Host immunity modulates the efficacy of microbiota transplantation for treatment of Clostridioides difficile infection.

Fecal microbiota transplantation (FMT) is a successful therapeutic strategy for treating recurrent Clostridioides difficile infection. Despite remarkable efficacy, implementation of FMT therapy is limited and the mechanism of action remains poorly understood. Here, we demonstrate a critical role for the immune system in supporting FMT using a murine C. difficile infection system. Following FMT, Rag1 heterozygote mice resolve C. difficile while littermate Rag1-/- mice fail to clear the infection. Targeted ablation of adaptive immune cell subsets reveal a necessary role for CD4+ Foxp3+ T-regulatory cells, but not B cells or CD8+ T cells, in FMT-mediated resolution of C. difficile infection. FMT non-responsive mice exhibit exacerbated inflammation, impaired engraftment of the FMT bacterial community and failed restoration of commensal bacteria-derived secondary bile acid metabolites in the large intestine. These data demonstrate that the host's inflammatory immune status can limit the efficacy of microbiota-based therapeutics to treat C. difficile infection.

Antibiotic-Induced Shifts in Fecal Microbiota Density and Composition during Hematopoietic Stem Cell Transplantation.

Dramatic microbiota changes and loss of commensal anaerobic bacteria are associated with adverse outcomes in hematopoietic cell transplantation (HCT) recipients. In this study, we demonstrate these dynamic changes at high resolution through daily stool sampling and assess the impact of individual antibiotics on those changes. We collected 272 longitudinal stool samples (with mostly daily frequency) from 18 patients undergoing HCT and determined their composition by multiparallel 16S rRNA gene sequencing as well as the density of bacteria in stool by quantitative PCR (qPCR). We calculated microbiota volatility to quantify rapid shifts and developed a new dynamic systems inference method to assess the specific impact of antibiotics. The greatest shifts in microbiota composition occurred between stem cell infusion and reconstitution of healthy immune cells. Piperacillin-tazobactam caused the most severe declines among obligate anaerobes. Our approach of daily sampling, bacterial density determination, and dynamic systems modeling allowed us to infer the independent effects of specific antibiotics on the microbiota of HCT patients.

Longitudinal changes in blood metabolites, amino acid profile, and oxidative stress markers in American Foxhounds fed a nutrient-fortified diet.

The objective of the present study was to evaluate the changes in blood metabolites, AA profile, and oxidative stress markers in American Foxhound dogs fed a nutrient-fortified endurance diet while undergoing unstructured endurance exercise over several months. Thirty-six adult American Foxhound dogs (mean age: 4.5, range 2 to 10 yr and mean BW: 34.7, range: 23.1 to 46.9 kg) were selected to participate in the study. Prior to the study, all dogs consumed a commercial diet for 16 wk. After collecting baseline blood samples, dogs were assigned to a standard commercial performance diet (control) or a nutrient-fortified dog food (test). Dogs were balanced by gender, age, body weight, and athletic performance between diets. During the study, dogs underwent 78 bouts of exercise, with approximately 22 km/bout. Blood samples were collected after 40, 75, 138, and 201 d on study (October 2012 to March 2013). All blood metabolites were similar at baseline and serum chemistry profile remained within normal ranges throughout the study. Over time, plasma taurine and vitamin E concentrations decreased (P < 0.05) in dogs fed the control diet but were maintained or increased (P < 0.05) in dogs fed the treatment diet. Also, plasma creatinine and triglycerides were lower (P < 0.05) and blood phosphorus and alkaline phosphatase were higher (P < 0.05) in dogs fed the treatment diet. Vitamin E and taurine status of dogs appear to be affected by extended endurance exercise. These data suggest dogs undergoing endurance exercise may benefit from supplementation of vitamin E and taurine to minimize oxidation and maintain taurine status.

Pathogenicity Locus, Core Genome, and Accessory Gene Contributions to Clostridium difficile Virulence.

Clostridium difficile is a spore-forming anaerobic bacterium that causes colitis in patients with disrupted colonic microbiota. While some individuals are asymptomatic C. difficile carriers, symptomatic disease ranges from mild diarrhea to potentially lethal toxic megacolon. The wide disease spectrum has been attributed to the infected host's age, underlying diseases, immune status, and microbiome composition. However, strain-specific differences in C. difficile virulence have also been implicated in determining colitis severity. Because patients infected with C. difficile are unique in terms of medical history, microbiome composition, and immune competence, determining the relative contribution of C. difficile virulence to disease severity has been challenging, and conclusions regarding the virulence of specific strains have been inconsistent. To address this, we used a mouse model to test 33 clinical C. difficile strains isolated from patients with disease severities ranging from asymptomatic carriage to severe colitis, and we determined their relative in vivo virulence in genetically identical, antibiotic-pretreated mice. We found that murine infections with C. difficile clade 2 strains (including multilocus sequence type 1/ribotype 027) were associated with higher lethality and that C. difficile strains associated with greater human disease severity caused more severe disease in mice. While toxin production was not strongly correlated with in vivo colonic pathology, the ability of C. difficile strains to grow in the presence of secondary bile acids was associated with greater disease severity. Whole-genome sequencing and identification of core and accessory genes identified a subset of accessory genes that distinguish high-virulence from lower-virulence C. difficile strains.IMPORTANCEClostridium difficile is an important cause of hospital-associated intestinal infections, and recent years have seen an increase in the number and severity of cases in the United States. A patient's antibiotic history, immune status, and medical comorbidities determine, in part, the severity of C. difficile infection. The relative virulence of different clinical C. difficile strains, although postulated to determine disease severity in patients, has been more difficult to consistently associate with mild versus severe colitis. We tested 33 distinct clinical C. difficile isolates for their ability to cause disease in genetically identical mice and found that C. difficile strains belonging to clade 2 were associated with higher mortality. Differences in survival were not attributed to differences in toxin production but likely resulted from the distinct gene content in the various clinical isolates.

Commensal microbes provide first line defense against Listeria monocytogenes infection.

Listeria monocytogenes is a foodborne pathogen that causes septicemia, meningitis and chorioamnionitis and is associated with high mortality. Immunocompetent humans and animals, however, can tolerate high doses of L. monocytogenes without developing systemic disease. The intestinal microbiota provides colonization resistance against many orally acquired pathogens, and antibiotic-mediated depletion of the microbiota reduces host resistance to infection. Here we show that a diverse microbiota markedly reduces Listeria monocytogenes colonization of the gut lumen and prevents systemic dissemination. Antibiotic administration to mice before low dose oral inoculation increases L. monocytogenes growth in the intestine. In immunodeficient or chemotherapy-treated mice, the intestinal microbiota provides nonredundant defense against lethal, disseminated infection. We have assembled a consortium of commensal bacteria belonging to the Clostridiales order, which exerts in vitro antilisterial activity and confers in vivo resistance upon transfer into germ free mice. Thus, we demonstrate a defensive role of the gut microbiota against Listeria monocytogenes infection and identify intestinal commensal species that, by enhancing resistance against this pathogen, represent potential probiotics.

Cooperating Commensals Restore Colonization Resistance to Vancomycin-Resistant Enterococcus faecium.

Antibiotic-mediated microbiota destruction and the consequent loss of colonization resistance can result in intestinal domination with vancomycin-resistant Enterococcus (VRE), leading to bloodstream infection in hospitalized patients. Clearance of VRE remains a challenging goal that, if achieved, would reduce systemic VRE infections and patient-to-patient transmission. Although obligate anaerobic commensal bacteria have been associated with colonization resistance to VRE, the specific bacterial species involved remain undefined. Herein, we demonstrate that a precisely defined consortium of commensal bacteria containing the Clostridium cluster XIVa species Blautia producta and Clostridium bolteae restores colonization resistance against VRE and clears VRE from the intestines of mice. While C. bolteae did not directly mediate VRE clearance, it enabled intestinal colonization with B. producta, which directly inhibited VRE growth. These findings suggest that therapeutic or prophylactic administration of defined bacterial consortia to individuals with compromised microbiota composition may reduce inter-patient transmission and intra-patient dissemination of highly antibiotic-resistant pathogens.

Bile acid sensitivity and in vivo virulence of clinical Clostridium difficile isolates.

Clostridium difficile is an anaerobic bacterium that causes diarrheal illnesses. Disease onset is linked with exposure to oral antibiotics and consequent depletion of secondary bile acids. Here we investigate the relationship between in vitro secondary bile acid tolerance and in vivo disease scores of diverse C. difficile strains in mice.

Innate Lymphocyte/Ly6C(hi) Monocyte Crosstalk Promotes Klebsiella Pneumoniae Clearance.

Increasing antibiotic resistance among bacterial pathogens has rendered some infections untreatable with available antibiotics. Klebsiella pneumoniae, a bacterial pathogen that has acquired high-level antibiotic resistance, is a common cause of pulmonary infections. Optimal clearance of K. pneumoniae from the host lung requires TNF and IL-17A. Herein, we demonstrate that inflammatory monocytes are rapidly recruited to the lungs of K. pneumoniae-infected mice and produce TNF, which markedly increases the frequency of IL-17-producing innate lymphoid cells. While pulmonary clearance of K. pneumoniae is preserved in neutrophil-depleted mice, monocyte depletion or TNF deficiency impairs IL-17A-dependent resolution of pneumonia. Monocyte-mediated bacterial uptake and killing is enhanced by ILC production of IL-17A, indicating that innate lymphocytes engage in a positive-feedback loop with monocytes that promotes clearance of pneumonia. Innate immune defense against a highly antibiotic-resistant bacterial pathogen depends on crosstalk between inflammatory monocytes and innate lymphocytes that is mediated by TNF and IL-17A.

TLR-7 activation enhances IL-22-mediated colonization resistance against vancomycin-resistant enterococcus.

Antibiotic administration can disrupt the intestinal microbiota and down-regulate innate immune defenses, compromising colonization resistance against orally acquired bacterial pathogens. Vancomycin-resistant Enterococcus faecium (VRE), a major cause of antibiotic-resistant infections in hospitalized patients, thrives in the intestine when colonization resistance is compromised, achieving extremely high densities that can lead to bloodstream invasion and sepsis. Viral infections, by mechanisms that remain incompletely defined, can stimulate resistance against invading bacterial pathogens. We report that murine norovirus infection correlates with reduced density of VRE in the intestinal tract of mice with antibiotic-induced loss of colonization resistance. Resiquimod (R848), a synthetic ligand for Toll-like receptor 7 (TLR-7) that stimulates antiviral innate immune defenses, restores expression of the antimicrobial peptide Reg3γ and reestablishes colonization resistance against VRE in antibiotic-treated mice. Orally administered R848 triggers TLR-7 on CD11c(+) dendritic cells, inducing interleukin-23 (IL-23) expression followed by a burst of IL-22 secretion by innate lymphoid cells, leading to Reg3γ expression and restoration of colonization resistance against VRE. Our findings reveal that an orally bioavailable TLR-7 ligand that stimulates innate antiviral immune pathways in the intestine restores colonization resistance against a highly antibiotic-resistant bacterial pathogen.

Innate Immune Defenses Mediated by Two ILC Subsets Are Critical for Protection against Acute Clostridium difficile Infection.

Infection with the opportunistic enteric pathogen Clostridium difficile is an increasingly common clinical complication that follows antibiotic treatment-induced gut microbiota perturbation. Innate lymphoid cells (ILCs) are early responders to enteric pathogens; however, their role during C. difficile infection is undefined. To identify immune pathways that mediate recovery from C. difficile infection, we challenged C57BL/6, Rag1(-/-) (which lack T and B cells), and Rag2(-/-)Il2rg(-/-) (Ragγc(-/-)) mice (which additionally lack ILCs) with C. difficile. In contrast to Rag1(-/-) mice, ILC-deficient Ragγc(-/-) mice rapidly succumbed to infection. Rag1(-/-) but not Ragγc(-/-) mice upregulate expression of ILC1- or ILC3-associated proteins following C. difficile infection. Protection against infection was restored by transferring ILCs into Ragγc(-/-) mice. While ILC3s made a minor contribution to resistance, loss of IFN-γ or T-bet-expressing ILC1s in Rag1(-/-) mice increased susceptibility to C. difficile. These data demonstrate a critical role for ILC1s in defense against C. difficile.

Distinct Contributions of Neutrophils and CCR2+ Monocytes to Pulmonary Clearance of Different Klebsiella pneumoniae Strains.

Klebsiella pneumoniae is a common respiratory pathogen, with some strains having developed broad resistance to clinically available antibiotics. Humans can become infected with many different K. pneumoniae strains that vary in genetic background, antibiotic susceptibility, capsule composition, and mucoid phenotype. Genome comparisons have revealed differences between K. pneumoniae strains, but the impact of genomic variability on immune-mediated clearance of pneumonia remains unclear. Experimental studies of pneumonia in mice have used the rodent-adapted 43816 strain of K. pneumoniae and demonstrated that neutrophils are essential for optimal host defense. It remains unclear, however, whether CCR2(+) monocytes contribute to K. pneumoniae clearance from the lung. We selectively depleted neutrophils, CCR2(+) monocytes, or both from immunocompetent mice and determined susceptibility to infection by the 43816 strain and 4 newly isolated clinical K. pneumoniae strains. The clinical K. pneumoniae strains, including one carbapenem-resistant ST258 strain, are less virulent than 43816. Optimal clearance of each of the 5 strains required either neutrophils or CCR2(+) monocytes. Selective neutrophil depletion markedly worsened infection with K. pneumoniae strain 43816 and three clinical isolates but did not increase susceptibility of mice to infection with the carbapenem-resistant K. pneumoniae ST258 strain. Depletion of CCR2(+) monocytes delayed recovery from infection with each of the 5 K. pneumoniae strains, revealing a contribution of these cells to bacterial clearance from the lung. Our findings demonstrate strain-dependent variation in the contributions of neutrophils and CCR2(+) monocytes to clearance of K. pneumoniae pulmonary infection.

Loss of Microbiota-Mediated Colonization Resistance to Clostridium difficile Infection With Oral Vancomycin Compared With Metronidazole.

Antibiotic administration disrupts the intestinal microbiota, increasing susceptibility to pathogens such as Clostridium difficile. Metronidazole or oral vancomycin can cure C. difficile infection, and administration of these agents to prevent C. difficile infection in high-risk patients, although not sanctioned by Infectious Disease Society of America guidelines, has been considered. The relative impacts of metronidazole and vancomycin on the intestinal microbiota and colonization resistance are unknown. We investigated the effect of brief treatment with metronidazole and/or oral vancomycin on susceptibility to C. difficile, vancomycin-resistant Enterococcus, carbapenem-resistant Klebsiella pneumoniae, and Escherichia coli infection in mice. Although metronidazole resulted in transient loss of colonization resistance, oral vancomycin markedly disrupted the microbiota, leading to prolonged loss of colonization resistance to C. difficile infection and dense colonization by vancomycin-resistant Enterococcus, K. pneumoniae, and E. coli. Our results demonstrate that vancomycin, and to a lesser extent metronidazole, are associated with marked intestinal microbiota destruction and greater risk of colonization by nosocomial pathogens.

Characterization of attenuated food motivation in high-fat diet-induced obesity: Critical roles for time on diet and reinforcer familiarity.

Prior work using animal models to study the effects of obesogenic diets on food motivation have generated inconsistent results, with some reporting increases and others reporting decreases in responding on food-reinforced tasks. Here, we identified two specific variables that may account for these discrepant outcomes - the length of time on the obesigenic diet and the familiarity of the food reinforcer - and examined the independent roles of these factors. Time on diet was found to be inversely related to food motivation, as rats consuming a 40% high-fat diet (HFD) for only 3weeks did not differ from chow-fed rats when responding for a sucrose reinforcer on a progressive ratio (PR) schedule, but responding was suppressed after 6weeks of ad lib HFD consumption. Explicitly manipulating experience with the sucrose reinforcer by pre-exposing half the rats prior to 10weeks of HFD consumption attenuated the motivational deficit seen in the absence of this familiarity, resulting in obese rats performing at the same level as lean rats. Finally, after 8weeks on a HFD, rats did not express a conditioned place preference for sucrose, indicating a decrement in reward value independent of motivation. These findings are consistent with prior literature showing an increase in food motivation for rats with a shorter time consuming the obesigenic diet, and for those with more prior experience with the reinforcer. This account also helps reconcile these findings with increased food motivation in obese humans due to extensive experience with palatable food and suggests that researchers engaging in non-human animal studies of obesity would better model the conditions under which human obesity develops by using a varied, cafeteria-style diet to increase the breadth of food experiences.

Shifting habitats, morphology, and selective pressures: developmental polyphenism in an adaptive radiation of Hawaiian spiders.

Particularly intriguing examples of adaptive radiation are those in which lineages show parallel or convergent evolution, suggesting utilization of similar genetic or developmental pathways. The current study focuses on an adaptive radiation of Hawaiian "spiny-leg" spiders in which diversification is associated with repeated convergent evolution leading to similar sets of ecomorphs on each island. However, two species on the oldest islands in the archipelago exhibit variability, occurring as two different ecomorphs. More derived species on the younger islands show much less variability, any one species displaying a single ecomorph. We measured ecomorphological features within individuals over time to determine the nature of the variability. Then, using transcriptomes, we conducted lineage-based tests for selection under varying models and analyses of gene tree versus species tree incongruencies. Our results provide strong evidence that variability in color in Tetragnatha kauaiensis and T. polychromata is associated with development within individuals (polyphenism). Moreover, a total of 28 loci showed a signature of selection associated with loss of the color-changing phenotype, and 37 loci showed a signature of selection associated with the colonization of a new environment. The results illustrate how developmental polyphenism might provide an avenue for the repeated evolution of ecomorphs during adaptive radiation.

Acute changes in blood metabolites and amino acid profile post-exercise in Foxhound dogs fed a high endurance formula.

Dogs participating in endurance exercise, including herding, hunting and racing have a greater energy requirement and may be more susceptible to nutrient depletion, electrolyte imbalance and metabolic stress. The objective of the present study was to investigate the acute response to unstructured mixed exercise in American Foxhounds fed a nutrient-fortified endurance diet. Thirty-nine adult Foxhound dogs (median age: 5·0, range: 2-10 years and median body weight (BW): 36·4, range: 24·9-49·5 kg) were allotted to a standard performance diet (Control) or nutrient-fortified endurance diet for adult dogs (Test). Dogs were balanced by sex, age, BW and athletic performance between diets. All male dogs were intact, whereas all the female dogs were spayed. After 80 d on diet, blood samples were collected via jugular puncture at baseline (0 h), and at 3 and 25 h post-exercise (mean: 17·7 (sem 0·92) km run over 2-3 h). Plasma taurine concentration and complete amino acid (AA) profile, serum chemistry and creatine kinase were measured. Serum chemistry profile remained within normal ranges throughout the study. A significant (P < 0·05) diet by time interaction was observed for calcium, alkaline phosphatase and most AA. Plasma taurine and most essential AA were increased (P < 0·05) after exercise and remained greater (P < 0·05) in dogs fed the Test diet, including the branched-chain AA (isoleucine, leucine and valine). Creatine kinase increased (P = 0·01) after 3 h and returned to baseline after 25 h post-exercise, but was not altered by diet. These data indicate that dogs undergoing a moderate bout of exercise did not suffer from electrolyte imbalance, and that a nutrient-fortified diet resulted in greater plasma taurine and essential AA concentrations.

Nutritional sustainability of pet foods.

Sustainable practices meet the needs of the present without compromising the ability of future generations to meet their needs. Applying these concepts to food and feed production, nutritional sustainability is the ability of a food system to provide sufficient energy and essential nutrients required to maintain good health in a population without compromising the ability of future generations to meet their nutritional needs. Ecological, social, and economic aspects must be balanced to support the sustainability of the overall food system. The nutritional sustainability of a food system can be influenced by several factors, including the ingredient selection, nutrient composition, digestibility, and consumption rates of a diet. Carbon and water footprints vary greatly among plant- and animal-based ingredients, production strategy, and geographical location. Because the pet food industry is based largely on by-products and is tightly interlinked with livestock production and the human food system, however, it is quite unique with regard to sustainability. Often based on consumer demand rather than nutritional requirements, many commercial pet foods are formulated to provide nutrients in excess of current minimum recommendations, use ingredients that compete directly with the human food system, or are overconsumed by pets, resulting in food wastage and obesity. Pet food professionals have the opportunity to address these challenges and influence the sustainability of pet ownership through product design, manufacturing processes, public education, and policy change. A coordinated effort across the industry that includes ingredient buyers, formulators, and nutritionists may result in a more sustainable pet food system.

Effects of exercise training on adiposity, insulin sensitivity, and plasma hormone and lipid concentrations in overweight or obese, insulin-resistant horses.

OBJECTIVE: To determine effects of exercise training without dietary restriction on adiposity, basal hormone and lipid concentrations and glucose and insulin dynamics in overweight or obese, insulin-resistant horses. ANIMALS: 12 overweight or obese (body condition score > or = 7), insulin-resistant (insulin sensitivity < or = 1.2 x 10(-4) L/min/mU) geldings. PROCEDURES: 4 horses remained sedentary, and 8 horses were exercised for 4 weeks at low intensity and 4 weeks at higher intensity, followed by 2 weeks of detraining. Prior to and after each training period, frequently sampled IV glucose tolerance tests with minimal model analysis were performed and baseline plasma insulin, glucose, triglycerides, non-esterified fatty acids, and leptin concentrations were analyzed. Adiposity was assessed by use of morphometrics, ultrasonic subcutaneous fat thickness, and estimation of fat mass from total body water (deuterium dilution method). RESULTS: Body weight and fat mass decreased by 4% (mean +/- SD, 20 +/- 8 kg) and 34% (32 +/- 9 kg), respectively, compared with pre-exercise values, with similar losses during low- and higher-intensity training. There was no effect of exercise training on subcutaneous fat thickness, plasma hormone and lipid concentrations, or minimal model parameters of glucose and insulin dynamics. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that moderate exercise training without concurrent dietary restriction does not mitigate insulin resistance in overweight or obese horses. A more pronounced reduction in adiposity or higher volume or intensity of exercise may be necessary for improvement in insulin sensitivity in such horses.

Effects of diet-induced weight gain on insulin sensitivity and plasma hormone and lipid concentrations in horses.

OBJECTIVE: To determine the effects of diet-induced weight gain on glucose and insulin dynamics and plasma hormone and lipid concentrations in horses. ANIMALS: 13 adult geldings. PROCEDURES: Horses were fed 200% of their digestible energy requirements for maintenance for 16 weeks to induce weight gain. Frequently sampled IV glucose tolerance tests were performed before and after weight gain to evaluate glucose and insulin dynamics. Adiposity (assessed via condition scoring, morphometric measurements, and subcutaneous fat depth) and plasma concentrations of insulin, glucose, nonesterified fatty acids, triglycerides, and leptin were measured on a weekly or biweekly basis. RESULTS: Mean + or - SD body weight increased by 20% from 440 + or - 44 kg to 526 + or - 53 kg, and body condition score (scale, 1 to 9) increased from 6 + or - 1 to 8 + or - 1. Plasma glucose, triglyceride, and nonesterified fatty acid concentrations were similar before and after weight gain. Leptin and insulin concentrations increased with weight gain. Mean + or - SD insulin sensitivity decreased by 71 + or - 28%, accompanied by a 408 + or - 201% increase in acute insulin response to glucose, which resulted in similar disposition index before and after weight gain. CONCLUSIONS AND CLINICAL RELEVANCE: Diet-induced weight gain in horses occurred concurrently with decreased insulin sensitivity that was effectively compensated for by an increase in insulin secretory response. Obesity resulted in hyperinsulinemia and hyperleptinemia, compared with baseline values, but no changes in lipid concentrations were apparent. Preventing obesity is a potential strategy to help avoid insulin resistance, hyperinsulinemia, and hyperleptinemia in horses.

Apparent adiposity assessed by standardised scoring systems and morphometric measurements in horses and ponies.

This study described a scoring system for the assessment of apparent neck adiposity and evaluated morphometric measurements for assessment of neck and overall adiposity. Twenty-one barren Thoroughbred mares, 13 Arabian geldings and 75 Welsh, Dartmoor, or crossbred pony mares, were clinically examined and blood samples analysed for insulin, glucose, leptin, and triglycerides. Bodyweight (BW), height, length, girth and abdominal circumferences, neck length, neck crest height and neck circumference were measured, and body condition scores (BCS) and cresty neck scores (CNS) were rated. Girth:height ratio had the strongest associations with BCS (r(s)=0.64, P<0.001 in horses; r(s)=0.83, P<0.001 in ponies) and blood variables, such as leptin (r(s)=0.39, P=0.024 in horses; r(s)=0.68, P<0.001 in ponies). Crest height and neck circumference:height ratio had the strongest association with CNS (r(s)>0.50, P<0.01) and blood variables, such as insulin (r(s)0.40, P<0.05). Cresty neck score was useful in the assessment of neck crest adiposity and had physiological relevance, as demonstrated by associations with blood variables. Girth:height was the most suitable morphometric for assessment of overall adiposity, and either crest height or neck circumference:height was a suitable morphometric for assessment of apparent neck adiposity.