Table 1.

Potential biomarkers for monitoring gut health in poultry.

Biomarkers	Biological samples for examination	Tools used to measure biomarker	Pros	Cons	References
Biomarkers in the intestinal wall
Microscopic examination of the intestine (measurement of villus length/crypt depth ratio)	Direct microscopic evaluation of intestinal wall	Histology	The gold standard method to evaluate intestine health. Reference values are available.	Requires skilled personnel to collect samples and a specialized laboratory for sample processing. For diagnostic purposes, additional tests are required increasing costs	(de Verdal et al., 2010; Chen et al., 2015; Ducatelle et al., 2018)
Intestinal inflammation and gut wall appearance	Direct macroscopic evaluation of intestine	Gross evaluation of tissues	Direct measurement of gut appearance, scores are available to determine the extent of intestinal inflammation for necrotic enteritis, coccidiosis, and dysbiosis	It is subjective, requires a skilled veterinarian for lesion scoring and terminal sampling of birds	(Johnson and Reid, 1970; Keyburn et al., 2006; De Gussem, 2010).
Acute-phase proteins
Ovotransferrin	Blood and excreta	Immunoassay	Cost-effective, no need to sacrifice chickens for sample collection	The biomarker is susceptible to proteolysis and samples should be tested from fresh excreta. Has only been reported to be increased in experimental meat chickens in a necrotic enteritis model, E. maxima, E. tenella, and E. coli infection	(Rath et al., 2009; Goossens et al., 2018)
Alpha-1 antitrypsin	Blood and excreta	Immunoassay	The enzyme is resistant to proteolysis, cost-effective technique, no need to sacrifice chickens for sample collection	The suitability of this biomarker in experimental meat chickens is debatable as the concentration of this protein in excreta or blood remained unchanged between control birds and birds with damaged intestinal integrity induced by fasting or by administration of dexamethasone and dextran sodium sulfate	(Gilani et al., 2017; Barekatain et al., 2020)
α1-acid glycoprotein	Blood	Immunoassay	Cost-effective, no need to sacrifice chickens for sample collection	No report of detection in excreta, it has been reported to be induced in blood after administration of dexamethasone in broilers	(Chen et al., 2015; Barekatain et al., 2020)
Host protein biomarkers
Citrulline	Blood	Immunoassay	Cost-effective, no need to sacrifice chickens, and has the potential to be used as a marker of small intestine barrier in poultry	Decreased level of plasma citrulline was correlated with reduced enterocyte mass in chickens fed a rye-based diet compared to chickens fed a corn-based diet after 20 d. Need to be tested in other conditions of intestinal disturbance.	(Baxter et al., 2019)
Fibronectin	Blood and excreta	Immunoassay	Cost-effective, no need to sacrifice chickens, and has the potential to be used as a marker of intestinal inflammation in poultry	Increased levels on gut leakage model induced by administration of dexamethasone and E. maxima and E. acervulina. Need to be tested in other conditions of intestinal disturbance	(Owen et al., 2008; De Meyer et al., 2019; Barekatain et al., 2020)
Diaminoxidase	Blood	Immunoassay	Cost-effective, no need to sacrifice chickens for sample collection	Only studied in laying hens as a marker of gut barrier failure after feed withdrawal for 12 h. Not reported in broilers, needs to be tested in other conditions of intestinal disturbance	(Lei et al., 2013)
Fatty acid-binding protein	Blood and excreta	Immunoassay, PCR	Cost-effective, no need to sacrifice chickens for sample collection	Due to the higher molecular weight of fatty acid-binding protein (15000 Da), severe damage of intestinal epithelium is required for its passage from the intestine to the blood. In excreta, one study showed no difference between gut barrier damage induced by dexamethasone using ELISA. However, in another study, the DNA of this biomarker was increased in blood after birds were administered twice the dose of a coccidiosis vaccine containing a mixture of E. acervulina, E. maxima, and E. tenella. Needs further investigation.	(Iizuka and Konno, 2011; Chen et al., 2015; Barekatain et al., 2020)
Myeloid protein-1, Alpha-actinin-4, Apolipoprotein A-1, Hemoglobin subunit beta, Nucleophosmin, Ovoinhibitor, Transthyretin	Colon contents	PCR	Cost-effective, colon contents can be collected without sacrificing chickens	Only tested in gut leakage model induced by coccidia administration (E. maxima and E. acervulina). Needs testing in other conditions of intestinal disturbance	(De Meyer et al., 2019)
Metabolome profiling	Blood	Ultra-high performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS)	No need to sacrifice chickens for sample collection. Characterizes biochemical and metabolic changes in the host, can be used for biomarker discovery and characterization of metabolites and metabolic changes of the host and microbiota-associated products in broilers after inoculation with Eimeria spp., eating restriction patterns, and supplementation with enzymes, antibiotics, prebiotics, and probiotics.	Expensive, time-consuming, requires specialized equipment and intensive bioinformatics	(Aggrey et al., 2019; Cao et al., 2020; Gonzalez-Uarquin et al., 2020; Wang et al., 2021; Zhang et al., 2021)
Microbial shift or detection of bacterial metabolites as biomarkers
Detection of microbial shift such as a decreased abundance of family Enterobacteriaceae as a marker of poor gut health	Gut contents and excreta	Sequencing based approach/PCR	Identification of microbial shifts could be used for testing of management interventions or occurrence of antibiotic resistance genes	Expensive, time-consuming, and difficult to find consistent microbial taxa associated with health and disease due to extensive bird to bird variation	(Ducatelle et al., 2018; Hernandez-Patlan et al., 2019a)
Bacterial metabolite ‘D-lactate’ in blood	Blood	Immunoassay	No need to sacrifice chickens for sample collection	Only studied in laying hens after feed restriction for 12 h. Needs testing in meat chickens and other conditions of intestinal disturbance.	(Lei et al., 2013; Gilani et al., 2016)
Bacterial count in liver	Liver	Culture	A more accurate approach to evaluate translocation of bacteria as a result of gut barrier failure	Need to sacrifice birds, time-consuming, and expensive. Has been reported to be increased in birds fed with the rye-based diet compared to the corn-based diet, not studied in intestinal disease models.	(Tellez et al., 2014; Ducatelle et al., 2018)
Genes encoding bacterial enzymes in the butyrate production pathway	Excreta and gut contents	PCR	Cost-effective, no need to sacrifice chickens for sample collection	Has not been studied in leaky gut models but reported to be increased amounts in improved feed conversion efficient birds fed with xylooligosaccharides and wheat-rye based diet compared to only wheat-rye based diet	(De Maesschalck et al., 2015; Ducatelle et al., 2018)
Endotoxin production by gram-negative bacteria	Blood	PCR	Cost-effective, no need to sacrifice chickens for sample collection	Has only been reported to increase in chickens administered twice the recommended dose of coccidiosis vaccine containing a mixture of E. acervulina, E. maxima, and E. tenella	(Chen et al., 2015)
Fatty acids and lactic acid profiles, other bacterial metabolites	Gut contents (cecum)	High-performance liquid chromatography (HPLC), Liquid chromatography-tandem mass spectroscopy (LC-MS/MS)	Monitor real-time, dynamic changes in bacterial metabolites, providing direct analysis of bacterial metabolic activity.	It is expensive and requires specialized equipment. If LC-MS/MS is used intensive bioinformatics is needed. Requires the sacrifice of chickens for sample collection, maintenance of cold chain after sample collection, and rapid processing of samples.	(Kers et al., 2019b; Niu et al., 2020; Park et al., 2020; Slizewska et al., 2020)
Others
Fluorescein isothiocyanate–dextran (FITC-d)	Blood	Immunoassay	Measures epithelial permeability	Limited to research settings since the compounds need to be gavaged and monitored in blood as a tool to determine gut barrier failure	(Niewold, 2014; Immerseel, 2019; Barekatain et al., 2020; Vuong et al., 2021)