Barrows and gilts respond differently as experimental models for Escherichia coli challenge during the nursery phaseABSTRACT.
The objectives of this study were to evaluate the effect of Escherichia coli (E. coli) F4 challenge in nursery piglets, and to develop an experimental model to be used in research. Ninety-six weaned piglets were divided in a 2 x 2 factorial design consisting of E. coli challenge (challenged or not challenged) and sex (barrows or gilts). Pig growth performance, fecal score, blood count, intestinal morphometry, and cecal content (microbiological and short-chain fatty acid analysis) data were collected and analyzed. Pigs in the non-challenged treatment had 6% greater average daily gain during day 8-28 (the period in which the health challenge was administered) and 1.2% greater average daily feed intake in over the entire experiment, when compared to challenged pigs. Gilts in the non-challenged group had a higher villus: crypt ratio when compared to piglets in the challenged group. These findings indicate that the pathogen challenge using Escherichia coli F4 strains, especially in gilts, proved to be an effective method to reproduce commercial health challenges in the first two weeks postweaning and may be used in experimental models.
Keywords:
health; disease challenge model; swine
Introduction
Modernization of the swine industry has increased the sanitary and environmental challenges for piglets, especially during the post-weaning and nursery phases (Oliveira Jr. et al., 2013). During these periods, piglets undergo biological stress resulting from physiological, environmental, and social challenges, which contributes to the occurrence of diseases and impairs piglet health, welfare and performance (Campbell et al., 2013).
During the nursery phase, piglets are in constant contact with several pathogenic microorganisms, such as Escherichia coli, which combined with stressors, predispose the piglets to infections (Kummer et al., 2009). Enterotoxigenic E. coli (ETEC) is a major bacterial agent that affects the health of piglets in the post-weaning period and is one of the main microorganisms related to the occurrence of diarrhea in the nursery phase (Che et al., 2017). The impairments on gastrointestinal tract caused by the pathogen affects the animal’s health, its growth and development throughout the production cycle (Wang et al., 2018; Che et al., 2017; Zhu et al., 2018).
The consequences of pathogenic E. coli in piglets are diverse such as damage intestinal integrity and morphology, trigger an infectious and inflammatory process, and result in intestinal dysfunction. Intestinal integrity is essential in defense against pathogens (Pi et al., 2014). When the small intestine is compromised, the digestion and absorption of nutrients, such as proteins and amino acids, are also diminished (Chen et al., 2014). This results in piglets becoming increasingly susceptible to other infections and enteric diseases, which affects pig growth performance and leads to economic losses.
Given the challenges the piglets face throughout the nursery phase, experimentally disease challenges that replicate conditions found in commercial productions are of the major importance. There are few studies that evaluate the effect of disease challenges in piglets. Thus, the objectives of the present study are to evaluate the effects of an E. coli F4 challenge on gilts and barrows during the nursery phase on parameters related to the intestinal health and productive outcome of piglets and to analyze the feasibility of using such a challenge under experimental conditions.
Material and methods
Animals, experimental design and procedures
The experiment was approved by the Ethics Committee on the Use of Animals of the School of Veterinary Medicine and Animal Science at the University of São Paulo, under protocol number: 3743220518. The study was carried out on at the Swine Research Laboratory of the University of São Paulo, located in the city of Pirassununga, São Paulo, Brazil (21° 59' 46'' S and 47° 25' 36'' W).
A total of ninety-six piglets (48 barrows and 48 gilts) of Choice Genetics terminal cross line, obtained from a commercial swine herd, were used. The piglets were weaned at an average age of 24 days and with an average weight of 6.7 ± 0.92 kg. The piglets were housed in a nursery unit equipped with 48 raised stalls, 50% slatted floor, with semi-automatic feeders and nipple drinkers. The temperature was controlled using side curtains and heating lamps.
The experimental design was carried out in randomized blocks (based on initial weight and sex) with four treatments and 12 repetitions, in a 2 x 2 factorial treatment design (challenged or not challenged with E. coli) X (barrow and gilt). The experimental unit was composed of the mean (BW 6.71 ±0.4 kg) of the two pigs present in each pen. The experimental period was 42 days, subdivided into pre-challenge (0 to 7 days), peri-challenge (8 to 28 days), and post-challenge (29 to 42 days). The diets were formulated to meet or exceed the nutritional requirements of the nursery phase according to the NRC (2012) and are presented the in supplementary materials.
The disease challenge with Escherichia coli F4 was performed on day 8, 9 and 17 of the experiment. The days were selected to mimic the most challenging days on a commercial farm. On the first and second challenge, piglets in the challenged group received 1 ml of a solution containing the bacteria (concentration of 106 CFU mL-1 of E. coli) and the piglets in the non-challenged group received 1 ml of saline solution. Another bacterial inoculation was performed on the 17th day of the experiment, in which piglets from the challenged group received 2 ml of the bacterial solution (concentration of 109 CFU mL-1 of E. coli) while piglets from the non-challenged group received 2 mL of saline solution. The challenges were performed with the aid of a nasogastric tube.
The bacterial inoculum was prepared from the field strain Escherichia coli F4 (LT+, Sta+ and STb+). The strain was cultivated in culture medium for 16 hours at 37ºC, and then washed sequentially in PBS (phosphate-buffered saline) until a concentration of 106 or 109 CFU mL-1 was reached according to the methodology of Rodrigues et al. (2020).
On days 11, 28, and 42 of the experiment, serial slaughters were performed to evaluate intestinal health in different times post-challenge. Nine piglets per treatment, piglets closest to the mean weight of each treatment, were selected for slaughter, totaling 54 animals during the experimental period (27 barrows and 27 gilts). The slaughter was carried out through electronarcosis followed by exsanguination, in the slaughterhouse of the University of São Paulo, located in Pirassununga, São Paulo, Brazil, using trained staff.
Performance, incidence of diarrhea, hemogram, microbiological and short chain fatty acids composition of the cecal content
To evaluate the productive performance, piglets were weighed on day 0, 7, 14, 21, 28, 35th, and 42 of experiment. The feed provided and leftovers were evaluated daily. Based on these data, average daily gain (ADG), average daily feed intake (ADFI) and feed conversion (FC, ADFI:ADG) were calculated.
Druing the experimental period, feces in all pens were evaluated and classified twice a day to estimate the incidence of diarrhea. Fecal score was based on the methodology of Pedersen and Toft (2011), in which stool scores range from 1 to 4 and are associated to the level of diarrhea and presence of liquid or pasty feces. The feces were evaluated and scored according to the following characteristics: score 1, feces firm and molded; score 2, soft and molded feces; score 3, loose feces; score 4, watery feces. Fecal scores of 1 and 2 were considered healthy and scores of 3 and 4 associated with diarrhea.
At slaughter, blood samples were collected to assess red blood cell count, leukocyte count, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). Additionally, platelet count and a morphological analysis of leukocytes were conducted using the May-Grunwald-Giemsa blood smear technique.
Post-slaughter samples of jejunum (2.0 cm) were collected to evaluate the integrity of the intestinal mucosa. The samples were prepared in accordance with the method outlined by Zhaxi and colleagues (2020). A microscope equipped with a camera and ImageJ software was utilized to measure parameters such as villi height, crypt depth, and the villus: crypt ratio.
Cecal content samples were obtained to assess the concentrations of short-chain fatty acids (SCFA) - acetate, propionate, isobutyrate, butyrate, isovalerate and valerate and to analyze the populations of intestinal bacteria, including Enterobacterium, Escherichia coli, Lactobacilli and Bifidobacterium. The samples were diluted in phosphate-buffered saline (PBS), with E. coli, Enterobacterium, and Bifidobacterium samples being fractionated from 10-1 to 10-3g, and Lactobacillus samples from 10-1 to 10-5. Selective culture media were employed for the cultivation of each bacterium group. Prior to statistical analysis, all colony counts (CFU g-1) underwent logarithmic transformation (log10).
For statistical analysis, the Shapiro-Wilk test was used to assess data normality. When the data did not follow a normal distribution, they were transformed using the PROC RANK procedure of SAS Institute Inc (SAS, 2009). Data was anaylzed using analysis of variance (ANOVA). When there was a statistical difference by the F test (p < 0.05) for the interactions, Tukey test was used to compare means. Data were submitted to the statistical package of the SAS software (2009) through the MIXED procedure. For the data “incidence of diarrhea”, SAS NPAR1WAY procedure was used. Dunn's test was applied as post-hoc for multiple paired comparisons, with p < 0.05 being considered significant, for variables that were rejected by the Kruskal-Wallis test at a 5% probability level.
Results and discussion
The performance of piglets was compared in periods related to experimental procedures: pre-challenge; peri-challenge and post-challenge (day 0-7; 8-28; and 29-42 respectively) and total experimental period (day 0-42). Results can be found in Table 1.
In the first period (day 0-7) and in total period (day 0-42) no significant interactions between sex and challenge were found (p > 0.05) and no significant differences were observed for any of the variables analyzed (p > 0.05). During the second period (day 8-28) no interaction was observed between sex and E. coli challenge (p > 0.05), but non-challenged piglets had a 6.0% higher ADG (P = 0.029) than challenged piglets. For the same phase, non-challenged piglets tended to have a 4.0% greater weight at 28 day (P = 0.055) and a 6.2% higher ADFI (P = 0.065) than piglets challenged with Escherichia coli. In the third phase (day 29-42), an interaction between sex and challenge was observed, in which challenged barrows had a greater ADFI (14.9% increase) when compared to non-challenged barrows, while gilts did not differ statistically (P = 0.014).
Unchallenged piglets had better growth performance when compared to piglets challenged by E. coli during the second experimental period (day 8-28), which corresponds to the period when the sanitary challenges were performed. The piglets that did not receive the bacterial inoculum had the greater ADG (day 8-28), tended to have a greater weight at 28 day, and ADFI (day 8-28) than those that were challenged. The systemic effects of inflammatory and infectious processes are known (Oliveira Jr. et al., 2013; Webel et al., 1997). Activation of the immune system triggers metabolic and neuroendocrine responses due to the action of inflammatory cytokines, which modulate immune-physiological interactions and modify nutritional requirements to allow reestablishment of homeostasis (Johnson, 1997; Oliveira Jr. et al., 2013). Among the cytokines involved in the process, there are Tumor Necrosis Factor α (TNF-α), Interleukin 1 (IL-1) and Interleukin 6 (IL-6), which lead to loss of muscle mass and proteolysis, increased secretion of hormones such as glucocorticoids that have a catabolic function in adipose and muscle tissues, promoting an anorexic effect and reducing the synthesis of anabolic hormones (Webel et al., 1997). Alterations observed in the present study coincide with effect of inflammatory processes associated with the experimental inoculation of E. coli F4. Other studies found similar results, such as Gao et al. (2013), who observed that animals of different breeds (Landrace and Jinhua) challenged with a solution containing E. coli (ETEC K88) had reduced growth and average daily feed intake during the study period.
Between day 29 and 42, challenged barrows had greater ADFI than unchallenged barrows. The increase in feed consumption by the barrows challenged with E. coli may have been due to their recovery after removal of the stimulus and source of infection since piglets were inoculated with the bacterial strains in the previous phase. Furthermore, the rapid recovery of barrows may be associated with the low concentration of E. coli inoculate used in the present study. Che et al. (2017) performed a disease challenge on piglets consisting of 100 mL doses with concentrations of 109 CFU mL-1 of E. coli. Owusu-Asiedu et al. (2003) used 6 mL doses of a bacterial solution containing 1010 CFU mL-1 of E. coli K88 (F4), while the present study used only 2 mL with 106 CFU mL-1 of E. coli (first and second challenge) and 2 mL with 109 CFU mL-1 of E. coli (third challenge). Additionally, the increased feed consumption observed in barrows (challenged with E. coli) when compared to gilts during the third phase of the experiment (between day 29-42) may be related to early life experiences (such castration), which influences and affects the way in which piglets cope with the weaning process and subsequently events, as suggested by Van Erp-Van der Kooji et al. (2000), not to mention hormonal status, since lower concentrations of testicular androgens and estrogens may have a suppressive effect on feed intake (Weiler et al., 1998; Santollo et al., 2021).
The percentages of each fecal score, mean fecal score and total fecal score, related to the treatment and sex of the animals, were compared in weeks of the experiment and periods of health challenges, as follows: week 1 (S1 - day 0 to 7), week 2 to week 4 (S2-S4 - day 8 to 28), week 5 to week 6 (S5-S6 - day 29 to 42). Analyzes were also compared in relation to total period of experiment (Total score - day 0 to 42). All results are presented in Table 2.
No significant interaction on fecal score analysis was observed between sex (barrow or gilt) and challenge with E. coli during all experiment (p > 0.05). In the first period (day 0 to 7), challenged animals had increased score 2 (32.0%; P = 0.019), while the control group tended to have a higher score 1 (8.4%; P = 0.058) and no interactions between sex and treatment were found. On the second period 4 (day 8 to 28), which corresponds to the period in which the health challenges were performed, non-challenge piglets had an increase of 7.0% in the score 1 (P = 0.034) and an increase of 42.3% in score 2 when compared to E. coli challenged group (P = 0.036). Challenged piglets had a mean fecal score 6.0% higher than the animals in the non-challenged group (P = 0.034). During the last period (S5-S6 - day 29 to 42), the E. coli challenged group had a higher score 4 (229.0%; P = 0.044) and a higher mean fecal score (7.5%; P = 0.032) when compared to the unchallenged group. Unchallenged piglets tended to have a 30.9% increase in score 1 (P = 0.070). In relation to the total experimental period (day 0 to 42), there was a 10.6% increase in Score 1 in the group not challenged by E. coli (P = 0.013). Piglets that received the E. coli challenge had higher scores 2 (16.6%; P = 0.018) and 4 (176.4%; P = 0.048), as well as a 6.4% increase in mean fecal score for the entire experimental period (P = 0.016).
When considering the total experimental period, the challenged group had a higher frequency of score of 4 (considered diarrhea) and total fecal score and a lower frequency of score 1 (considered normal feces). Furthermore, average fecal score for the period in which the sanitary challenges occurred (S2-S4) was greater for animals challenged with E. coli; in the subsequent period (S5-S6), challenged piglets also showed higher frequency of Score 4 (diarrhea). These results may demonstrate the effects of the bacteria Escherichia coli F4 (enterotoxigenic), which is associated with diarrheal conditions in the nursery phase. This enteric pathogen is considered as one of the main causes of diarrhea in weaned piglets due to colonization in the gastrointestinal tract (Zhu et al., 2018). Bacteria recognizes and adheres to specific receptors present in the gastrointestinal tract of piglets, with the help of fimbriae and adhesins, to finally start the production of enterotoxins, causing diarrhea (Moonens et al., 2015; Zhu et al., 2018). Piglets at this stage are also more susceptible to diseases due to the various stressors to which they are exposed in the post-weaning period (Campbell et al., 2013). The fecal score results obtained in the study demonstrate the effectiveness of the Escherichia coli F4 challenge.
Other studies corroborate with our findings. Che et al. (2017) observed that piglets challenged with solutions containing 109 CFU mL-1 of ETEC (E. coli O149) had a higher fecal score (diarrhea) during the first 24 hours after the challenge. SØrensen et al. (2009) observed variable effects on fecal characteristics and consistencies after a E. coli O149 challenge, demonstrating that controlled models of challenge with E. coli can be used to induce conditions like those present in commercial swine facilities that lead to diarrhea.
Data referring to the hemogram are presented in Table 3 and were divided by slaughter date due to the collection of samples. Slaughter 1 occurred during the first phase of nursery period (day 11), Slaughter 2 during the second phase of nursery period (day 28), and Slaughter 3 during the third phase of nursery period (day 42).
For the first nursery period (Slaughter 1), no significant effects were found for the variables analyzed (p > 0.05) and no interaction between sex and challenge was observed (p > 0.05). Samples collected during Slaughter 2 indicated a significant interaction between sex and treatment for the variable “Eosinophils count”, in which unchallenged gilts had 375.0% greater eosinophil numbers compared to challenged gilts, while the barrows did not differ statistically (P = 0.011). As for the third phase of the nursery period (Slaughter 3), there was a significant interaction between sex and treatment for variables: “Red Cells”, in which challenged barrows had a 10.6% higher value compared to unchallenged barrows (P = 0.024) and “Monocytes”, in which unchallenged gilts and challenged barrows showed an increase of 507.0 and 398.0%, respectively, when compared to challenged gilts (P = 0.006). Challenged piglets tended to have 136.5% greater value for “Erythroblasts/100 Leukocytes” in relation to the non-challenged group (P = 0.058).
In the present study, it has been noted that the responses of some blood parameters to E. coli challenge were variable, including interactions with sex. Modifications on red cell count, eosinophil count and monocyte count can be related to E. coli challenge and a weaning-induced systemic inflammatory response in piglets (Sugiharto et al., 2014). It is also known that some differences and modifications on the total blood count can be influenced by other factors, such as piglet age (Davis et al., 2006). Davis et al. (2006) demonstrated that weaned piglets had increased total white blood cell count following weaning and increased age, consequently. Despite the significant effects found in some variables of the blood tests, the values observed in this study remained within the physiological intervals considered normal for the swine species and for the age group of the animals in the study, according to Kaneko, Harvey, and Bruss (1997) and Friendship and Henry (1992).
The results regarding intestinal morphometry are shown in Table 4 and dived by slaughter age. For the Slaughter 1 period, there was no interaction between sex and challenge (p > 0.05), but the non-challenged group tended have a 30.2% greater value for the variable “Crypt Depth” in relation to the challenged group (P = 0.062). Furthermore, gilts tended to have a 29.6% increase in Crypt Depth when compared to barrows (P = 0.058). During the period covered by Slaughter 2, there was a significant interaction between sex and treatment for “Villus height” (P = 0.004) and “Villus: Crypt Ratio” (P = 0.044). Unchallenged gilts had an increase of 107.6% on villus height when compared to challenged gilts, while barrows had similar statistical behavior. Furthermore, unchallenged gilts showed an 85% higher villus: crypt ratio compared to not challenged barrows, while piglets in the challenged group, both barrows and gilts, were statistically similar. In the third slaughter period, there were no significant effects between groups for all parameters analyzed (p > 0.05) and no interaction between the factors analyzed (sex and challenge with E. coli).
Our findings suggest compromised intestinal integrity and morphology, which are associated with the activation of innate immunity and the local inflammatory process (Che et al., 2017). Such alterations in organism's homeostasis may be related to the presence of Escherichia coli F4, which is an enterotoxigenic bacterium. It is known that the maintenance of intestinal integrity is extremely important in the post-weaning period, due to the vital function of this organ, which is responsible for the final digestion and absorption of nutrients (Chen et al., 2014). Thus, both the length and width of the villi and the depth of the crypt are related to the maintenance of intestinal integrity and health of the animals, and the greater the villus: crypt ratio, the better the intestinal digestive and absorptive capacity (Bontempo et al., 2006). Gao et al. (2013) observed that pigs challenged with E. coli K88 had greater villus atrophy (i.e., lower villus height) and reduced crypt depth when compared to unchallenged pigs and Che et al. (2017) observed that piglets challenged by E. coli had lower villus height, evaluating jejunum and ileum samples, compared to unchallenged pigs, which corroborates with our findings. The compromised intestinal integrity, demonstrated by the results regarding intestinal morphometry, may be correlated to the impairments observed on the piglet’s growth performance and incidence of diarrhea. Furthermore, unchallenged gilts had superior villus: crypt ratio compared to barrows not challenged by E. coli.
The results of the of the populations of intestinal bacteria and concentrations of short-chain fatty acids (SCFA) are presented in Tables 5 and 6, respectively. The results were divided accordingly the Slaughter dates.
In the microbiological analysis, no significant differences and interactions were observed between the variables analyzed at the first slaughter. At Slaughter 2, challenged piglets had an 8.9% higher Bifidobacterium value (P = 0.039) and tended to have a Lactobacilli value 6.9% greater (P = 0.089) when compared non-challenged piglets. No interactions between sex and challenge were found at the second slaughter (p > 0.05). In the third nursery phase (Slaughter 3), there was a significant interaction between sex and treatment for the Bifidobacterium variable (P = 0.029), in which unchallenged gilts showed an 24.4% increase in relation to unchallenged barrows, while piglets in the challenged group were statistically similar.
At Slaughter 2, challenged animals had higher Bifidobacterium content and tended to have higher Lactobacilli content when compared to animals not challenged with E. coli. These microorganisms (Lactobacilli and Bifidobacterium) are considered one of the main components of the intestinal microbiota of swine (Tsuchida et al., 2017), and thus, increases in their counts in pitlets challenged with E. coli F4 may be associated with host response to the presence of pathogenic bacteria in the intestinal environment. Intestinal microbiota is an important defense mechanism against pathogenic bacteria since commensal bacteria increase host resistance to the pathogen colonization in turn promoting host’s health. (Shen et al., 2009). Also, I 1n humans, Bifidobacterium have beneficial effects on the physiology and pathology of some diseases, such as enterohemorrhagic caused by Escherichia coli - EHEC. Bifidobacterium produce acetate, increasing its concentration in the intestines and helping with the response against infectious agents by acting in vivo to promote defensive functions of host epithelial cells (Fukuda et al., 2011). In the present study, an increase in acetate levels was also observed in the same period (Slaughter 2), corroborating the results presented by Fukuda et al. (2011).
The fatty acids evaluated were acetate, propionate, isobutyrate, butyrate, isovalerate and valerate. No interactions were observed between sex and challenge for the first and last periods (Slaughter 1 and 3, respectively). For the Slaughter 2, there was a significant interaction between sex and treatment of animals for the variable acetate (P = 0.048), in which challenged gilts had a 113.5% higher acetate value when compared to unchallenged gilts, while barrows of both treatments were statistically similar.
In the period of the second slaughter, challenged gilts had higher acetate value when compared to unchallenged gilts. This result suggests that challenged gilts could be reacting to the infection and the presence of the Escherichia coli inoculated in the sanitary challenge. In humans, it is known that increases in the production and concentration of acetate modulates the gut's defensive response and improves defense mediated by epithelial cells, which leads to protection against infectious agents (Fukuda et al., 2011). In addition, short-chain fatty acids are responsible for providing energy to colonocytes, having essential roles in the reabsorption of water and sodium in the body and in the individual's intestinal health (Bergam, 1990; Zlotowski et al., 2008).
Conclusion
The Escherichia coliF4 challenge was effective mainly in the first two weeks post-challenge, as indicated by changes in the incidence of diarrhea, growth performance and intestinal morphometry. The effect of the challenge differed between the sexes, with gilts showing a greater response than the barrows, indicated by the differences observed mainly on the analysis of intestinal morphometry. Therefore, gilts may be recommended for use in experimental conditions with E. coli O149 challenge, as demonstrated in this study.
Data availability
In accordance with the principles of Open Science, the data generated and/or analyzed during the present study are available from the corresponding author upon reasonable request.
Acknowledgements
Thanks to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (FCPES) - Finance code 001, and Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) - protocol numbers: 2019/19720-0; 2019/17453-4; 2020/05069-2 for partially funding this study
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