Acute phase proteins and biomarkers for health in chickens

Acute phase proteins (APPs) are proteins synthesised predominantly in the liver, whose plasma concentrations increase (positive APP) or decrease (negative APP) as a result of infection, inflammation, trauma and tissue injury. They also change as a result of the introduction of immunogens such as bacterial lipopolysaccharide (LPS), turpentine and vaccination. While publications on APPs in chickens are numerous, the limited availability of anti-sera and commercial ELISAs has resulted in a lot of information on only a few APPs. Disease is a threat to the poultry industry, as pathogens have the potential to evolve, spread and cause rapid onset of disease that is detrimental to the welfare of birds. Low level, sub-acute disease with non-specific, often undiagnosed causes can greatly affect bird health and growth and impact greatly on productivity and profitability. Developing and validating methods to measure and characterise APPs in chickens will allow these proteins to be used diagnostically for monitoring flock health. Using immune parameters such as APPs that correlate with disease resistance or improvements in production and welfare will allow the use of APPs as selection parameters for breeding to be evaluated. For APPs to be useful parameters on which to evaluate chicken health, information on normal APP concentrations is required. Ceruloplasmin (Cp) and PIT54 concentrations were found to be much lower in healthy birds form commercial production farms than the reported normal values obtained from the literature. These APPs were found to be significantly higher in culled birds from a commercial farm and Cp, PIT54 and ovotransferrin (Ovt) were significantly higher in birds classified as having obvious gait defects. Using quantitative shotgun proteomics to identify the differentially abundant proteins between three pools: highly acute phase (HAP), acute phase (AP) and non-acute phase (NAP), generated data from which a selection of proteins, based on the fold difference between the three pools was made. These proteins were targeted on a individual samples alongside proteins known to be APPs in chickens or other species: serum amyloid A (SAA), C-reactive protein (CRP), Ovt, apolipoprotein A-I (apo-AI), transthyretin (Ttn), haemopexin (Hpx) and PIT54. Together with immunoassay data for SAA, Ovt, alpha-1-acid glycoprotein (AGP) and Cp the results of this research reveal that SAA is the only major APP in chickens. Ovotransferrin and AGP behave as moderate APPs while PIT54 and Cp are minor APPs. Haemopexin was not significantly different between the three acute phase groups. Apolipoprotein AI and Ttn were significantly lower in the HAP and AP groups and as such can be classed as negative APPs. In an effort to identify CRP, multiple anti-sera cross reacting with CRP from other species were used and a phosphorylcholine column known to affinity purify CRP were used. Enriched fractions containing low molecular weight proteins, elutions from the affinity column together with HAP, AP and NAP pooled samples were applied to a Q-Exactive Hybrid Quadrupole–Orbitrap mass spectrometer (Thermo Scientific) for Shotgun analysis and CRP was not identified. It would appear that CRP is not present as a plasma protein constitutively or during an APR in chickens and as such is not an APP in this species. Of the proteins targeted as possible novel biomarkers of the APR in chickens mannan binding lectin associated serine protease-2, α-2-HS-glycoprotein (fetuin) and major facilitator superfamily domain-containing protein 10 were reduced in abundance in the HAP group, behaving as negative biomarkers. Myeloid protein and putative ISG(12)2 were positively associated with the acute phase being significantly higher in the HAP and AP groups. The protein cathepsin D was significantly higher in both HAP and AP compared to the NAP indicating that of all the proteins targeted, this appears to have the most potential as a biomarker of the acute phase, as it was significantly increased in the AP as well as the HAP group. To evaluate APPs and investigate biomarkers of intestinal health, a study using re-used poultry litter was undertaken. The introduction of litter at 12 days of age did not significantly increase any APPs measured using immunoassays and quantitative proteomics at 3, 6 and 10 days post introduction. While no APP was found to be significantly different between the challenged and control groups at anytime point, the APPs AGP, SAA and Hpx did increase over time in all birds. The protein apolipoprotein AIV (apo-AIV) was targeted as a possible APP and because of its reported role in controlling satiety. An ELISA was developed, successfully validated and used to measure apo-AIV in this study. While no significant differences in apo-AIV plasma concentrations between challenged and control groups were identified apo-AIV plasma concentrations did change significantly between certain time points in challenged and control groups. Apoliporotein AIV does not appear to behave as an APP in chickens, as it was not significantly different between acute phase groups. The actin associated proteins villin and gelsolin were investigated as possible biomarkers of intestinal health. Villin was found not to be present in the plasma of chickens and as such not a biomarker target. Gelsolin was found not to be differentially expressed during the acute phase or as a result of intestinal challenge. Finally a proteomic approach was undertaken to investigate gastrocnemius tendon (GT) rupture in broiler chickens with a view of elucidating to and identify proteins associated with risk of rupture. A number of proteins were found to be differentially expressed between tendon pools and further work would enable further detailing of these findings. In conclusion this work has made a number of novel findings and addressed a number of data poor areas. The area of chicken APPs research has stagnated over the last 15 years with publications becoming repetitive and reliant on a small number of immunoassays. This work has sought to characterise the classic APPs in chickens, and use a quantitative proteomic approach to measure and categorise them. This method was also used to take a fresh approach to biomarker identification for both the APR and intestinal health. The development and validation of assays for Ovt and apo-AIV and the shotgun data mean that these proteins can be further characterised in chickens with a view of applying their measurement to diagnostics and selective breeding programs.

The Shrine of ʿAbd al-Qādir al-Jīlānī in Baghdad & the Shrine of ʿAbd al-ʿAzīz al-Jīlānī in ʿAqra: Mapping the multiple orientations of two Qādirī Sufi shrines in Iraq

This thesis charts the stakeholder communities, physical environment and daily life of two little studied Qādiriyya Sufi shrines associated with Shaikh ʿAbd al-Qādir al-Jīlānī (1077 – 1165 AD), a 12th century Ḥanbalī Muslim theologian and the posthumous founder of one of the oldest Sufi orders in Islam. The first shrine is based in Baghdad and houses his burial chamber; and the second shrine, on the outskirts of the city of ‘Aqra in the Kurdish region of northern Iraq, is that of his son Shaikh ʿAbd al-ʿAzīz (died 1206 AD). The latter was also known for lecturing in Ḥanbalī theology in the region, and venerated for this as well as his association with Shaikh ʿAbd al-Qādir. Driven by the research question “What shapes the identity orientations of these two Qādiriyya Sufi shrines in modern times?” the findings presented here are the result of field research carried out between November 2009 and February 2014. This field research revealed a complex context in which the two shrines existed and interacted, influenced by both Sufi and non-Sufi stakeholders who identified with and accessed these shrines to satisfy a variety of spiritual and practical needs, which in turn influenced the way each considered and viewed the two shrines from a number of orientations. These overlapping orientations include the Qādirī Sufi entity and the resting place of its patron saint; the orthodox Sunnī mosque with its muftī-imams, who are employed by the Iraqi government; the local Shīʿa community’s neighbourhood saint’s shrine and its destination for spiritual and practical aid; and the local provider of welfare to the poor of the city (soup kitchen, funeral parlour and electricity-generation amongst other services). The research findings also revealed a continuously changing and adapting Qādirī Sufi scene not immune from the national and regional socio-religio-political environments in which the two shrines exist: a non-Sufi national political class vying to influence and manipulate these shrines for their own purposes; and powerful national sectarian factions jostling to do the same. The mixture of stakeholders using and associating with the two shrines were found to be influential shapers of these entities, both physically and spiritually. Through encountering and interacting with each other, most stakeholders contributed to maintaining and rejuvenating the two shrines, but some also sought to adapt and change them driven by their particular orientation’s perspective.