Despite being preventable, foodborne diseases remain a global health challenge. Poor food hygiene practices such as improper handling of kitchen utensils are among the major causes of diarrhea transmission. A formative study was conducted in Malawi to inform an intervention design to promote complementary food hygiene practices. An assessment of contextual and psychosocial factors for behavior change was conducted using Risk, Attitude, Norms, Ability, and Self-regulation model. We conducted 323 household surveys with caregivers of children aged 6 to 24 months. Analysis of variance was used to estimate difference between doers and non-doers of three targeted behaviors: washing utensils with soap, keeping utensils on a raised place, and handwashing with soap. Analysis of variance analyses revealed that literacy level, ownership of animals, and presence of handwashing facility and dish racks were contextual factors predicting storage of utensils on an elevated place and handwashing frequencies. Psychosocial factors, such as time spent to wash utensils with soap, distance to the handwashing facility, and cost for soap, had an influence on washing utensils and handwashing practices. Perceived vulnerability determined effective handwashing and storage of utensils. Perceived social norms and ability estimates were favorable for the three targeted behaviors. Promotion of already existing targeted beneficial behaviors should be encouraged among caregivers. Risk perceptions on storage of utensils and handwashing practices should be increased with motivational exercises such as paint games. Caregivers’ technical know-how of local dish rack and tippy tap construction is essential. This corresponds with reports that at least 70% of diarrhea-related pathogens among children could be caused by contaminated food.8Worldwide, the lives of approximately 525,000 children are lost each year from 1.7 billion cases of childhood diarrhea with the highest mortality rates reported among children aged less than 2 years in south Asia and sub-Saharan Africa.7 Nevertheless, such protection is temporary because children are subsequently exposed to pathogens when introduced to complementary food between the ages of 4 and 6 months.10 This exposure together with increased environmental interaction have been linked to the high incidence of diarrhea among children aged between 6 and 24 months.13 To reduce diarrhea among children, the WHO has indicated important parameters that need to be implemented at the household level, including access to safe water, improved sanitation facilities, exclusive breast feeding, hygienic weaning practices, and improved personal and household hygiene.14If children aged between 0 and 6 months are exclusively breastfed, they are expected to be free from pathogens.Escherichia coli, Salmonella, and Vibrio cholerae) in food.16 Contamination of utensils was attributed to the method of cleaning, resulting from repeated use of wash water and dirty cloths. Because of the risk of post-cooking contamination, the cleaning of utensils before eating, particularly for high-risk groups, is integral to food safety, as demonstrated by studies in Thailand and Mali.18 As such, effective cooking of food cannot be considered as a sole critical control point, but must be combined with washing of utensils with soap and handwashing with soap at critical times.18Food can become microbiologically contaminated if prepared under unhygienic conditions, and studies have shown that utensils, such as spoons, cups, pots, baby bottles, and plates, are potential sources of pathogens , social , and physical parameters .32Changing an individual’s behavior is a process that requires change in contextual and psychosocial factors that predict human behavior in a given setting, such as attitudes, norms, and self-regulation attributes.33 which was developed based on psychological theories35 and has been applied in this formative study was designed to understand contextual and psychosocial parameters of individuals associated with their water, sanitation, and hygiene (WASH) practices. The model presents five “factor blocks” that should be applied to understand psychosocial factors of a study population to determine a specific behavior.The Risk, Attitude, Norms, Ability, and Self-regulation (RANAS) modelThe risk factors respond to the level of understanding and awareness of the person’s vulnerability and severity of diseases. They also include health knowledge about disease transmission, prevention options, and personal consequences.Attitude factors include beliefs about the costs and benefits of a particular behavior and feelings associated with the behavior.The norm factors address the perception of what behavior is performed in the society and the level of personal obligation to a specific behavior. It includes how family and community members, including leaders, approve or disapprove a particular behavior.Ability factors assess an individual’s capacity to perform a certain behavior, which includes its uptake, maintenance, and recovery from drawbacks.The self-regulation factors check on an individual’s plan on how to sustain a specific behavior, and they include the element on how to deal with barriers to the implementation of the behavior.37 The RANAS model is applied in two stages: 1) determine the behavior factors for the individuals under study and 2) select behavior change techniques (BCTs) that should be applied to the identified gaps.38 Consequently, this can provide scientific guidance on which strategies to follow during an intervention. Because human behavior occurs in an environmental setting where a number of factors come into play, understanding of psychosocial factors alone may not be enough to bring about behavior change. As such, this must be complemented with details of the contextual factors in which the behavior occurs.To our knowledge, the RANAS model has not been previously applied in a food hygiene assessment. However, it has been successfully used to evaluate water treatment, sanitation, and handwashing behavior.The objective of this formative study was to describe the situation and behavior, and to determine the contextual and psychosocial factors associated with 1) washing of household utensils with soap, 2) storing of household utensils on an elevated area, and 3) washing hands with soap at critical times. This study was a component of a larger body of work to understand behavioral factors related to complementary food hygiene in the development of an intervention trial.39 and Chichewa is the main language of the area. Chikwawa is in a low-lying area and, therefore, prone to flooding in the rainy season. Similar to other districts of Malawi, Chikwawa has two seasons per year, that is, rainy/farming season that lasts from November to April and dry/off farming season from May to October. The district has an annual average temperature of 25.7°C and an average rainfall of 797 mm.40The formative study was conducted in three rural administrative Traditional Authorities of Masache, Ngowe/Ngabu and Maseya in Chikwawa district in southern Malawi. During the 2018 population census, the Chikwawa district population was 564,684,Three Traditional Authorities were chosen taking into account their geographic location , socioeconomic variability (low-income communities), and access to protected water sources and high sanitation coverage (declared open defecation free), but with a continued high risk of cholera and diarrhea. Households in the targeted villages are very close to each other, and this provides an opportunity for communities to have common values and share WASH, including food hygiene issues without social resistance.41 Taking into account of nonresponse rate and missing data, the sample size was increased to 323.This formative study targeted caregivers and their children aged 6 to 24 months. In this article, the word caregiver includes any household member, including parents who are responsible for daily care of the targeted child. This includes feeding and preparing the child’s food, bathing, and assisting the child during defecation. With the use of information from the community health workers’  register, a list was drawn up of households with children aged between 6 to 24 months, having a pit latrine, and with access to safe water within a distance of 500 m. A sample size of 295 was calculated based on Chikwawa district diarrhea prevalence of 26.3% with an acceptable error margin of 5%.The research team collected data from February to July 2017. As behaviors are determined by a wide range of factors, it was necessary to use different data collection methods to reveal the complexity of the socioeconomic, cultural, and other factors that influence the child caregiver’s decision on the “what,” “how,” “when,” and “why” of infant and child feeding practices. Therefore, formative data were collected from four complementary phases which included checklist and structured observations, in-depth interviews, household surveys , and focus group discussions. This article presents only findings from household survey. All households (323) undertook the combined demographic and RANAS model–based household questionnaire. Initially, before conducting household surveys, observations were conducted which identified three critical behaviors: 1) washing utensils with soap, 2) keeping utensils on an elevated area, and 3) handwashing with soap at critical times, where critical times in this article mean handwashing with soap before food preparation; before eating, including child feeding; after changing child’s nappy; and after latrine use by the caregiver.n = 323) which was translated into the local language of Chikwawa district (Chichewa). Responses to the RANAS questions were recorded on a 5-point Likert scale . The household survey questionnaire was mainly composed of closed questions that captured information about demographics, child feeding, health status and awareness, psychosocial factors related to washing utensils with soap, keeping utensils on elevated area, and handwashing with soap at critical times . Pretesting of the questionnaire was conducted before data collection where the research team identified and eliminated irrelevant questions, whereas key questions were further edited for easy understanding.11 For each targeted behavior, the significant factors among those noted with ANOVA calculation were further analyzed  with effect size, d, where Cohen’s d values mean small for those = or < 0.20, medium = or < 0.50, and large = or > 0.80.Demographic household and RANAS data were collected using Open Data Kit software  on android tablets and exported to Microsoft Excel  and quality checked before being exported to Statistical Package for Social Sciences (SPSS) where frequency distribution of demographic characteristics using descriptive statistics was plotted. IBM SPSS version 25, the PROCESS macro for SPSS, was used to undertake all statistical tests . The household RANAS model–based data were analyzed using ANOVA mean comparison analysis to determine the differences between doer and non-doer contextual and psychosocial factors for the targeted behaviors. To measure the three targeted behaviors, data collectors asked caregivers how often they washed utensils with soap, how often they kept utensils on a raised place, and how often they washed hands with soap at critical times. Frequencies were measured on a 5-point scale. All factors falling at or below the mid 3-point value on a scale of 1–5 were considered non-doers of the targeted behaviors, whereas those factors at or above 4 were doers of the behavior, and the mean score for each targeted behavior was calculated. Washing utensils with soap, keeping utensils on an elevated area, and handwashing with soap were dependent variables, whereas behavioral factors of the RANAS model were independent variables. Three questions were asked to caregivers to assess knowledge about diarrheal disease causation, signs, and preventive measures. The ratio of correct answers from the caregivers to all possible answers formed the health knowledge constructs. A single item was used to measure perceived severity, whereas perceived vulnerability of diarrhea and other psychosocial factors were measured with multiple items. The WHO and United Nations Children’s Fund definition of diarrhea was used when assessing diarrhea incidence among targeted children.The formative study protocol was approved by the University of Malawi’s College of Medicine Research Ethics Committee (P.04/16/1935). Permission was obtained from the local authorities, that is, Chikwawa district council, Chikwawa district health office, and the traditional chiefs. The participants were informed of the research objectives and were advised that they had the freedom to refuse participation or withdraw from the study at any time. Participants’ written informed consent was obtained before inclusion in the study. Participants were provided with a unique identifying number, and data were anonymized during data analysis. Data were accessed only by the authors. The study was registered with the Pan African Clinical Trials Registry (PACTR201703002084166).All respondents of the household questionnaire were females whose age ranged from 18 to 53 years (mean 26.72 with SD 6.78). The majority of them (71%) attended primary education, whereas 16% had never been to school. Income was primarily from subsistence farming (67%), and majority of the households (74%) earned at most $14 per month. As such, households reported some levels of uncertainty about food supply. The age range of targeted children was 6–24 months (mean 14.27 with SD 5.72) of which 49% were females. Forty percent of children were introduced to complementary food  when they were between 3 and 6 months old, and 27% of the targeted children were reported to have had diarrhea in the 2 weeks before the survey. No participating households were connected to an electrical power supply, and therefore, none owned a refrigerator. Domesticated animals, such as pigs, dogs, goat and poultry, were observed roaming freely in household yards. Human and animal feces were observed in 2% and 52.9% of the household yards, respectively.Caregivers accessed safe water through boreholes (93%) and piped water supply (3%). Latrines were owned by 95% of the households, whereas 5% either depended on their neighbors’ latrine or practiced open defecation. Despite high coverage, most toilets were unimproved traditional latrines (64%) subject to collapse during the rainy season and offering minimal privacy. Soap was available in 61% of the households, and it was prioritized in the following order: washing clothes, bathing, washing kitchen utensils, and handwashing.Contextual factors were compared between doers and non-doers of the three targeted behaviors. Statistical analysis identified significant variables related to handwashing with soap and keeping of utensils on an elevated place, whereas no significant variables were observed for washing utensils with soap . Factorsd values were found on others’ behavior , others’ approval (d = 0.74), and confidence in performance , where non-doers reported highly that they could not wash utensils with soap because of inadequate water at the household (d = 0.45) and self-regulation , meaning that doers found it more pleasant to wash utensils with soap than non-doers. Similarly, the doers were less likely to forget to wash their utensils with soap than the non-doers. Slightly significant differences between doers and non-doers were noted on others’ behavior , intention (d = 0.33), and communication (d = 0.3). This implies that non-doers do not desire much to wash their utensils with soap than the doers. In addition, the non-doers do not discuss much with their friends or relatives about the practice of washing utensils with soap compared with the doers.From the household spot checks, the study noted that 29% of the caregivers washed their utensils with soap. Risk, Attitude, Norms, Abilities, and Self-regulation model–based questions were asked to understand psychosocial factors that contributed to caregivers not using soap when washing utensils. As shown in ousehold . This med values were noted on others’ behavior , others’ approval (d = 0.6), and confidence in performance which included “difficult” (d = 0.44), “hurry” (d = 57), and “restart” (d = 0.65) (d = 0.47) about using an elevated surface to keep or dry their utensils and felt less vulnerable (d = 0.58) to the risk of diarrheal disease than doers, which is related to the non-doers perception that keeping utensils on a raised place is not a pleasant practice (d = 0.35).The study found that 31% of the caregivers kept their utensils on an elevated place that could not easily be reached by animals. On psychosocial factors related to storage of utensils on an elevated place, significant differences between doers and non-doers could not be found on severity, health knowledge, attitude (time and effort), and personal obligation . Hence,  = 0.65) . This imd = 1.17) and confidence in continuation (d = 0.81). This implies that non-doers show a lower preference to washing their hands with soap and are less likely to continue using soap when washing hands at critical times. Furthermore, the non-doers found it expensive  and time-consuming  to wash hands with soap compared with the doers. The factor attitude (distance) was also found to be significant (d = 0.34). As such non-doers perceived that the handwashing facility located near the latrine was too far for them to wash hands with soap during other critical times of handwashing, such as before preparing food. The caregivers found it hard to have another handwashing facility within the cooking area because they had no technical know-how on handwashing facility construction. They depended on their husbands to construct the handwashing facilities, but they, in most cases, were reportedly engaged with food-fetching activities for the home. Other significant factors included cost (d = 0.27), others’ behavior , remembering (d = 0.7), and communication (d = 0.62). Furthermore, risk factors (vulnerability and severity) were slightly significant for handwashing with soap practice . This means that doers found it more probable that they would suffer from diarrhea and its severity would be more, compared with the non-doers, although health knowledge, time, effort, confidence in performance (water), and commitment (importance) were insignificant.A specific place for handwashing was found in 51% of the households, of which 62% were located near the latrine. However, only 19% of the handwashing facilities had soap and water. The study explored psychosocial factors that contributed to nonuse of soap when washing hands at critical times. As shown in 33 provided guidance on which BCTs should be applied for the behavioral interventions. Evidence-based decisions in the choice of BCTs to promote complementary food hygiene practices were derived from analysis of contextual and psychosocial factors. Furthermore, household spot checks noted that only 29% and 31% of the visited households had soap for washing utensils and had an elevated place for keeping kitchen utensils, respectively, whereas handwashing facilities with soap and water were noted in 19% of the households. The formative data provided a platform for developing interventions with an overall aim of promoting child caregivers toward improved complementary food hygiene practices. As shown in 42 In addition, their confidence in performance would be enhanced through demonstrations such as “Glo germ gel” and “hand and utensil painting exercise” (see the following paragraphs) that would lead to an increased perception of self-efficacy. 3) Indicate that others are already performing the desired practices. Thus, public commitment to show that others are performing the targeted behaviors would be performed through open days where caregivers would also sing songs about targeted behaviors. Public pledges would enhance normative factors and posters to be placed outside caregivers’ houses would show community members that others are performing targeted behaviors that would boost descriptive norms. 4) Reinforce the action self-efficacy through use of attractive posters with key messages to remind caregivers to always wash utensils and hands with soap will enhance their confidence to practice the behavior.Based on the results from formative data, the RANAS model fact sheet45 and high prevalence of diarrhea among children in this study suggests that food hygiene practices such as these may play an important role in child health. However, motivators and barriers for food hygiene improvements in this setting were not clearly understood. For the first time, our study assessed the contextual and psychosocial factors related to caregivers’ food hygiene practices in rural Malawi. Such data were necessary for the development of population-tailored behavior change interventions. In this formative study, data were collected from child caregivers who had children aged between 6 and 24 months in Chikwawa, Malawi. Normative factors about others’ behavior and ability factors were identified as the main factors for all three behaviors. In addition, the self-regulation factor (remembering) was found to be a strong predictor of handwashing with soap at critical times. Guided practice, memory aids, information about others’ behavior, and model behavior are being considered in a behavior change intervention for improved practices on washing of utensils with soap, keeping utensils on a raised place and handwashing with soap at critical times.As reported in other developing countries, complementary food hygiene is suboptimal in Malawi,16 As such, the creation of effective promotion strategies to encourage the use of soap to wash utensils is important. The practice of placing utensils on the ground before, during, and after washing utensils is common in Malawi and may increase the risk of childhood diarrhea contaminating utensils with pathogens in soil and animals (directly and via feces). Nevertheless, this study found that the presence of a dish rack at a household influenced the doers to keep their utensils on an elevated place compared with the non-doers. Previous research has shown that promoting existing beneficial behavior is important in addressing local needs.19 Thus, the safe practice of using locally made dish racks which is already performed by a few (31%) in the study area should be promoted.Although we acknowledged that washing utensils without soap is not the only risk factor for diarrhea among children, the practice of washing utensils without soap could increase the risk of food contamination as this is a proven route of pathogen transmission.46 purchase of soap in this study was found to slightly influence handwashing with soap practice. However, usage of soap greatly depended on influence from others and the availability of a convenient place for handwashing. Failure of caregivers to wash hands because of the lack of a handwashing facility confirms what has been previously reported that a specific place for handwashing is a predictor of household handwashing frequency.48 Generally, 62% of the handwashing facilities were located near the latrine (behind and away from the cooking area) which affected the frequency of caregivers’ washing hands at other critical times . Furthermore, it has been shown in this study that the presence of a handwashing facility at a household increased the handwashing practice among the doers compared with the non-doers. Thus, constructing additional handwashing facilities within the cooking area could improve the frequency of handwashing practice.In this study, use of soap for handwashing was uncommon. Soap was found to be prioritized for other household usage such as washing clothes and bathing. Contrary to what was reported by Seimetz et al.,Three knowledge sections in the questionnaire showed no significant difference between doers and non-doers about diarrhea causation, signs/symptoms, and prevention as knowledge was found to be high in both groups. However, significant differences in risk perception between doers and non-doers were noticed on keeping utensils on a raised place and handwashing with soap practices. Thus, practical strategies to sensitize the caregivers to the health risks associated with storage of utensils and handwashing with soap should be incorporated in an intervention.49 Therefore, corresponding normative BCTs should be applied to facilitate behavior change. As community meetings have been reported to strengthen normative elements,51 group meetings with caregivers would be essential where a positive group identity would be reinforced and role models would be identified to promote the behaviors. In addition, communication about the behaviors among caregivers would be strengthened through the group meetings. Household visits would be conducted as follow-up to group meetings to prompt guided and behavioral practice at an individual level. Importantly, BCTs related to personal commitment would be appropriate to address personal norms toward the three behaviors. Such commitment should be made in public by caregivers together with their husbands as they have been found to have a major role in the construction of handwashing facilities and dish racks. As reported in other behavior change studies,49 public pledges would also help to reach out to more people and, thus, change descriptive norms. Having adequate water at the household increased the confidence of caregivers to wash utensils with soap. This suggests that promoting adequate water availability at households is a potential strategy for washing utensils with soap. Role models on this practice should be encouraged to demonstrate to others how they manage to have adequate water in their homes for washing utensils.Washing utensils with soap, keeping utensils on an elevated surface, and handwashing with soap strongly interdepended on the normative factor—others’ behavior . A study in Nepal showed that influence from others plays a major role in one’s behavior about sanitation and hygiene.52 Practical demonstrations on dish rack and hand washing facility construction should be promoted to strengthen the perception of self-efficacy, thus reinforcing ability factors. In addition, the use of behavioral cues should be incorporated to remind caregivers’ abilities to wash utensils and hands with soap. The use of such interesting and innovative approaches has proven to be effective in behavior change initiatives.55Caregivers’ abilities (confidence in performance) to keep utensils on an elevated place and wash utensils and hands with soap were a very strong predictor for the practice of these behaviors. The lower perceived self-efficacy in washing utensils with soap, keeping utensils on an elevated place, and handwashing with soap among the non-doers requires the implementation of a corresponding BCT. Demonstrations such as “Glo germ gel” and “hand and utensil painting exercise” could be applied to strengthen caregiver’s belief and ability to continuously use soap when washing hands and utensils as its effectiveness would be appreciated. Hand painting exercises show the potential movement of pathogens from one person to another through hand shaking and being in contact with household items, for example, utensils. Participants put paint in their palm and then shake hands among themselves and touch household items to represent spreading of germs. While having paint in the hands, some are asked to wash hands with soap, whereas others without and notice the difference. Similarly, the utensil painting exercise demonstrates the effectiveness of soap in removing dirt and germs from utensils such as plate. Handwashing Glo germ gel reveals areas in the hands that are concentrated with germs.Self-reported findings are prone to bias as the participants may report what the researcher wants to hear. However, this was controlled by conducting spot checks on some of the variables that were reported by the participants. Food hygiene practices cover additional practices to those covered in this article, such as storage conditions and reheating of leftover food. However, further analysis of formative research findings assessed these parameters in the same study setting. Sociocultural practices and geographical conditions across Malawi may differ; hence, the results of this study may not be applied to all the rural areas without further study. In addition, during recruitment, all study households had a latrine and access to safe water within a distance of 300 m. This is not the case with other households in rural settings of Malawi. However, despite the stated limitations, this research provides a good platform for understanding the contextual and psychosocial factors related to complementary food hygiene practices for the design of an effective food hygiene intervention in rural Malawi.This study for the first time has applied the RANAS model to assess contextual and psychosocial factors influencing child caregivers’ behavior relating to food hygiene practices in rural Malawi. This research provides evidence-based results as a basis for the development and implementation of food hygiene interventions to contribute toward prevention of diarrheal diseases. Selected contextual  and psychosocial factors which include normative, ability, and self-regulation (remembering) factors have been identified as strong predictors for the success of an intervention that focuses on washing of utensils with soap, keeping of utensils on an elevated place, and hand washing with soap at critical times. Therefore, they should be considered for promotion in future initiatives.


Inhibition of AMPK with SBI-0206965 had no effect on the inhibition of insulin-stimulated Akt phosphorylation by A769662, suggesting the inhibitory action of A769662 is AMPK-independent. A769662 decreased IGF1-stimulated Akt phosphorylation yet had no effect on VEGF-stimulated Akt signalling in HUVECs, suggesting that A769662 attenuates early insulin/IGF1 signalling. The effects of A769662 on insulin-stimulated Akt phosphorylation were specific to human ECs, as no effect was observed in the human cancer cell lines HepG2 or HeLa, as well as in mouse embryonic fibroblasts (MEFs). A769662 inhibited insulin-stimulated Erk1/2 phosphorylation in HAECs and MEFs, an effect that was independent of AMPK in MEFs. Therefore, despite being a potent AMPK activator, A769662 has effects unlikely to be mediated by AMPK in human macrovascular ECs that reduce insulin sensitivity and eNOS activation.Protein kinase B (Akt) is a key enzyme in the insulin signalling cascade, required for insulin-stimulated NO production in endothelial cells (ECs). Previous studies have suggested that AMP-activated protein kinase (AMPK) activation stimulates NO synthesis and enhances insulin-stimulated Akt activation, yet these studies have largely used indirect activators of AMPK. The effects of the allosteric AMPK activator A769662 on insulin signalling and endothelial function was therefore examined in cultured human macrovascular ECs. Surprisingly, A769662 inhibited insulin-stimulated NO synthesis and Akt phosphorylation in human ECs from umbilical veins (HUVECs) and aorta (HAECs). In contrast, the AMPK activators compound  Endothelial cells (ECs) are essential for modulation of vascular homeostasis and signal transduction , includiIn healthy subjects, insulin acts as a vasodilator, stimulating calcium-independent NO synthesis in cultured human aortic ECs (HAECs) through Akt-mediated phosphorylation of endothelial nitric oxide synthase (eNOS) Ser1177 and Ser615 .AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine protein kinase sensitive to intracellular AMP levels which acts as a master regulator of energy metabolism ,10. AMPKPrevious studies have reported that AMPK activation improved insulin sensitivity and energy homeostasis, and attenuated inflammatory signalling in insulin-sensitive tissues, such as muscle , liver ,17 and a991—a compound 5–10-fold more potent than A769662 [In the last 15 years, there has been considerable effort to develop AMPK-selective small-molecule activators, resulting in the development of A769662 , the dis A769662 . A769662 A769662 ,25. Sinc A769662 ,27,28,29Stimulation of HUVECs with concentrations of insulin above 0.1 µM robustly stimulated phosphorylation of Akt at Ser473 and Thr308 , similar991, which allosterically activates AMPK at the same site as A769662 [A769662  increased AMPK activity, assessed by immunoblotting of AMPK-specific ACC (acetyl CoA carboxylase) Ser79 phosphorylation B,C, yet  A769662 , had no To examine the relationship between AMPK activation and inhibition of insulin-stimulated Akt phosphorylation by A769662, the concentration dependence of either effect of A769662 was assessed. Significant A769662-mediated stimulation of ACC phosphorylation was achieved with 50–100 μM A769662 A,B. A769Furthermore, the time dependence of the inhibitory effect of A769662 on insulin-stimulated Akt phosphorylation was assessed. A769662 (50 µM) rapidly stimulated ACC phosphorylation in HUVECs within 5 min, an effect that was sustained for at least 2 h E,F. The To determine whether the inhibition of insulin-stimulated Akt phosphorylation in ECs was AMPK-dependent, similar experiments were conducted after prior incubation in the SBI-0206965, which has recently been described as a selective inhibitor of AMPK . PreincuTo determine whether this inhibition of insulin signalling was conserved in ECs from other regions of the vasculature, insulin-stimulated NO synthesis and signalling were assessed in HAECs. A769662 (50 μM) markedly inhibited insulin-stimulated NO synthesis A, an eff991  significantly increased ACC Ser79 phosphorylation in both HeLa  or compound oth HeLa  and HepGoth HeLa  to a simoth HeLa . CompounG2 cells  yet intrG2 cells . The inhTo further assess the AMPK-dependence of the inhibition of insulin-stimulated Akt and Erk1/2 phosphorylation by A769662, SV40-immortalised wild-type (WT) or AMPK knockout (KO) mouse embryonic fibroblasts (MEFs)  were stiTo examine whether A769662 inhibits Akt phosphorylation in response to growth factors other than insulin in ECs, the effect of A769662 on IGF-1 and VEGF were assessed in HUVECs. VEGF  stimulated a significant increase in Erk1 Thr202/Tyr204 phosphorylation, yet there was only a trend towards an increase in Akt Ser473 phosphorylation . In cont991 selectively activate AMPK complexes containing the β1 regulatory subunit [A769662 and compound  subunit . It has  subunit , yet the subunit   has been demonstrated previously to inhibit basal Akt Ser473 phosphorylation in prostate cancer cell lines, yet stimulated Akt Ser473 phosphorylation at a lower concentration (50 µM) . Indeed,991 increased insulin-stimulated Akt phosphorylation. These data argue for a cell-type specific/selective effect of A769662 on insulin signalling, whereby only A769662 markedly reduces insulin signalling in ECs and insulin-stimulated Erk1/2 phosphorylation in ECs and MEFs. This cell-type selectivity could be related to differential expression of IR/IGF1R between cell types. HUVECs have been previously reported to have approximately 400,000 IGF1R/cell and 40,000 IR/cell [https://www.proteinatlas.org/) [In addition, we demonstrate that preincubation of HeLa cells, but not HepG2 cells or ECs, with compound  IR/cell . Furtheras.org/) , HepG2 aas.org/) . Higher 991 [+ channels in rat neurons and the Na+/K+ ATPase in L6 cells [2+ levels, by an endothelium-dependent yet AMPK-independent manner [It is unlikely that the differential effects of A769662 in the different cell types can be explained by differences in AMPK isoform expression, due to the selectivity of A769662 and compound 991 . Both co991 . This fuL6 cells ,28,29 haIn conclusion, A769662 decreases insulin-stimulated NO synthesis and Akt Ser473 phosphorylation in HUVECs and HAECs in a manner likely to be independent of AMPK. Furthermore, A769662 decreases insulin-stimulated Erk1/2 phosphorylation in a manner that is AMPK-independent in MEFs and HAECs. Taken together, these data demonstrate that caution should be exercised when interpreting data obtained using A769662 as a tool in cultured human endothelial cells.991 was synthetised by MRC Technology. A769662 and mouse anti-β-tubulin (#11307) antibodies were purchased from Abcam . Rabbit anti-phospho-Akt Thr308 (#13038), anti-phospho-Akt Ser473 (#4058), anti-ERK1/2 (#9102), anti-phospho-ACC Ser79 (#3661), anti-ACC (#3676), anti-α-tubulin (#2144) and mouse anti-Akt (#2920) and anti-phospho-p44/42 Erk1/2 (Thr202/Tyr204) (#9106) antibodies were from New England Biolabs UK . Sheep anti-AMPKα1, anti-AMPKα2, anti-AMPKβ1 and anti-AMPKβ2 antibodies used for immunoprecipitation of AMPK complexes containing specific subunit isoforms were a kind gift from Professor D.G. Hardie (University of Dundee) and have been described previously [Cryopreserved HUVECs, HAECs and MV2 medium were purchased from Promocell . SV40-immortalised wild-type and AMPKα1 and AMPKα2 knock-out MEFs were kindly provided by Dr. B. Viollet  and have been described previously . HeLa aneviously ,63,64. Iv/v) serum (Promocell). HUVECs and HAECs were utilised between passages 3 and 6. MEFs and HeLa cells were cultured in DMEM supplemented with 10% (v/v) foetal calf serum. HepG2 cells were cultured in RPMI 1640 supplemented with 10% (v/v) foetal calf serum, 1 mM sodium pyruvate. Once cells reached 90–100% confluence, HUVECs/HAECs were incubated in serum-free Medium 199 and HeLa cells and HepG2 cells were incubated with serum-free DMEM for 2 h and then with AMPK activators prior to stimulation with insulin (1 μM), VEGF (10 ng/mL) or IGF1 (25 ng/mL). Cells were placed on ice and washed with PBS prior to lysis in Triton X-100-based lysis buffer  Triton-X-100, 250 mM mannitol, 1 mM DTT, 1 mM Na3VO4, 0.1 mM benzamidine, 0.1 mM PMSF, 5 μg/mL SBTI). Cell lysates were scraped into microcentrifuge tubes and incubated on ice for 20 min, centrifuged  and the subsequent supernatants stored at −20 °C. Lysate protein concentrations were determined using Bradford or BCA methods, as previously described [HAECs and HUVECs were cultured in MV2 medium supplemented with EC growth factor mix, 5% (v/v) LI-COR blocking buffer in TBS containing 0.1% (v/v) Tween-20 as described previously [w/v) milk powder prior to probing with primary antibodies. In all other cases, immunoblots for phospho- and total protein levels of ACC, Akt and Erk1/2 were obtained on immunoblots conducted concurrently in parallel.Cell lysate proteins were resolved by SDS-PAGE and immunoblotted with antibodies diluted in 50% , and supernatants were stored at −20 °C. NO concentration was determined using a Sievers 280A NO Meter  as described previously [HAECs and HUVECs were cultured in 6-well plates until 100% confluent and serum-starved for 2 h in Medium 199 and then in the presence of Krebs-Ringer-Hepes (KRH) buffer for 20 min. The medium was replaced and cells incubated in the presence or absence of stimuli in KRH for a further 15–20 min. Samples of medium (50 μL) were taken at different intervals and 200 μL methanol added to each. Samples were centrifuged  Triton-X-100, 1% (v/v) glycerol, 1 mM DTT, 0.1 mM benzamidine, 1 mM PMSF, 5 μg/mL SBTI, 1 mM Na3VO4). Immunoprecipitates were then washed into HBD buffer (50 mM HEPES-NaOH (pH 7.4), 0.02% (v/v) Brij-35, 1 mM DTT) and assayed for AMPK activity using SAMS substrate peptide as described previously [AMPK specific isoforms were immunoprecipitated from HAECs using sheep anti-AMPKα1, anti-AMPKα2 , anti-AMeviously ,35.p < 0.05 as significant.All data is expressed as a relative change (mean ± SEM) from control baseline or normalised to group of reference for each experiment. Prism software  was used to perform one or two-way ANOVA (with Tukey’s or Dunnett’s post hoc multiple comparison tests), where appropriate, using


Global inequalities in economic access and agriculture productivity imply that a large number of developing countries rely on working equids for transport/agriculture/mining. Therefore, the understanding of hoof conditions/shape variations affecting equids' ability to work is still a persistent concern. To bridge this gap, using a multi-scale interdisciplinary approach, we provide a bio-physical model predicting the shape of equids’ hooves as a function of physical and biological parameters. In particular, we show (i) where the hoof growth stress originates from, (ii) why the hoof growth rate is one order of magnitude higher than the proliferation rate of epithelial cells and (iii) how the soft-to-hard transformation of the epithelium is possible allowing the hoof to fulfil its function as a weight-bearing element. Finally (iv), we demonstrate that the reason for hoof misshaping is linked to the asymmetrical design of equids' feet (shorter quarters/long toe) together with the inability of the biological growth stress to compensate for such an asymmetry. Consequently, the hoof can adopt a dorsal curvature and become ‘dished’ overtime, which is a function of the animal's mass and the hoof growth rate. This approach allows us to discuss the potential occurrence of this multifaceted pathology in equids. Owning a working equid provides financial benefits to families [Equids  are ‘ungulates’ or single-digit hoofed mammals. As the function of the hoof is to sustain the animal's weight and to balance external stresses during locomotion, hoof pathologies and associated shape variations have important repercussions on equids' health and have puzzled humankind for centuries, with reference being made in Aristotle's writings around 350 BC . While efamilies  and playfamilies ,5 and tifamilies .As perissodactyls, or odd-toed ungulates, horses have a strong hoof capsule, protecting the internal structures of each single-digit foot. This horny structure encloses the distal part of the second phalanx, the distal phalanx and the navicular bone. The adhesion of the hoof to the distal phalange is warranted by the dermo-lamellar junction that, through its hierarchical design, allows a strong adhesion to take place during an abrupt rise in mechanical stresses, e.g. gallop –8  develop over a long period of time and are commonly perceived as a warning sign underlying a past or present disorder associated with biological factors  and sometimes, physical factors, including the specific loading of a limb –11. Howe−1 [−1 (approx. 0.01 mm day−1)? (ii) How can a hoof capsule with an elastic modulus approximately 108 Pa [3 Pa [Biomechanical studies of the equid's hoof capsule were the first to gather essential information regarding the stress/strain relationships and viscoelastic properties of the hoof capsule in relation to its morphology by considering the adult hoof as a static piece of tissue –23. Howe−1  when kery 108 Pa  emerge fPa [3 Pa ,26?  and biology (e.g. proliferation and differentiation of keratinocyte progenitor cells) to demonstrate the weakness of this system.2.2.1.2.Hooves were obtained from horses that were not euthanized for research purposes. Horse hooves were collected from the abattoir 1 h post euthanasia following ethical approval by The School of Veterinary Medicine and Science, University of Nottingham. For three-dimensional imaging and histological sampling, PBS was injected through the medial and lateral palmar digital arteries to remove the blood, and tissues were fixed by replacing PBS with a 4% PFA/PBS  fixative solution under manual pressure. When needed, biopsies were taken from the dorsal and quarter parts of the coronary band and placed into a 4% PFA/PBS solution fixative solution prior processing. For primary cell isolation, hooves were aseptically cleaned and progenitor keratinocyte cells obtained as described below. All primary cell cultures were performed at 37°C in 5% CO2.2.et al. [To investigate details of the circulatory system and tissues surrounding and within the papillae, a hoof specimen was imaged on Beamline I12-JEEP at the Diamond Light Source, UK  using 0.et al.  to improet al. . Three-d2.3.Individual hooves were scanned using a Phoenix v|tome|x m industrial scanner . A maximum X-ray energy of 125 kV, 320 µA current and a 0.5 mm thick copper filter was used to scan each sample, consisting of 2160 projection images, with a detector exposure time of 333 ms, acquired over a 360° rotation. The magnification and spatial resolution achieved were ×1.82 and 120 µm respectively. Data were reconstructed for visualization using datos|x software and were visualized using VGStudio MAX 2.2 . The average dorsal curvature of the hoof was estimated from a ventro-dorsal sagittal section using Fiji .2.4.N-butanol prior to DPX-mounting .Formalin-fixed biopsies were embedded in wax oriented and sectioned every 8 µm. The haematoxylin and eosin (H&E) staining was performed to enable measurement of cell size. For immunostainings, sections were deparaffinized in xylene, rehydrated and incubated in a 10% trisodium citrate buffer (pH6) solution at 60°C for 10 min for antigens retrieval. Blocking solutions, i.e. 5%FBS/PBS (S1) for immunohistochemistry or 2%BSA/PBS (S2) for immunofluorescence, were used for 30 min prior to incubating the primary antibodies for 1 h . For immunohistochemistry, the final step was performed using the Novolink Polymer Detection System . For immunofluorescence, Alexa-fluor 594 or Alexa-fluor 488  secondary antibodies were used (1/1000 dilution in S2) for 30 min and mounted using VectorShield . DNA fragmentation was determined using ApopTag following the manufacturer's instructions . A 0.5% methyl green in a 0.1 M sodium acetate (pH4) solution  was used to counterstain the nucleus of non-apoptotic cells. Slides were finally washed in 100% 2.5.3 pieces and transferred into a 0.25% trypsin–EDTA  solution for 18 h. Epidermal tissues containing progenitor keratinocytes were then transferred into a 0.1% collagenase type-1/1% BSA/DMEM  solution for 2 h at 37°C. Single keratinocytes were obtained by passing the solution through a 40 µm cell strainer  prior to centrifugation at 300g for 5 min. Cells were re-suspended in 10% FBS-DMEM-F12 (Gibco) and plated at a density of 5 × 103 cells cm−2 on collagen-I  pre-coated culture flasks. Cells were passaged using trypsin when reaching 80% confluence. The osteogenic differentiation was performed using the StemPro® Osteogenesis Differentiation Kit  following the manufacturer's instruction. Cells were then fixed in 4% PFA/PBS for 10 min and stained with 1% Alizarin Red solution (pH 4.2) for 10 min. The proportion of osteogenic cells was assessed using light microscopy. For immunofluorescence staining, cells were fixed for 10 min in 4% PFA/PBS prior to be incubated in 0.15% (w/v) saponin/PBS solution  for 1 h. The S2 blocking solution (see § 2.4.) was used for 1 h prior to adding primary antibodies for 1 h . Secondary antibody Alexa-fluor 594 or Alexa-fluor 488  were used at a dilution of 1 : 1000 in S2 for 30 min. Fluorescence intensities were measured using Image Pro .Incisions were made dorsally in the coronary band region allowing the proximal hoof wall to be lifted to expose the underlying soft tissue which was then cut into approximately 0.1 cm2.6.® bridge and the mass recorded to the nearest kilogram. General adiposity was assessed by body condition scoring (BCS) using the Henneke nine-point scale [One hundred and twenty-nine horses from 12 different yards in the southeast of England were evaluated from 19 September 2016 to 3 October 2016. Eligibility criteria for inclusion in the study included an age greater than 5 years and height under 144 cm with shoes. All horses were healthy at the time of evaluation and none had a history of laminitis/hoof conditions, pre-existing health conditions or were treated for pituitary pars intermedia dysfunction. There were no selection criteria on breed, sex, management or exercise regime. Hoof diameters were measured using a 40 cm ruler across the widest part of the hoof. Horses were walked onto an electronic Horse Weightnt scale ,32. Late2.7.p-Values less than 0.05 were taken as statistically significant.All statistical analysis and linear regressions were performed using Prism6 . 3.3.1.a) is indicative in equids, of a past or present pathology. Provided a more or less circular hoof a simple geometric argument suggests that such dorsal curvature is possible because the heel and dorsal regions do not grow at the same rate. This argument is further intuitively confirmed by the occurrence of diverging growth bands on the hoof capsule from the dorsal to the heel regions (see arrow in figure 1a), which underlines a differential growth between these regions. To better understand the occurrence of diverging growth bands, a thorough investigation of the initial stages of hoof growth was completed.The presence of a dorsal curvature . Between papillae, it was possible to differentiate three interpapillary regions (c). The blue ‘proximal nuclear region’, comprising cells with little cytoplasm and where the nucleus occupies most of the cellular volume. The red ‘cytosolic region’, in which the cytoplasmic volume of cells enlarges prior to its dissipation under the form of a ‘transition’, after which there is no visible cytoplasm nor nucleus but only remains that are expected to be keratin proteins as the hoof is a keratinized tissue. While the location of the ‘transition’ within the interpapillary space did vary slightly between hooves (d), the near-linear rate of cytoplasmic accumulation before the transition (c), which may suggest the presence of a pressure within the interpapillary space, possibly linked to the ability of cells to increase their size via accumulation of cytosolic material (figure 1d). To relate the changes in the cell surface area (figure 1d) to any concomitant variation in the physical appearance of papillae, high energy X-rays were used to reconstruct a three-dimensional image of the papilla in the dorsal region (figure 1e(i)). This technique allowed us to extract the papillae from the stratum medium involved in the synthesis of the hoof capsule (figure 1e(ii)) and measure their diameter along their proximo-distal axis, i.e. along the axis of hoof growth (figure 1e(iii)). The result confirmed that the average diameter of the papilla decreases to become minimal at a typical distance of approximately 2 mm that corresponds to the position of the transition (figure 1d).The synthesis of the hoof capsule starts from the coronet, i.e. the papillary region, and upon dissection and H&E staining of this region dorsally, soft digit structures a.k.a. papillae became visible b. Betwee regions c. The bln hooves d, the nef). This last result suggests that perpendicular to the direction of hoof growth the size of cells is relatively homogeneous within the interpapillary space.Perpendicular to the direction of growth, the cells were visibly smaller close to the papillae in a region corresponding to approximately three cell-thickness f. This la) [figure 2b) [figure 2c) [figure 2d) [d), the probabilities that cells were still expressing Ki-67 or p63 in the interpapillary space were at most approximately 1% of what was seen in the basement membrane. This result suggests that the proliferation process is negligible in the interpapillary space. Interestingly, although Ki-67 expression was visible over the entire length of the papillae (figure 2a(ii)), cells emerged in the interpapillary space only proximally (figure 2a(i)) suggesting that cells from the papillae move in the opposite direction of the hoof growth prior to entering in the papillary space. This counter-flow mechanism can be potentially rationalized taking into consideration the interpapillary pressure linked to the changes in cell volume applying a normal pressure against the surface of the papillae . DNA fragmentation became clearly visible in the transition region  and peaked when cell size reached minimal value after the transition . The keratinocyte progenitor stem cell marker K14 existed at a much higher expression proximally compared to K10, a later stage marker of keratinocyte differentiation (figure 2d); and both expressions decreased from the proximal to the distal locations.Regarding key molecular determinants, Ki-67  , p63 (orgure 2b) , DNA fragure 2c) , K14 andgure 2d)  were labHoof synthesis needs to be seen as a dynamical process and the remarkable changes in cell size as a function of their progression in the interpapillary space, concomitant with the variations in the diameter of papillae, are indicative that physical stresses linked to changes in pressure may be at play to initiate and synchronize the early stage of hoof morphogenesis . This pressure would be required in any case for the hoof to grow to balance the weight of the animal and the adhesion of the capsule on the distal phalange. Furthermore, the near-constant cell surface area of transverse cells when the cells move along the direction of growth suggests that a one-dimensional model as a leading approximation is potentially valid to describe the growth stress.3.3.b) by, d correspond to a) and assuming that the interpapillary space volume does not depend on the angular positioning, by noting f), it is possible to compare the difference in pressures between two angular positions by determining the number of cells that are present in the interpapillary space at these locations. In the remaining text one shall note, Histological pictures represent a steady-state of the early stage of hoof morphogenesis. To model cell volume changes anywhere at the coronet level, we note by, position b by, Rθ the balance of stresses vm is the proportionality constant representing the rate of inward flow of mass across the membrane of cells and; (iii) a change in the target size as a function of the position in the interpapillary space written as, The variation of the cell size along the d provides Radj)2 greater than 0.98 for each fit prior to the transition (inset figure 1d). From the numerical constants, the typical hoof growth rate can be determined. Assuming that the growth rate is driven by the variation in cell size prior to the transition located at the position d), by using a nominal cell size d) and a typical proliferation time Concentrating on the dorsal region and assuming the histological pictures as being initial states, i.e. a, i.e. when d, to estimate that the pressure at transition, By virtue of the balance of stresses, the interpapillary pressure can also be determined under the form:As beyond the transition there is no possibility for cells to actively generate any further growth, the pressure at the transition, 3.4.c,d. In this context, one assumes that the transition is driven by the pressure involved at the transition. Secondly, let us assume also that prior to collapsing, each non-aggregated keratin filament occupies a volume V0 within living cells that is their individual degree of freedom. Thus upon cell death, by neglecting the binding energy between keratins, the entropy gained by forcing the formation of one thick fibril containing ‘n’ non-aggregated intermediate filaments is, n approximately 20. As the bending stiffness of filaments is proportional to the fourth power of their radius, the mechanical resilience of the keratin bundle gained through this reorganization would be approximately 204 or equivalently approximately 105. This means that the persistence length determined when the bending energy equals the thermal energy should be increased by a similar factor [5 order of magnitude difference between the elastic moduli of keratinocytes and the solid keratinized hoof capsule.The reason for which the transition in size corresponds to the formation of the hard horn can be understood as follows. Firstly, let us assume that cells have to be in a certain biological state when cell death is ongoing to collapse, as observed in figure 1ly 14 nm  at room r factor . Let us r factor , then thPassed this transition, a mechanical description of the hoof capsule treated as a solid can provide a better understanding of the underlying bio-physical mechanisms regulating hoof shape.3.5.r0 that is curved dorsally modelled as a two-dimensional object of constant thickness e and of longitudinal length C is the dorsal curvature  describes the set of hoof shapes, the ideal case of a straight hoof can now be discussed. Let us assume that 3.7.a, the gradient in growth rate is key to define the dorsal curvature of a hoof. As equation (3.1) defines the angular growth rate of the hoof it is possible to relate the gradient in growth rate to the dorsal curvature. In order to achieve this, let us consider that a); by considering the first order in r0 ∼ 5 cm typically, one finds a numerical slope approximately 2.4 m−1.Following figure 1a, to estimate To validate the model, as the interpapillary space and papillae form a closed system, namely that cells proliferating from the papillae should populate the interpapillary space in due course, the experimental relationship between the ability of cells to proliferate locally from the papillae at the quarter and dorsal regions and the average dorsal curvature of the hoof were determined using equine feet. The average dorsal curvature of the capsule was calculated using a dorsoventral sagittal section of the hoof using µCT scanning. The feet were then dissected to estimate the rates of progenitor cell proliferation from papillae  at dorsal , 4 < BCS ≤ 7 (n = 187) and BCS ≥ 8 (n = 20) where ‘n’ is the number of feet. The stress linked to the weight was estimated on the forefeet knowing the animals' mass and foot circumference. The hoof dorsal curvature was calculated using a lateral picture of the hoof. Given that the weight appears as a second-order term in the quadratic equation related to the stresses balance, only extreme cases were plotted, i.e. BCS less than or equal to 4 and BCS greater than or equal to 8, demonstrating a negative relationship between the stress linked to weight and hoof curvature .In order to address these points, 129 ponies were selected and their body condition score (BCS) assessed using the Henneke nine-point scale ,32, withb. In this context, one can estimate that a null curvature occurs for The concordance between the theory and measurements can be underlined from the fit figure 3As Thus, it is the magnitude of the pressure load linked to the equids’ weight applied onto the distal edge of its hoof that drives the dorsal curvature, which means also that for the equids studied the straight hoof condition, 4.In developing countries, owing to the fact that good husbandry and veterinarians are expensive to afford , between4.1.Owing to the fact that a three-dimensional histologic resolution at the cellular scale was not feasible and that no specific cellular organizations were observed in the interpapillary space, a minimalist model considering the interpapillary cells as being spherical was used. The concepts applied would not be affected if cells had different geometry as only geometric constants would change. Finally, the coherence between the theory and the experimental data provides a guarantee that this assumption is sound. As a result, the hoof growth rate is principally related to the number of keratinocytes duplicating from the papillae and how well these can swell while differentiating in the interpapillary space. The process whereby soft tissues make hard ones is essential in ungulates as the hoof is a weight-bearing element and the mechanical resilience of the hoof has to be adjusted to the horse weight. This process is in theory possible by the ability of dead soft structures to reorganize single keratin filaments into large connected bundles under pressure. Indeed, the role of the local pressure in the interpapillary space, namely the presence of two opposed pressures applied to differentiating cells that are the growth stress and ground reaction to the weight, seems to mark the main difference between the hoof and the hair follicle. In the hair follicle where no distal stress is present, the keratinization process is continuous . In the Therefore, provided a more or less constant hoof growth rate, with quarters shorter than the toe and an adhesion stress proportional to the longitudinal length of the hoof capsule, the asymmetry of the horse's foot means that unless the asymmetry is properly adjusted, its quarter regions have a tendency to grow slightly faster when compared to the toe region leading to the occurrence of a dorsal curvature. However, the tendency for a dorsal curvature can be modulated, i.e. rendered invisible or exacerbated, by the weight of the animal. Said differently, in an imaginary world devoid of gravity, equids hooves should curve automatically. To demonstrate this point, we have concentrated on extreme BCSs whereby the impact of gravity/weight becomes physically and mathematically significant. However, we have observed disparities in the dorsal curvature of hooves analysed in equids with normal BCS. These disparities are very likely linked to the different horse genotypes or gene sets involved in hoof growth. Therefore, although physics can describe hoof shape, the underlying biology of hoof growth remains an essential ingredient regarding hoof deformities (see thereafter EMS/ECD cases).The conclusion deduced from our study regarding the BCS of equids does not imply that overloading an animal excessively over long period of time is necessarily a good thing. On the contrary, it is possible to define a critical loading blocking the dorsal growth of the hoof that can reignite the formation of a dished hoof. Indeed as equids feet are asymmetrical and that the growth stress is homogeneous along the coronet, this critical loading would not block the growth of the quarter regions (as the adhesion stress is lower in these regions). Thus, beyond this critical loading, the formation of a dished hoof should reappear. Numerically, this critical loading can be estimated as being 2.3 times the optimal weight of an animal required to have straight hooves. With regard to the leisure equids used in this study, this would correspond numerically to a loading 4.2.b).Finally, it is also worth noting that the physical parameter that defines the low and high BCS regions is given by  or ECD) . Both EM or ECD)  and EMS- or ECD) . In ECD, or ECD) –55 resul or ECD) . As insu4.3.Taken together the results provide: (i) an understanding of the bio-physical contradictions between synthesizing a hoof using soft matter, its resulting mechanical properties and foot asymmetry and, as a result; (ii) a potential connection between the different bio-physical aetiologies leading to the occurrence of a dorsally curved hoof, explaining why Aladdin's slippers are ‘natural’ and possible in Equids. Finally, this work underlines also the importance of providing a prescriptive understanding of the physics of animal health, prior to starting any live animal experiments.


Natural wetlands are known to play an important role in pollutant remediation, such as remediating acid mine drainage (AMD) from abandoned mine sites. However, many aspects of the microbiological mechanisms underlying AMD remediation within wetlands are poorly understood, including the role and composition of associated microbial communities. We have utilized an AMD-polluted river-wetland system to perform rRNA sequence analysis of microbial communities that play a role in biogeochemical activities that are linked to water quality improvement. Next-generation sequencing of bacterial 16S rRNA gene amplicons from river and wetland sediment samples identified variation in bacterial community structure and diversity on the basis of dissolved and particulate metal concentrations, sediment metal concentrations and other water chemistry parameters (pH and conductivity), and wetland plant presence. Metabolic reconstruction analysis allowed prediction of relative abundance of microbial metabolic pathways and revealed differences between samples that cluster on the basis of the severity of AMD pollution. Global metabolic activity was predicted to be significantly higher in unpolluted and wetland sediments in contrast to polluted river sediments, indicating a metabolic stress response to AMD pollution. This is one of the first studies to explore microbial community structure dynamics within a natural wetland exposed to AMD and our findings indicate that wetland ecosystems play critical roles in maintaining diversity and metabolic structure of sediment microbial communities subject to high levels of acidity and metal pollution. Moreover, these microbial communities are predicted to be important for the remediation action of the wetland. Pollution from abandoned mine sites is a major environmental problem that has deleterious consequences for aquatic and terrestrial ecosystems . FreshwaThe characterization of microorganisms in such polluted environments is important in order to understand the impacts of AMD on microbial ecology and evolution, to identify microorganisms that may have bioremediation properties, and to understand the mechanisms of microbial AMD tolerance and remediation . The abi-1 of Fe) over many decades, indicating long-term adaptation to AMD stress . Following changes in drainage routes from the mine, this river has experienced substantially greater pollution in the last 15 years such that it is a recently impacted ecosystem that has not fully adapted to AMD with no abundance of wetland plants . Samples were also taken at an unpolluted wetland (UW) site at Cefni Reservoir that is 13 km due South of Parys Mountain. Exact locations are provided in Supplementary Table Figure 1) two upstream of the adit (site N1 and N2), the Dyffryn Adda adit itself (site NA), and two sites downstream of the adit (N3 and N4). Sampling was carried out on four (at site N1) or six (sites N2–N4 and NA) occasions on June 2010 , July 2010 , August 2013 , October 2013 (not site N1), March 2014 , and October 2014 (not site N1).Water and sediment samples were collected from five sites along the northern Afon Goch river and three sites within the wetland through which the southern Afon Goch river flows . Approximately 500 m below the mine source at the Mona adit, the river flows through a natural wetland of ∼0.1 km2. The river then runs east and enters the Irish Sea at Dulas Bay. Three sites were sampled within the wetland (sites S1–S3). Site S1 is an entry point for AMD runoff at the start of the wetland, while sites S2 and S3 are in the middle and at the end of the wetland, respectively . Sampling was carried out on four (site S2) or seven (sites S1 and S3) occasions on June 2010 , July 2010 , November 2011 , August 2013 , October 2013 (not site S2), March 2014 , and October 2014 (not site S2).The southern Afon Goch is 11 km in length and runs south of Parys Mountain , and dissolved, sediment, and particulate metals were collected at each site on each sampling occasion. A YSI 556 probe  was used to monitor water pH, conductivity, and dissolved oxygen. Samples for metal analysis  were collected at each site and processed within 5 h following collection. For dissolved metals, a known volume of river water  was filtered through a 0.45-μm cellulose acetate filter and the filtrate was stored in an acid-washed polypropylene bottle and was acidified to 2% with ultra-pure nitric acid. The pre-weighed filters were retained for analysis of metal particulates. Sediment samples were collected to approximately 1 cm depth using a plastic scoop and sealed in a plastic bag until analysis. On return to the laboratory, the filters containing suspended particulates were dried (60°C for 48 h), and the sediment samples were dried (60°C for 48 h) and then passed through a 250-μm filter. The particulate filter papers and 100 mg of sediment samples were digested in 67% ultra-pure nitric acid for 24 h at 100°C. Digests were then diluted to 2% nitric acid in deionized Milli-Q water (Millipore). Samples were refrigerated before metal concentrations in water samples and digests were analyzed by inductively coupled plasma atomic emission spectroscopy (ICP-AES) using a Perkin-Elmer Optima 5300 for Al, As, Cd, Cu, Fe, Mn, Pb, and Zn. The spectroscope was calibrated using an internal standard, which was a matrix matched serial dilution of Specpure multi element plasma standard solution 4 (Alfa Aesar).Juncus sp. and samples were taken from sediment surrounding Juncus sp. roots from sites S1, S2, S3, and UW. In addition, a non-vegetated sediment sample was taken from site S1 (S1R), and a sediment sample surrounding cotton grass (Eriophorum angustifolium) roots was also taken from site S2 (S2C). Samples were taken in sterile 50-mL Falcon Tubes.For microbial community analysis, three sediment samples were collected from each site in March 2014 for DNA extraction. The southern Afon Goch wetland and Cefni Reservoir wetland are both dominated by Table 1). Sequence data were deposited in the European Nucleotide Archive (ENA), study accession number: PRJEB23187, sample accession numbers: ERS1983433 (site UW), ERS1983434 (site S1), ERS1983435 (site S2), ERS1983436 (site S3), ERS1983438 (site S2C), ERS1983439 (site N1), ERS1983440 (site N2), ERS1983441 (site NA), ERS1983442 (site N3), ERS1983443 (site N4), and ERS1983444 (site S1R).DNA was extracted from 100 mg sediment samples (three independent samples per site) using a Powersoil DNA isolation kit  and quantified using a Nano-drop 3300 . DNA from each site was pooled and then amplicons for Illumina MiSeq sequencing were generated from PCR reactions using primers Bakt_341F (S-D-Bact-0341-b-S-17) and Bakt_805R (S-D-Bact-0785-a-A-21) as previously described and validated , modifiep < 0.05) and Tukey’s multiple comparison post hoc test using GraphPad Prism. Hierarchical clustering (HC) of environmental data was performed in R and statistical comparison of nodes performed by a Similarity Profile (SIMPROF) test  of environmental data was performed using the R vegan package v.2.4.2. The environmental data (except pH) was log-transformed prior to PCA. The PCA comprised a matrix of 46 datasets (replicates) from the six samples with 24 variables used. Statistical comparison of environmental data was performed using one-way ANOVA (OF) test .de novo picked at 97% similarity using UCLUST (H), Pielou’s evenness index (H/maxH), and Chao1 species richness estimate values were calculated using QIIME. Before the calculations, samples were resampled to an even depth of 310,833 sequences per sample, the sequence depth size of the smallest sample (N2). Rarefaction curves were also generated by QIIME.Raw de-multiplexed sequence reads of the 16S rRNA V3–V4 region were trimmed and paired and further filtered using QIIME v.1.9.0 . Chimerig UCLUST . Taxonomg UCLUST . In ordeThe assigned taxa dataset was statistically analyzed by HC and by non-metric multidimensional scaling (NMDS) both performed using R vegan package v.2.4.2. Taxa relative abundance was square-root-transformed and a distance matrix based on Bray–Curtis dissimilarity was obtained for HC. Complete linkage was used as an agglomerative clustering method and SIMPROF was used for identifying genuine groups between samples in HC. Samples were classified as “unpolluted” and “polluted” groups according to the environmental data. Analysis of similarity ANOSIM;  was used2 fold-change abundance values, normalized enzyme abundance values were converted to logarithmic scale (base 2) after adding a value of 10 to allow consideration of zero values. HC of normalized metabolic pathway abundances was performed using the Euclidean distance method and heatmaps were created using R vegan package v.2.4.2.Metabolic structure was predicted from the 16S rRNA gene amplicon library of each sample site using the PAPRICA v.0.4.0 metabolic inference pipeline . For thiJuncus sp. and some E. angustifolium (cottongrass) and Phragmites sp. The northern Afon Goch has neutral pH and low conductivity water at sites N1 and N2 upstream of the adit entering the northern river, while highly acidic (pH 2.3–2.5), high conductivity water was downstream, although this was slightly diluted at site N4 . An increase of pH from 2.4 to 5.7 and a decrease of conductivity from 1.1 to 0.3 mS were observed along the wetland from site S1 to S3, indicative of the remediation process previously described . Many particulate metals including Al, Cu, Fe, Pb, and Zn show high concentrations within the middle of the wetland at site S2  although sediment metal profiles vary depending on the element . In contrast, the less polluted sites N1, N2, and S3 correlate with high water pH (pH 5.1–6.4), high concentration of Mn in sediments, and low concentration of As and Fe in sediments. S2 clustered separately from the other sites  and correlates with particulate metals in the water column . It was previously observed that the wetland remediation process is mediated in part by precipitation of metals in the middle of the wetland . Natural wetlands such as this one can function as long-term metal sinks by reducing flow rate, reducing acidity, and promoting microbial activities such as sulfate reduction . All samples showed a consistent level of saturation of OTU richness . At the start of the wetland (site S1), all diversity parameters were lower than for all other wetland sites (Table 1). Equivalent values were seen at an S1 site that was not directly associated with a wetland plant  in contrast to cottongrass (site S2C) did not lead to substantial variation in bacterial diversity . Examination of proteobacterial sequences in more detail showed dominance of an unclassified Xanthomonadaceae sequence particularly at site NA (17.5%) but also abundant at site S1, and an unclassified Methylophilales sequence with extremely high abundance (46.2%) at site N3 . This unclassified Methylophilales taxon had reduced abundance at site N4 compared to N3; however, abundance of a Gallionella sp. was high (16.8%). Members of some of these taxa have previously been associated with Fe oxidation. For example, a Xanthomonadales taxon was observed in Fe(III)-rich sediments . The dominant taxa observed within the wetland were equivalent to those seen previously in slightly acidic, non-metal polluted sediments of natural wetlands . There was no significant distinction between S1 and S2 and between N3 and N4, but discrimination between wetland sites S1 and S2 compared to sites NA, N3, and N4 was significant (p < 0.05). On the basis of the physicochemical data, sites UW, S3, N1, and N2 were classified as an unpolluted group while sites S1, S2, N3, and N4 plus site NA were classified as a polluted group. ANOSIM demonstrated that there was a significant separation between the two groups  on the basis of community structure. NMDS ordination further demonstrated similar clustering of polluted and unpolluted sites . Inclusion of the 1608 assigned taxa in the ordination indicated a greater density of taxa associated with the unpolluted sites, as shown by the taxa clustered on the left side of the plot. Of these taxa, 139 were uniquely present in the unpolluted group compared to 176 unique to the polluted group of sites. SIMPER analysis indicated that the unclassified Methylophilales taxon, which was dominant at sites NA, N3, and N4 , was the highest ranked contributor to the assemblage of the sites . A Methylophilales taxon associated with Cu tolerance was previously reported as one of the most abundant taxa within a microbial mat involved in natural remediation of heavy metal-contaminated mine water .Hierarchical clustering on the basis of community structure similarity found that unpolluted sites UW, N1, N2, and S3 clustered together, while a second cluster included sites S1, S2, N3, and N4, with the adit site NA as an outlier , BIO-ENV analysis found that pH alone was not the key environmental variable for explaining the variation in community structure between the sites. Conductivity and metal concentration characteristics are strongly correlated with species assemblage, indicating that these factors may be particularly important (Table 2). Dissolved metal concentrations entering the northern Afon Goch river were extremely high (up to 628 mg L-1 of dissolved Fe at site NA) in comparison to many other studied AMD environments . Organizing the sites as a river group and a wetland group allowed the comparison of assigned taxa on the basis of wetland plant presence. Most of the assigned taxa (73%) were shared between the river and wetland groups, while only 316 taxa were unique to the river group and 118 were unique to the wetland group. Some taxa had high abundance within wetland sites, but low abundance within non-wetland river sites  likely due to altered biogeochemical processes at this location , due to a substantial predicted decrease in abundance of many enzymes specifically at these three sites within the ochre-rich sediment . Metabolic and biochemical reactions with various elements including sulfur, arsenic, carbon, hydrogen, and nitrogen are important for adaptation by prokaryotes in AMD habitats . The overall metabolic prediction profile is particularly significant as this indicates a clear-cut distinction between the wetland and non-wetland AMD sites. The sediment at sites NA, N3, and N4 has very high Fe and As concentrations with high concentrations of other metals in the surface water. Therefore, we might speculate that the extreme metal-rich and acidic environment leads to inhibition of some enzymatic activities within the microbial communities. The reduced metabolic potential within these sites is not due to reduced bacterial abundance because the presented data are normalized on the basis of 16S rRNA gene amplicon sequence read number . In contrast, the metabolic potential remains stable within the wetland sediment sites despite AMD exposure. This clear difference in the metabolic potential of bacterial communities at the polluted wetland sites compared to the polluted river sites  contrasts with the more subtle discrimination between these sites on the basis of bacterial community structure . We therefore suggest that wetland environments with their associated AMD impacts influence the metabolic structure of sediment microbial communities. These selected communities are sufficiently tolerant of these conditions that the microbial mediated processes of immobilization of metals in the sediment are sustained during the wetland evolution.Although wetland plants directly promote some bulk extraction of dissolved metals into their tissues , the prilocation , for exalocation  and sulflocation , as welllocation . We mighlocation  of each location . Clusterhabitats . The PAPFigure 7). For most enzyme classes, there was a relatively strong positive correlation (R2 > 0.4) in predicted enzyme abundance change between the northern and southern river sites, such that the majority of the enzymes that increased or decreased in abundance at site NA also increased or decreased at site S1. A second analysis compared changes along each river system, between the site of highest source of AMD pollution (site S1 or NA) and the furthest downstream site (site S3 or N4). This provides an evaluation of metabolic potential change moving through the wetland (x-axis) or moving down the northern Afon Goch river . These plots displayed no significant correlation  indicating that there are very different profiles of metabolic potential depending on whether the flow is through a wetland or not. Using >2 and <-2 log2 fold-change as the threshold of significant enzyme abundance change, the plots predict that there are a number of significant increases and decreases in enzyme abundance through the wetland with no change along the northern river, and likewise predicted changes in enzyme abundance along the northern river that are not seen through the wetland . A number of significant increases or decreases in enzyme abundance  are specific to the northern river and result in no significant change through the wetland. In contrast, a few enzymes, especially of the EC1 oxidoreductase and EC2 transferase classes, have increased abundance through the wetland but are not predicted to increase abundance between site NA and N4 . This prediction is in line with expected activities within AMD impacted wetland sediment, such as mechanisms that direct the oxidation of Fe(II) . Breakdown and assimilation of organic carbon products, such as cellulose and other polysaccharides from plants, will likely require increased hydrolytic activities  and northern Afon Goch (site NA relative to site N1) was performed (f Fe(II) , althougf Fe(II) . A clusttivities , while mtivities . As metaWetlands provide significant ecological functions and can provide beneficial ecosystem services such as promoting bioremediation of contaminated water, which may be replicated in constructed wetlands . The potWe show that a long-term AMD-impacted river, which has established a pollution-adapted wetland ecosystem, is clearly distinguished from a less adapted, non-vegetated river on the basis of variation in microbial community structure and hence predicted function. AMD river sediment in the absence of a wetland is characterized by the prediction of significantly reduced metabolic activity, indicative of stress response, while more specific metabolic changes are predicted within the wetland and which contribute toward the reduction in acidity and removal of dissolved metals. Our work suggests that wetlands can maintain high sediment microbial community diversity despite high levels of acidity and metal pollution, and this microbial community structure will in turn influence biogeochemical activities that can take place within the wetland. Future investigations of these biogeochemical processes will validate the predictions of the metabolic modeling by functional analysis. Furthermore, examination of other AMD impacted natural wetlands and constructed wetlands will be needed to identify conserved functional traits within these systems, and in particular determine whether characteristics of a long-established wetland ecosystem are also seen or can be replicated in a recently developed constructed treatment wetland.PRJEB23187 (https://www.ebi.ac.uk/ena).The datasets generated for this study can be found in the European Nucleotide Archive (ENA), Study accession number: OA, AM, AD, and JP generated the data and performed the data analysis. KW, AD, and JP conceived the project. OA, KW, AD, and JP interpreted the results and wrote the paper. All authors read and approved this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Infectious bronchitis virus (IBV) is one of the leading causes of mortality and morbidity in chickens. There are numerous serotypes and variants, which do not confer cross protection resulting in failure of currently used IBV vaccines. Although variant IBV isolates with major genetic differences have been subjected to comparative studies, it is unknown whether minor genetic differences in IBV variants within a serotype are different in terms of pathogenesis and eliciting host responses. Two Massachusetts (Mass) variant IBV isolates recovered from commercial layer flocks in the Western Canadian provinces of Alberta (AB) and Saskatchewan (SK) were compared genetically and evaluated for their pathogenicity, tissue distribution and ability to recruit and replicate in macrophages.Although whole genome sequencing of these two Mass IBV isolates showed low similarity with the M41 vaccinal strain, they had an identical nucleotide sequence at open reading frames (ORFs) 3a, 3b, envelop (E), matrix (M), 5a and 5b. The rest of the ORFs of these 2 IBV isolates showed 99.9% nucleotide similarity. However, upon experimental infection, we found that the IBV isolate originating from AB was different to the one that originated in SK due to higher tracheal lesion scores and lower lung viral replication and lower genome loads in cecal tonsils. Nevertheless, both IBV isolates elicited host responses characterized by significant macrophage recruitment to the respiratory tract and there was evidence that both IBV isolates replicated within tracheal and lung macrophages.Overall, this study shows that Mass variant IBV isolates, although possessing minor genetic variations, can lead to significant differences in pathogenicity in young chickens. Further studies are required to investigate the pathogenicity of these two Mass variant IBV isolates in laying hens.The online version of this article (10.1186/s12917-018-1720-9) contains supplementary material, which is available to authorized users. Coronaviridae. Traditionally, IBV is considered to be a host-specific respiratory pathogen in chickens and IBV initially replicates at the route of entry, the tracheal mucosa [Infectious bronchitis virus (IBV) belongs to the family l mucosa , 2. Howel mucosa –10, gastl mucosa , 11, 12,l mucosa , 13 and l mucosa . IBV is l mucosa , 16. Fall mucosa , 18. IBVl mucosa .An array of serotypes and strains of IBV infect chickens throughout the world . GeneticPreviously, comparative studies have been conducted to elucidate differential pathogenicity and host responses between variant IBV isolates with larger genome variability such as nephropathogenic and Massachusetts (Mass) IBV isolates recovered from various geographical areas , 42–46. A wide array of IBV variants are affecting commercial broiler, layer and breeder flocks in Canada , 50, 51.The present study was conducted with the aim to elucidate the genetic differences of two Mass variant IBV isolates (15AB-01 and 15SK-02) that originated from layer flocks in Western Canada, Alberta (AB) and Saskatchewan (SK). Additionally, we aimed to describe whether these genetic changes in Mass isolates 15AB-01 and 15SK-02 lead to differential pathogenicity, tissue distribution and macrophage responses in the respiratory tract.For the isolate 15AB-01, total of 525,998 and 908,860 read sets were obtained from the direct allantoic fluid sample and layered virus sample, respectively from the Illumina MiSeq workflow. From these, a total of 418,446 and 825,147 reads were aligned with the reference IBV genomes. For isolate 15SK-02, a total of 4,355,902 and 3,731,578 reads were obtained from the direct allantoic fluid sample and layered virus sample, respectively, of which, a total of 3,146,584 and 3,152,622 reads were aligned with the IBV reference genomes. The number of reads obtained from Illumina MiSeq for 15SK-02 was 10 times higher than for 15AB-01 and the viral genome concentration of the submitted complimentary (c)DNA was also much higher for 15SK-02 than for 15AB-01 . Entirely based on the NGS output, the complete genomes obtained for the Mass IBV isolates, 15AB-01 and 15SK-02 were 27.6 kb in length. As determined by the open reading frames (ORF) predictor, both isolates had similar genome organization with 11 ORFs and they were 5′-1a-1b-S-3a-3b-E-M-5a-5b-N-3′ .From 2 to 8 days post-infection (dpi), there were two birds with mildly increased respiration (more labored and higher frequency) from the IBV (15AB-01) infected group. On 9 and 10 dpi, there were 3 birds with mildly increased breathing. Only one bird continued to show a mild breathing increase by 11 dpi and thereafter all birds were bright and alert. In contrast, 2 birds from the 15SK-02 infected group started showing increased breathing as late as 7 dpi and continued until 11 dpi. The control birds were alert and active throughout. The highest body weight gain was achieved by the uninfected control group followed by the group infected with the isolate 15AB-01 and 15SK-02  and 15SK-02 (p < 0.01) were significantly higher than that of trachea from uninfected controls   . There was no difference in genome loads in the kidney tissue between two time points in both strains. The uterine genome loads were available only at 14 dpi for both strains. None of the uninfected control birds had detectable IBV genome loads (results not shown).The highest genome loads in tissue were found in trachea and lung tissues of chicken infected with either isolate at 4 dpi, however, the genome load in lung tissue was significantly lower for IBV isolate 15AB-01 . Macrophage infiltration was significantly higher (p < 0.05) in tracheas infected with both Mass IBV isolates compared to the tracheas of uninfected controls  than lungs from uninfected controls  and Mass type IBV isolates recovered from various geographical areas , 45, 46.Literature that describe pathogenicity using Canadian IBV isolates are scarce  AlthoughIt appears that host responses, characterized by respiratory macrophages following IBV infection is dependent on the IBV strain. For example, it has been shown that M41 but not Australian T strain increases macrophages in respiratory tract lavage . Previou50 [In spite of the novel aspect of our work comparing two Mass type IBV isolates with minor genetic variations for pathogenicity and host responses, there were some limitations to this study. First, we did not include a control group infected with the standard M41 IBV strain in our experiment and this was due to the limited infrastructure availability at our animal facility. However, in a separate study, we investigated the M41 IBV induced macrophage recruitment to respiratory tract . Second,50 . AlthougIn conclusion, Mass IBV variants recovered from layer flocks had identical nucleotide sequence at ORFs 3a, 3b, E, M, 5a and 5b. The remainder of the ORFs showed 99.9% nucleotide similarity. Although, the genetic changes were minor, the pathogenicity in vivo of the Mass IBV variant originating from AB was different to the one that originated in SK, whereby the AB variant caused significantly higher tracheal lesions and replicated to a lower titer in the lung and lower genome loads in cecal tonsils. Additionally, both Mass IBV variants elicited host responses characterized by significant macrophage recruitment to the respiratory tract and there was evidence that both Mass IBV variants replicated within macrophages in the respiratory tract. Studies are underway to evaluate the pathogenicity of these two Mass type IBV isolates in laying hens.Day old white leghorn (SPF) layer chickens (White Leghorn) were obtained from the Canadian Food Inspection Agency (CFIA), Ottawa, ON, Canada and housed in high containment poultry isolators at the Prion- Virology Animal Facility of the University of Calgary’s Foothills Campus with ad libitum access to feed and water.50) according to the method described by Reed and Muench [Mass IBV isolates originating from layer flocks in AB (15AB-01) and SK (15SK-02) were used in the study. Viral titers were determined using 9-day old SPF embryonated chicken eggs and expressed as 50% embryo infectious dose . Ribonucleic acid (RNA) was extracted from the sixth fraction and allantoic fluid directly. cDNA was synthesized, and quantitative polymerase chain reaction (PCR) assay was performed to quantify the IBV genome load.n = 7 per group) were infected with 50 μL inoculum containing 2.75× 104 PFUs of IBV isolates 15AB-01 and 15SK-02 intra-tracheally under isoflurane anesthesia. Another 5 of 6-day old SPF chickens were kept as uninfected controls. The chickens were weighed at 0, 2, 3, 4, 10, 12 and 14 dpi. At 2, 3, 10 and 12 dpi, oropharyngeal and cloacal swabs were obtained using Puritan® Unitranz-RT transport system  and stored at − 80 °C until further processing. At 4 dpi, 4 chickens from each infected and 3 chickens from the uninfected group were euthanized and trachea, lung, kidneys, cecal tonsils and reproductive tract samples were collected into RNASave®  and stored at -20 °C. In addition, a portion of trachea, lung, kidneys, cecal tonsils and reproductive tract samples were collected into 10% neutral buffered formalin for histopathological examination and in Optimum Cutting Medium  for immunostaining. The remainder of chickens were continued to be observed and tissue samples were collected in RNASave® for genome load quantification at 14 dpi.Two groups of 6 day-old unsexed-SPF chickens . A total of 10–12 ng of cDNA from each isolate was sent for whole genome sequencing to the Swine and Poultry Infectious Diseases Research Center (CRIPA), University of Montreal, Canada where library preparation and sequencing performed on Illumina® Miseq  next generation sequencing (NGS) platform generating 150 bp paired-end reads.Formalin-fixed terminal tissue samples were processed at the Diagnostic Services Unit of the University of Calgary’s Faculty of Veterinary Medicine including staining of sections with stained hematoxylin and eosin (H&E). Sections were examined under the light microscope and photomicrographs were taken under 40x magnification. The histological changes observed in the trachea  and lungTracheal and cloacal swab samples, which were in the transport media were vortexed and medium was transferred to 1.5 mL centrifuge tubes and centrifuged at 1000 g for 20 mins at − 4 °C. A total of 250 μL of supernatant was used for RNA extraction using the Trizol LS® reagent  following manufacturer’s instructions. Approximately 50 mg piece of tissue preserved in RNA Save® was homogenized in 1 mL of Trizol® reagent  using a Pro200 Power homogenizer  on ice and RNA was extracted following the manufacturer’s protocol. The RNA pellet was re-suspended in 20 μL of RNase-free water and quantified using the Nanodrop 1000 spectrophotometer at 260 nm wavelength . A total of 2000 ng of RNA was transcribed using 10X RT random primers  as has been instructed by the manufacturer.The IBV genome load quantification was carried out following the products and protocol as described previously . BrieflyFrozen tissues in OCT blocks were sectioned at 5 μm thickness using a cryotome  and adhered on to positively charged slides. The sections were air dried on the bench for 20 min, fixed by immersing in ice-cold acetone for 5 min and stained for macrophage and IBV antigens sequentially as has been described previously . Brieflyhttps://www.ncbi.nlm.nih.gov/orffinder/) and the genome annotation was carried out using Geneious software, version 10.1.3 .All reads were assembled to get a single contig. The resulting full genome sequences were aligned, and identity matrix was performed with 43 published sequences of IBV reference serotypes retrieved from the National Center for Biotechnology Information (NCBI) GenBank database using the BLAST® research tool and the Custal Omega 1.2.3 package with automated settings (Geneious® 10.1.3). The phylogenetic tree inferred based on the full-length IBV genomes using Randomized Axelerated Maximum Likelihood (RAxML 8.2.7) method with GTR GAMMA nucleotide substitution model with 1000 bootstrap iterations . The resulting proportional tree of the two Mass IBV isolates of this study with 43 reference sequences  and DyLight® 488 (IBV antigen) fluorescent signals and corresponding nuclear stained (DAPI) areas were captured under 40x magnification using an epifluorescent microscope . The obtained images were transferred to the Image-J® software  and the areas of each micrograph covered with DyLight® 550 (macrophages), DyLight® 488 (IBV) signals, combined DyLight® 550 and DyLight® 488 signals (IBV positive macrophages) and the combined DyLight® 550 and DyLight® 488 signals with DAPI  were quantified. The areas covered with macrophage and IBV antigens and the combined signals were expressed as a percentage of the total area covered by the section.p < 0.05. The significance on graphs are labelled as the following: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.Two-way analysis of variance (ANOVA) followed by Benferroni post-hoc test was used to determine the significant time points of body weight among the infected and control groups. Tracheal and lung histopathological lesion score data was analyzed using Kruskal-Wallis test followed by Dunn’s multiple comparison test. The genome load data was analyzed by the two-way ANOVA followed by Bonferroni post-test. One-way ANOVA test followed by Tukey’s posttest was used to determine the significance of immunofluorescence data. Grubbs’ outlier test was performed in order to identify outliers before the data was analyzed. All the statistical procedures were performed using in-built statistical analysis feature of the GraphPad™ Prism 5 . Differences were considered significant at Additional file 1:Figure S1. Specificity of avian macrophage staining. The power point slide contain lung sections stained with anti-chicken macrophage antibody (KUL-01) and isotype control. The sections were also stained with nuclear staining (DAPI). (DOCX 271 kb).Additional file 2:Table S1. List of reference IBV sequences used in this study. This table contain names and accession numbers of IBV whole genome reference sequences retrieved from the GenBank repository. (DOCX 15 kb).