels of T-cell infiltration were higher than those of B cells, with more CD8+ than CD4+ cells throughout SLV. Elevated leukocyte infiltration in early and active SLV was accompanied by increased levels of cytokine expression, especially in IFN-γ, IL-10, and IL-21. Low expression of IL-4 and IL-17 did not suggest important roles of Th2 and Th17 cells in SLV pathogenesis. Taken together, SLV appears to be a Th1-polarized autoimmune disease, whereby IFN-γ expression is strongly associated with parallel increases in IL-10 and IL-21, particularly during early and active stages of SLV.
[Show abstract][Hide abstract] ABSTRACT: Cellular immune responses, chemokine, and cytokine profiles were investigated in 20-d-old turkey poults following an oral infection with 12.5 × 10(3) oocysts of Eimeria adenoeides, a protozoan parasite of the genus Eimeria that develops in the ceca. Large numbers of oocysts were produced in the feces of infected birds from d 5 after infection followed by a rapid decline by d 7. Local immune activities were characterized by observing the extent of leukocyte infiltration in the ceca by histology, measuring subsets of the lymphocyte population by immunohistochemistry, and determining the relative expression of cytokines by real-time, reverse-transcription PCR. Inflammation, assessed by scoring the extent of cellular infiltration of leukocytes in sections of ceca, was significantly higher in infected poults compared with uninfected poults on d 4, 7, 9, and 11 following infection. The percent area occupied by CD4+ and CD8+ cells in the ceca was significantly greater on d 9 and 11 (CD4+) and d 11 (CD8+) in infected poults compared with uninfected controls. The relative expression of the chemokine CXCLi2 and the cytokines interleukin (IL) 1β, interferon (IFN) γ, IL13, and IL10 was investigated in tissue samples taken from the ceca. Increased expression of CXCLi2 occurred on d 4 and 7. Increased expression of IL10 and IFNγ occurred on d 4 and of IL1β and IL13 occurred on d 7 postinfection. The increased leukocyte infiltration in the ceca, alterations in the lymphocyte subpopulations, and changes in expression of chemokines and cytokines are an indication of the cell-mediated immune mechanisms occurring in the host as a result of exposure to E. adenoeides.
[Show abstract][Hide abstract] ABSTRACT: Plexiform lesions develop in the pulmonary arteries of humans suffering from idiopathic pulmonary arterial hypertension (IPAH). Plexogenic arteriopathy rarely develops in existing animal models of IPAH. In this study, plexiform lesions developed in the lungs of rapidly growing meat-type chickens (broiler chickens) that had been genetically selected for susceptibility to IPAH. Plexiform lesions developed spontaneously in: 42% of females and 40% of males; 35% of right lungs, and 45% of left lungs; and, at 8, 12, 16, 20, 24, and 52 weeks of age the plexiform lesion incidences averaged 52%, 50%, 51%, 40%, 36%, and 22%, respectively. Plexiform lesions formed distal to branch points in muscular interparabronchial pulmonary arteries exhibiting intimal proliferation. Perivascular mononuclear cell infiltrates consistently surrounded the affected arteries. Proliferating intimal cells fully or partially occluded the arterial lumen adjacent to plexiform lesions. Broilers reared in clean stainless steel cages exhibited a 50% lesion incidence that did not differ from the 64% incidence in flock mates grown on dusty floor litter. Microparticles (30 μm diameter) were injected to determine if physical occlusion and focal inflammation within distal pulmonary arteries might initiate plexiform lesion development. Three months postinjection no plexiform lesions were observed in the vicinity of persisting microparticles. Broiler chickens selected for innate susceptibility to IPAH represent a new animal model for investigating the mechanisms responsible for spontaneous plexogenic arteriopathy.
The Anatomical Record Advances in Integrative Anatomy and Evolutionary Biology 05/2011; 294(5):739-55. DOI:10.1002/ar.21369 · 1.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interleukin-12 (IL-12), an important immunomodulator for cell-mediated immunity, shows significant potential as a vaccine adjuvant and anticancer therapeutic in mammals. Therapeutic strategies to develop mammalian IL-12 as a vaccine adjuvant/immunomodulator for promoting cellular immunity and establishing a Th1-biased immune response further support the potential value of ChIL-12. Transgenic plants show promise as scalable bioproduction platforms for challenging biopharmaceutical proteins. We have expressed, characterized, and purified biologically active ChIL-12 in plants using a rapid Agrobacterium-mediated tobacco plant-based transient expression system. To ensure the stoichiometric expression and assembly of p35 and p40, we expressed a single-chain version of chicken IL-12 (ChIL-12). A histidine 6x tag was used for identity and purification of ChIL-12(His) protein. Our results demonstrated precise cleavage of the endogenous chicken p40 signal peptide in plants as well as addition of N-linked glycans. Biological activity was confirmed in vitro by interferon-gamma secretion of ChIL-12-treated chicken splenocytes. In addition, splenocytes treated with ChIL-12 expressed with or without the His tag demonstrated comparable ChIFN-gamma induction. These studies indicate that plant-based platforms for bioproduction of complex pharmaceutical proteins produce functional ChIL-12 and provide key advantages in safety, scale, and cost-effective platform for veterinary vaccine and therapeutic applications.
Journal of interferon & cytokine research: the official journal of the International Society for Interferon and Cytokine Research 03/2010; 30(3):143-54. DOI:10.1089/jir.2009.0040 · 2.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic autoinflammatory/autoimmune disorders typically are multifactorial in nature, requiring several components such as genetic susceptibility, immune system components, and environmental factors for expression. Appropriate experimental animal models have become an essential tool to delineate and dissect the relative contributions of these components.
The spontaneous autoimmune vitiligo described in the Smyth-line chicken recapitulates the entire spectrum of clinical and biological manifestations of human vitiligo, providing a unique opportunity to examine processes prior to expression and throughout the progression of the disease.
Induction of autoimmune vitiligo in the mouse by the administration of plasmids encoding melanocyte differentiation antigens in the context of stress/danger signals provides a defined in vivo model to examine immune recognition of melanocyte proteins.
[Show abstract][Hide abstract] ABSTRACT: A primary infection of 12.5 x 10(3) oocysts of Eimeria adenoeides , given to 20-d-old turkey poults, resulted in depression of weight gain, and the production of large numbers of oocysts in the feces, compared with uninfected controls. Poults were raised under conditions to prevent possible reinfection to determine the ability of the primary infection to confer protective immunity against a challenge infection of 5 x 10(4) oocysts given at 34 d of age. Using weight gain and oocyst production after challenge as criteria for protection, the results indicated that immunity had developed. The concentration and proportions among white blood cell (WBC) populations in peripheral blood were determined at different times after the primary infection. The WBC concentration of infected poults was elevated on d 7 and 11, primarily due to elevated levels of lymphocytes and monocytes on d 7 and eosinophils on d 11. There were no differences in heterophil and basophil concentrations between infected and uninfected poults at any of the time points examined. With the exception of increased percentages of eosinophils on d 11, infection was not associated with alterations in the proportions among WBC populations. Comparison of CD4+ and CD8+ defined lymphocyte subpopulations in the blood of infected versus uninfected poults revealed higher concentrations of CD4+ lymphocytes on d 11, lower concentrations of CD8+ cells on d 4, and higher concentrations of CD8+ cells on d 11 of infection, as well as elevated ratios of CD4+:CD8+ lymphocytes in infected birds on d 4 and 11. These alterations in WBC profiles after primary E. adenoeides infection in turkey poults suggest initiation of both innate and adaptive cellular immune activities designed to effectively cope with a parasitic, intracellular pathogen.
[Show abstract][Hide abstract] ABSTRACT: Pulmonary hypertension syndrome (PHS; ascites) in fast growing meat-type chickens (broilers) is characterized by the onset of idiopathic pulmonary arterial hypertension (IPAH) leading to right-sided congestive heart failure and terminal ascites. Intravenous microparticle (MP) injection is a tool used by poultry geneticists to screen for the broilers that are resistant (RES) or susceptible (SUS) to IPAH in a breeding population. MPs occlude pulmonary arterioles and initiate focal inflammation, causing local tissues and responding leukocytes to release vasoactive mediators such as serotonin (5-HT), endothelin-1 (ET-1), and nitric oxide (NO). RT-PCR was used to examine the differences between RES and SUS broilers in terms of gene expression of ET-1, ET receptor types A and B (ET(A) and ET(B)), the serotonin transporter (SERT), serotonin receptors (5-HT(1A), 5-HT(2A), 5-HT(1B), 5-HT(2B)), endothelial NO synthase (eNOS), and inducible NOS (iNOS) in the lungs of these broilers before (0 h) and after (2, 6, 12, 24, and 48 h) MP injection. In SUS broilers MP injection elicited higher (P < 0.05) pulmonary expression of 5-HT(1A), 5-HT(2B), and ET-1, which promote vasoconstriction and proliferation of pulmonary arterial smooth muscle cells (PASMC). In RES broilers the MP injection elicited higher expression of eNOS, iNOS, and ET(B), which promote vasodilation and inhibit PASMC proliferation. These observations support the hypothesis that the resistance of broiler chickens to IPAH may be due to the higher expression of vasoactive mediators that favor enhanced vasodilation and attenuated vasoconstriction during MP injection challenges to the pulmonary vasculature.
[Show abstract][Hide abstract] ABSTRACT: Infectious and metabolic disorders are common in poultry and cause stress, poor performance, and mortality that results in considerable economic loss. Identifying the nature of stress in chickens will assist the development of appropriate measures to improve health and welfare. Acute phase proteins are hepatic proteins, the blood concentrations of which change significantly in the event of many health problems including inflammation and physical injuries. Thus, acute phase proteins are used as nonspecific diagnostic markers for various health disorders. Our previous studies showed that serum ovotransferrin (OVT) is an acute phase protein in chickens. Therefore, in the present study, we investigated whether OVT concentration can be a marker of physiological stress using sera from chickens with different infectious and metabolic disorders. A competitive enzyme immunoassay was developed to measure serum OVT concentrations. The results show that with experimentally induced pulmonary hypertension syndrome and tibial dyschondroplasia, there were no significant changes in OVT levels compared with matched controls. In contrast, when chickens were infected with microbes such as the bacterium Escherichia coli, or protozoan parasites such as Eimeria maxima and Eimeria tenella, there was a significant increase in the levels of OVT in the serum. Chickens with spontaneous autoimmune vitiligo also showed a significant increase in blood OVT levels. These studies suggest that blood OVT concentration is modulated under inflammatory and microbial stress and can therefore be used as a diagnostic marker of infection and inflammation in chickens.
[Show abstract][Hide abstract] ABSTRACT: Intravenous injection of microparticles (MPs) is a tool to reveal susceptibility to pulmonary hypertension (PH) syndrome (PHS, ascites) in broilers. After injection MPs get lodged in pulmonary arterioles and cause localized inflammation. To examine the expression of chemokines/cytokines during the MP-induced pulmonary inflammatory response, lungs were collected from 4-week-old broilers (6/line/time point) from the PHS-resistant (RES) and -susceptible (SUS) broilers before (0h) and after (2, 6, 12, 24, and 48h) MP injection and analyzed using quantitative RT-PCR. In both lines, expression of interleukin-1beta (IL-1beta), IL-6, IL-8, and K60 increased from 0 to 6h, reached peak levels at 6 and 12h, and decreased thereafter, whereas IL-4 and interferon gamma (IFN-gamma) expression remained elevated past 12h. Lungs from the RES line broilers had higher expression (P<0.05) of IL-1beta and IL-6 at 2, 6, and 12h; higher IL-8 at 6 and 12h; higher K60 at 6, 12, and 24h; higher IL-4 at 12, 24, and 48h and higher IFN-gamma expression at 6 and 48h post-MP injection than SUS line broilers. Higher expression of chemokines/cytokines in RES compared to SUS line lungs may explain the ability of RES line broilers to effectively counteract the MP-induced PH and resolve the vascular occlusion.
[Show abstract][Hide abstract] ABSTRACT: Lipopolysaccharide (LPS) is a Gram-negative bacteria cell wall component that activates monocytes and macrophages to produce nitric oxide (NO) from inducible nitric oxide synthase. Nitric oxide production in the plasma of chickens peaks 5-6-h post-i.v. LPS injection reflecting iNOS activation. To determine monocyte responsiveness after an i.v. LPS injection, a time course study was conducted examining the concentrations among peripheral blood leukocytes post-i.v. LPS injection in male and female chickens, the proportions among peripheral mononuclear leukocyte (PBMC; containing lymphocytes, thrombocytes, and monocytes) populations isolated from the blood samples collected at various times post-i.v. LPS treatment, and the ability of monocytes to produce NO with and without further LPS stimulation in vitro using the PBMC NO production assay. Additionally, monocyte extravasation activity was determined by analyzing macrophage proportions after the i.v. LPS injection in spleen, lung, and liver tissues. Blood was collected from male and female chickens at 0 h (pre-LPS injection control) and at 1, 3, 6, 24, and 48 h post-LPS injection, and additionally, at 72 h from female chickens. Tissues were collected 0, 1, 6, and 48 h post-i.v. LPS injection from male chickens. Monocyte concentrations dropped substantially by 1h in both males and females. In males, monocyte concentrations returned to control concentrations by 6h and increased at 24- and 48-h post-LPS injection, whereas in females, monocyte concentrations recovered more slowly, returning to near control concentrations by 24-48-h and increasing above control levels by 72 h. Lipopolysaccharide stimulated NO production by PBMC cultures established from blood samples obtained at various times post-LPS injection in vivo followed the same pattern as monocyte concentrations in the blood. Hence, NO concentrations within PBMC cultures were dependent upon the number of monocytes that were in the PBMC cultures isolated at different times post-i.v. LPS injection. Furthermore, macrophage proportions in spleen tissues responded similarly to monocyte concentrations in the blood, decreased in lung tissue, and varied widely in liver tissue throughout 48 h after an LPS injection. Monocytes and other leukocytes may attach to the endothelium post-i.v. LPS injection preventing the monocytes from entering the needle during blood collection resulting in what seems to be leukopenia in blood and in PBMC cultures attenuating NO production in PBMC cultures. Furthermore, monocyte differentiation and recruitment from the bone marrow is a likely contributor to the reconstitution and rise of monocyte concentrations in blood samples post-i.v. LPS injection.
[Show abstract][Hide abstract] ABSTRACT: Previously, we reported that intratracheal administration of lipopolysaccharide elicited pulmonary hypertension (PH) in broilers reared under commercial conditions and in broilers reared in environmental chambers and pretreated with aerosolized red food colorant # 3 and propylene glycol (Red#3+PG), but not in control broilers reared in environmental chambers. The objective of the present experiment was to determine possible changes in the number or proportion of airway leukocytes that could contribute to the magnitude of the PH responses elicited in broilers. Birds were aerosolized for 40 min with a saturated mixture of Red#3+PG. After 24 h, a blood sample was taken, the broilers were killed, and a pulmonary lavage process was conducted in each bird. Leukocyte concentration (white blood cells/microL) and differential leukocyte counts (%) were measured in blood and lavage fluid. Leukocyte concentration in blood did not differ between groups, but the percentage of blood lymphocytes was lower in broilers from the Red#3+PG group compared with birds from the control group (52.4+/-2.9 and 56.9+/-2.9%, respectively). Cells recovered from the lavage fluid from both groups were primarily heterophils. The concentration of leukocytes was greater in the lavage fluid of broilers from the Red#3+PG group compared with broilers from the control group (763.2+/-158.7 and 402.9+/-62.6 white blood cells/microL, respectively), but the proportions among leukocytes were not different between the 2 groups. We propose that the increased concentration of leukocytes present within the airways was one of the components that enabled broilers pre-treated with aerosolized Red#3+PG to exhibit PH responses to intratracheal lipopolysaccharide.