Novel multi-strain probiotics reduces Pasteurella multocida induced fowl cholera mortality in broilers

Pasteurella multocida causes fowl cholera, a highly contagious poultry disease of global concern, causing significant ecological and economic challenges to the poultry industry each year. This study evaluated the effects of novel multi-strain probiotics consisting of Lactobacillus plantarum, L. fermentum, Pediococcus acidilactici, Enterococcus faecium and Saccharomyces cerevisiae on growth performance, intestinal microbiota, haemato-biochemical parameters and anti-inflammatory properties on broilers experimentally challenged with P. multocida. A total of 120 birds were fed with a basal diet supplemented with probiotics (108 CFU/kg) and then orally challenged with 108 CFU/mL of P. multocida. Probiotics supplementation significantly (P < 0.05) improved growth performance and feed efficiency as well as reducing (P < 0.05) the population of intestinal P. multocida, enterobacteria, and mortality. Haemato-biochemical parameters including total cholesterol, white blood cells (WBC), proteins, glucose, packed cell volume (PCV) and lymphocytes improved (P < 0.05) among probiotic fed birds when compared with the controls. Transcriptional profiles of anti-inflammatory genes including hypoxia inducible factor 1 alpha (HIF1A), tumor necrosis factor- (TNF) stimulated gene-6 (TSG-6) and prostaglandin E receptor 2 (PTGER2) in the intestinal mucosa were upregulated (P < 0.05) in probiotics fed birds. The dietary inclusion of the novel multi-strain probiotics improves growth performance, feed efficiency and intestinal health while attenuating inflammatory reaction, clinical signs and mortality associated with P. multocida infection in broilers.

www.nature.com/scientificreports/ the NC+ and PC− groups were significantly lower when compared with other groups. Also, although the dressing carcass of the PC− group was significantly (P < 0.05) higher than all other groups (except Pro+), the dressing carcass of birds in the Pro+ group similarly had significantly (P < 0.05) higher relative weight when compared with other challenged groups (PC+ and NC+) ( Table 3). The relatively weights of carcass and visceral organs from the P. multocida challenged groups generally had the least values especially from birds in the NC+ group.
Enumeration of intestinal digesta intestinal bacteria, yeast and P. multocida. There was no significant (P > 0.05) differences in the numbers of total aerobes from the ileal and caecal contents on day 21 of the trial among all the treatments. However, the LAB counts in the gizzard contents of probiotics supplemented birds (groups Pro− and Pro+) was significant (P < 0.05) higher on day 28 when compared with other groups (Table 4). Also, the LAB counts in the ileum and caecum of birds from PC+ and NC+ were lower (P < 0.05) than probiotics supplemented birds (groups Pro− and Pro+). The numbers of enterobacteria recorded from the gizzard, ileum and caecum on day 21 of the trial were significantly (P < 0.05) higher in P. multocida challenged groups, PC+ and NC+ , when compared with other treatments, and this trend persist till the end of the trial. The numbers of yeast cells tended to be higher in the ileum and caecum of birds supplemented probiotics than other groups. However, the least yeast counts were recorded from the gizzards of all the birds across the treatments with birds in PC+ and NC+ groups having a significantly (P < 0.05) lowered counts on day 21. Throughout this experimental trial, birds in the probiotic control, Pro+ treatment had significantly (P < 0.05) lowered counts of P. multocida in their gizzards, ilea and caeca when compared with those of birds in the other challenged treatments, PC+ and NC+ respectively (Table 4). Whereas the gizzards of birds in the probiotic control treatment, Prob+ had the least counts of P. multocida, 0.50 log 10 CFU/g on day 21, significantly higher counts of P. multocida ranging between 6.62 and 7.01 log 10 CFU/g were recorded from the ilea and caeca of birds in the PC+ and NC+ treatments on day 28 of the trial. The numbers of P. multocida in the ileal and caecal contents were significantly (P < 0.05) reduced in Pro+ group when compared with other challenged groups, PC+ and NC+ on days 21 and 28 (i.e., 7 and 14 days post-challenged) respectively. Birds from all the treatments were confirmed to be culture-negative for Pasteurella before inoculation with P. multocida on day 14 of the experiment. Also, the non-P. multocida challenged treatments (i.e., NC−, PC− and Pro−) were Pasteurella negative throughout the experimental period.
Intestinal digesta pH. Generally, this study recorded an increasing pH concentration from acidity to neutrality with the gizzard digesta of birds having the lowest pH levels followed by the ileal and then the caecal contents respectively ( Table 5). The pH of the ileal contents from birds in PC+ groups were significantly (P < 0.05) lowered when compared with other challenged groups, PC+ and NC+ on days 21 and 28 respectively.
Haemato-biochemical parameters. The total cholesterol concentration in birds from the probiotics supplemented groups, Pro− and Pro+ were significantly (P < 0.05) lower than P. multocida challenged groups, PC+ and NC+ on days 21 and 28 of the experiment. Nevertheless, there was no significant (P > 0.05) difference in the HDL cholesterol and LDL cholesterol concentrations across the treatment all through the experiment. On day 28, while triglyceride levels were reduced (P > 0.05) in probiotics supplemented groups but higher (P < 0.05)  1  0  0  0  0  0  0   2  0  0  0  0  2  3   3  0  0  0  0  4  2   4  0  0  0  1  4  3   5  0  0  0  0  0  3   6  0  0  0  0  1  www.nature.com/scientificreports/ in NC−, glucose levels were significantly(P < 0.05) reduced in the same groups. Similarly, protein levels were significantly (P < 0.05) higher in probiotics supplemented groups when compared with other groups on day 28 ( Table 6). The results of the haematogical parameters analyzed are shown in Table 7. Whereas no difference (P > 0.05) was recorded in total RBC, haemoglobin, ESR, PCV, basophiles, monocytes, total platelet count and MPV across the treatments on day 21, statistical difference were recorded for total WBC and neutrophils between the probiotics supplemented groups when compared with other treatments. Nevertheless, on day 28 of the experiment MCV and RDW were significantly (P > 0.05) higher in probiotics supplemented groups Pro-and Pro+ when compared with non-P. multocida challenged negative control group, NC−. Also, probiotics supplemented birds with or without P. multocida challenge significantly (P < 0.05) increased total WBC counts at the end of the trial. Also, the concentrations of lymphocytes and monocytes were higher in probiotics supplemented groups while PC− and Prob− had higher (P > 0.05) total platelet counts on day 28 of the experiment.
Anti-inflammatory gene expression. On 14-day post P. multocida challenged, dietary supplementation of birds with probiotics significantly (P < 0.05) upregulated the mRNA profiles of anti-inflammatory genes including HIF1A (hypoxia inducible factor 1 alpha) and TSG-6 (Tumor necrosis factor-(TNF) stimulated gene-6) on the caecal mucosa when compared to the birds in the control group (Table 8). However, when both antiinflammatory genes are compared, probiotic effect in the upregulating the expression of HIF1A was higher than for PTGER2. There was no difference in the expression of both anti-inflammatory genes in birds supplemented with antibiotic and the negative control except for TSG-6 ( Table 8).

Discussion
Avian cholera caused by P. multocida is a highly contagious poultry disease of global concern, causing significant ecological and economic challenges to the poultry industry each year 3,24,25 . The ability of P. multocida to survive asymptomatically in carrier birds for a longer period of time even after the disappearance of clinical signs have often led to frequent recurrence of P. multocida outbreaks with high mortality 4,25 . Also, P. multocida has been reported to persist for several months in the environment, water supplies, and insects 24,26,27 . In this study, we proposed that the supplementation of poultry with multistrain probiotics containing L. plantarum, L. fermentum, P. acidilactici, E. faecium and S. cerevisiae can control P. multocida infection in broiler chickens through mitigating the manifestation of clinical signs and the reduction of mortality associated with P. multocida while improving the overall performance. www.nature.com/scientificreports/ Although some studies have previously tried the effectiveness of vaccines as antibiotic alternatives in the control of P. multocida in poultry, studies evaluating the possible role of probiotics in the control of P. multocida colonization and infections in poultry production are lacking. With the successful reports of probiotics effectiveness in the control and mitigation of the colonization and infection by poultry pathogens including Salmonella 14,28,29 , Campylobacter 15,30,31 , E. coli 32,33 , Eimeria spp. 34,35 , L. monocytogenes [36][37][38] and Clostridium perfringens 39,40 , the trial of probiotics in the control of P. multocida would further unravel probiotics effectiveness against this devastating poultry pathogen.
Generally, the dietary supplementation of probiotics has been reported to positively influence animal health and productivity. The results obtained from this study shows that dietary inclusion of probiotics significantly improved the performance and feed efficiency in broiler chickens with beneficial impact on the intestinal microbiota composition and health, hence decreasing the severity of the FC in birds. The significant improvement in BW, BWG and FCR recorded among broilers supplemented with probiotics in comparison with the controls from this study confirmed the positive impact of probiotics supplementation on the performance of broilers. This finding is significant not only to confirm the improvement of intestinal health after probiotic supplementation, but also to mitigate the economic losses due to P. multocida infections in poultry production.   14,15,28,30 . Furthermore, apart from the adoption of biosecurity measures and vaccination (which are most potent preventive measures) in endemic regions of the world, the routine use of probiotics in the control and prevention of enteric infections in poultry production could further help in reducing the severity of cases of fowl cholera hence, diminishing the spread of the pathogen. Changes in the relative weight of visceral organs and carcass in broilers is one principal effect mostly attributed to probiotic supplementation in poultry. The symptoms of pasteurellosis in the P. multocida challenged positive control (PC+) and negative control (NC+) were accompanied by decrease in the live BW of birds, visceral organs and dressing carcass in these treatments with higher increase in gizzard weights on day 28 of the experiment. These trends were similarly reported previously by Olnood et al., and Park and Kim, after experimentally infecting broiler chickens with Salmonella spp. 41,42 . The improved growth performance of birds due to  44 . Probiotics effect on the weight of visceral organs and intestines of animals is inexplicit, and can also be determined by the nature and amount of microbial strains used as probiotics. It has been reported that probiotics consistently influence the intestinal morphology and micro-structure which often increases the absorptive function of the ileum 14,45 . Also, Pelicano et al. reported significant improvement in the leg yield and breast of birds fed with probiotics 46 . A significantly higher mortality was recorded in P. multocida challenged birds supplemented with antibiotic and challenged negative control when compared with the P. multocida challenged birds supplemented with probiotic. The change in behavior and the manifestation of clinical signs in the control groups were consistent with the reported signs characterizing fowl cholera in poultry such as diarrhoea, depression, listlessness, severe weakness, nasal discharge, recumbency and moribund status, isolation, anorexia combined with reduction in feed and water consumption, ruffled feathers, immobility and lameness 4,22,23 . Within 24 h post-P. multocida infection, birds in the control groups showed mild to moderate signs with mortality which increased in severity till about 92 h post infection. A similar trend was reported in experimental birds challenged with P. multocida 25,47,48 . Depending on the strain, the incubation period of P. multocida usually varied between 12 and 48 h with 100% mortality majorly between 24 and 72 h post infection 49 . There is generally limited information about the clinical pathology of pasteurellosis in poultry. Also, Wilkie et al. reported that broiler chickens experimentally challenged with P. multocida died within 22-72 h post infection 2 . Furthermore, the rapid death of the host animal including broilers due to acute form of fowl cholera is a characteristic of septicaemia induced by P. multocida 47 . The persistence of P. multocida strains at the site of infection as well as their migration to other host tissues and organs, and the eventual time of the host death depend primarily on the host immune response and the characteristics of P. multocida strain causing the infection which may also influence the shedding and isolation of the pathogen 2,47,50 . The inhibitory effect of each of the probiotic strain i.e. L. plantarum, L. fermentum, P. acidilactici, E. faecium and S. cerevisiae (used in this study) against P. multocida and other poultry pathogens have been evaluated previously and their probiotic potentials elucidated 17,18 .
Information regarding in vivo antimicrobial activity of probiotics strains including LAB and Saccharomyces against P. multocida and P. multocida infections are lacking. However, the inhibitory activity of probiotics consisting of strains of LAB and S. cerevisiae in broilers as recorded in this study indicates that these probiotic strains can be successfully used as alternatives for growth-promoting antibiotics in poultry production. The numbers of enterobacteria in the P. multocida challenged control groups were higher in the ileum and ceacum than the unchallenged groups both on days 21 and 28 of the experiment. Probiotic supplemented groups showed higher number of LAB and yeast in the gizzard, ileum and caecum which significantly decreased the numbers of P. multocida on both sampling days during the experiment. The reduction of enterobacteria by beneficial gut microflora may be attributed to the bacteriostatic effect of volatile fatty acids (VFA) secreted in the GIT of birds 14 . In vitro evaluation has demonstrated that VFA inhibited enterobacterial growth at the pH of 6 51 . Therefore, probiotics supplementation may have increased the concentration of VFA in the gut of the birds examined. This finding is in agreement with the results of Lan et al. 52 who reported significant decrease in the number of enterobacteria after broiler chickens were supplemented with multi-strain probiotics containing a mixture of L. agilis, L. acidophilus/gallinarum and L. salivarius. The inclusion of strains of Bacillus, Clostridium and Lactobacillus as multi-strain probiotics at the level of 10 6 to 10 9 CFU/kg of diet reportedly suppressed the growth of enterobacteria [53][54][55] . Also, probiotics' antimicrobial effects come from their secretion of antimicrobial compounds including bacteriocins, organic acids (acetic, lactic, propionic, succinic acid, etc.), short-chain fatty acids, hydrogen peroxide and other low molecular weight substances 56 . The combination of the strains of LAB and yeast used as multi-strain probiotic in this study possibly synergized to form a robust antimicrobial activity against P. multocida and enterobacteria in the gut of the birds supplemented with the study probiotics. Table 8. Effect of probiotics supplementation on anti-inflammatory gene expression in caecal mucosa of Pasteurella multocida challenged broilers. Values are means of two replicates and standard errors of means. Within each variable, values with the same superscript letter are not significantly different according to Duncan's multiple range test (P > 0.05). Pro+: challenged probiotic control; PC+: challenged positive control; NC+: challenged negative control. HIF1A hypoxia inducible factor 1 alpha, PTGER2 prostaglandin E receptor 2, TSG-6 tumor necrosis factor-(TNF) stimulated gene-6, Pro+ challenged probiotic, PC+ challenged antibiotic; NC+ challenged control. www.nature.com/scientificreports/ Furthermore, these probiotic strains are able to competitively exclude pathogens, hence preventing their attachment to intestinal walls thereby improving intestinal microbial balance. In agreement with Olnood et al. there was a gradual increase in pH concentration from the proximal to the distal GIT regions with probiotic supplemented birds having a more lowered pH level especially in the gizzards 14,41 . The reduced pH among probiotic supplemented broiler chickens also contributed in the reduction of the numbers of P. multocida and enterobacteria.

Anti-inflammatory gene
Dietary conditions and pathological stress commonly determine the haematological changes and health status of birds 57 . The reduction in major haematological parameters including Hb, PCV, ESR and total RBC in P. multocida challenged control groups clearly depicts the onset of anaemia. The occurrence of anaemia in avian cholera infection in poultry has been properly reported 48 . The cause of anaemia in P. multocida challenged birds as recorded in this study may be attributed to bacterial septicaemia. The concentration of total WBC and lymphocytes were also higher in probiotics supplemented birds. Probiotics are known to modulate host immune system response primarily through balance between anti-inflammatory and proinflammatory cytokines 36 . Similarly, after dietary supplementation of B. subtilis-based probiotics, Park and Kim and Lee et al. showed the reduction of coccidiosis clinical signs and improved immune response in broiler chickens challenged with Eimeria maxima 42,58 . The improvement of gut health through the modulation of gut microflora and the modulation of intestinal inflammatory and immune response may significantly inhibit the P. multocida colonization and proliferation within the gut hence influencing haptoglobin concentration. The dietary inclusion of probiotics positively influenced haematopoiesis which among others increase the WBC counts, hence enhancing immune cells synthesis which further protects the host against invading pathogens 59,60 . The presence of congested blood vessels and haemorrhages observed in the lungs, livers, hearts and intestines of P. multocida infected birds as a result of fowl cholera is similar to the findings of Shivachandra et al. and Sonone et al. 9,48 . The supplementation of probiotics as revealed in this study significantly reduced the severity of P. multocida infection throughout the experiment, hence, reflecting in the improved haematological parameters as clearly shown.
The reduction in the concentration of total cholesterol, triglycerides, glucose and LDL cholesterol which are major biochemical parameters as reported in our study due to probiotic supplementation agrees with the report of Arun et al. and Al-Kassie et al. who separately reported a significant reduction in total cholesterol, triglycerides and glucose by dietary inclusion of 100 mg/kg diet of L. sporogene probiotic and the combination of probiotic (Aspergillus niger) and prebiotic (Taraxacum officinale) in broilers 61,62 . Total cholesterol reduction in probiotic supplemented birds could be as a result of direct assimilation of cholesterol by bacterial cells (which causes reduction in the cholesterol absorption and synthesis in the GIT), 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibition and bile salt hydrolysis 63,64 . Furthermore, triglyceride reduction in probiotic treated birds may be as a result of increased hydrolysis of bile salt which causes inadequate lipid absorption in the small intestine 10 . Strains of Lactobacillus are known to show high hydrolytic activity on bile salt which consequently leads to bile salts deconjugation within the GIT 65 . Also, the concentration of total protein was significantly higher in probiotics fed birds. This corroborated with the findings of Dimcho et al. and Alkhalf et al. who reported probiotic effects on total protein concentration in chickens 10,66 .
Maintaining intestinal health and the integrity of intestinal barrier function is essential for the growth and wellbeing of animals. Several pathogenic factors such as stress and pathogenic bacteria challenges can cause inflammation and damage to the intestinal barrier 67 . On day 14 post P. multocida challenge, greater effects were found for probiotic supplementation on HIF1A, and TSG-6 (P < 0.05). The data obtained from this study implies that probiotics supplementation can attenuate inflammatory reactions through the upregulation of the secretion of anti-inflammatory factors.
In the gut of animals, pathogens including P. multocida readily cause different degree of inflammatory damage of intestinal epithelial cells by establishing hypoxic microenvironments 68 . One of the major transcriptional factors that dampen hypoxia-induced inflammation in the gut is HIF1A. HIF1A enhances the synthesis and signaling effects of anti-inflammatory signaling molecules 69 . TSG-6 is multifunctional protein that has been implicated as having important anti-inflammatory and tissue protective properties 70 . From this study, HIF1A and TSG-6 in the control treatment with P. multocida infection expressed the lowest mRNA profiles, whereas they were upregulated with probiotics supplementation, depicting that these 2 genes are collaboratively associated with either P. multocida or probiotics. This is the first work that assessed the expression of these 2 genes on probiotics supplemented chickens infected with P. multocida. Unfortunately, no information exists about the expression of these genes in the presence of probiotics and P. multocida. However, in a recent work Deng et al. reported the upregulation of HIF1A in probiotic supplemented chickens infected with Listeria monocytogenes 36 . Therefore, further study on the mechanisms responsible for the dampening of inflammation and the upregulation of antiinflammatory factors, HIF1A and TSG-6 in probiotics supplemented birds is needed. The higher population of Enterobacteria and P. multocida in the control group could be associated with the invasion of these bacteria and the pathogenesis of P. multocida resulting in the clinical manifestation of P. multocida infection and high mortality. A pool of P. multocida strains could be evaluated to determine their persistence, spread and multiplication in host tissues and shedding in future research.

Materials and methods
Ethical approval of the study. The field trial was approved by the Animal Care and Use Committee of the Faculty of Biological Science and Technology, Jashore University of Science and Technology, Jashore, Bangladesh (certification number: ERC/FBST/JUST/2019-32). The health status of birds in the field were routinely monitored by a veterinarian. The birds were kept under controlled environmental conditions in the animal house of Jashore University of Science and Technology, Jashore, Bangladesh, throughout the experimental period. Strains used for the study and diets. Five potential probiotic strains previously isolated from broiler chicken and raw milk, and identified using 16S rRNA sequencing as Lactobacillus plantarum, L. fermentum, Pediococcus acidilactici, Enterococcus faecium and Saccharomyces cerevisiae with suitable probiotic properties including antagonistic activity against broad range of poultry pathogens including P. multocida, survivability in simulated gastric juice, bile salts and phenol tolerance, adhesion to ileum epithelial cells, aggregation and hydrophobicity abilities, α-glucosidase inhibitory activity, and competitive exclusion of pathogens were selected for this field trial 17,18 . Basal diets (starter and grower/finisher) formulated in our laboratory were provided as pellets all through the trial and were based on wheat, soybean meal and corn as shown in Table 9. The five probiotic strains were mixed at equal ratio and added into respective experimental treatments at dose of 10 8 CFU/kg of diet.
Experimental design and treatments. A total of 120 one-day-old Cobb 500 broiler mixed-sex chicks were purchased from a commercial hatchery (NOURISH FARMS, DHAKA, BANGLADESH), weighed individually and randomly assigned to 6 experimental treatments with 2 replicate groups containing 10 chicks each after they were allowed to acclimatize for 2 days. Birds in each treatment were housed in a floor pen containing sawdust litter. Twenty-three hours of light was provided during the first week and then reduced to 18 h throughout the 28 days of the experiment. The 6 experimental treatments adopted in this trial included: (1) negative control (NC−), non-probiotic and unchallenged with P. multocida; (2) positive control (PC−), supplemented with doxycycline HCL (0.5 g/mL), non-probiotic and unchallenged with P. multocida; (3) probiotic control (Pro−), probiotics supplemented and unchallenged with P. multocida; (4) probiotic challenged (Pro+), as probiotics supplemented and challenged with P. multocida; (5) positive challenged (PC+), as doxycycline HCl, supplemented and P. multocida challenged; and (6) negative challenge (NC+), as non-antibiotic, non-probiotic and challenged with P. multocida. In all the treatments, feed and water were provided ad libitum according to the experimental design. Birds in the antibiotic treatments were administered 1 g/L of the doxycycline HCL following the manufacturer's instructions. Both probiotics and antibiotic were administered between days 3-21 of the experiment (Fig. 2).
Pathogen challenge. For the pathogen challenge experiment, strain P-931of P. multocida subsp. Multocida ATCC 12945 (capsular Type A) isolated from fowl and known to cause fowl cholera was obtained and used for the experimental infection of birds. Stock culture of P. multocida stored at − 80 °C was revived and grown overnight at 37 °C in brain heart infusion broth (LIOFICHEM, ABRUZZI, ITALY) with shaking at 150 rpm. Cells were harvested by washing (8000×g, 10 min) three times with sterile PBS and finally reconstituted in PBS to Sample collection and processing. The individual weight of all the chickens were measured before grouping them into respective treatment pens. Individual bird and leftover feed from each treatment were weighed weekly and the feed intake (FI) and body weight gain (BWG) recorded. Also, feed conversion ratio (FCR; feed intake/weight gain) and mortality (when it occurred) for each treatment were also calculated 41,71 . On days 21 and 28 of the experiment, four birds from each pen were selected at random and sacrificed by cervical dislocation after exposing them to overdose of isoflurane anesthesia. All efforts were made to minimize suffering. Visceral organs of each of the sacrificed bird were carefully removed and weighed after opening the abdominal cavity. After emptying the contents into sterile plastic containers, the weight of gizzard, ileum and caecum were recorded. Also, the weight of heart, liver, bursa, spleen, thigh, drumstick, breast, wing and dressing were recorded and expressed as the percentage of the body weight 43 . Statistical analysis. Data were collected and analyzed by analysis of variance as a completely randomized design using the GLM procedure as described by GRAPHPAD PRISM version 5.0 for Windows (GRAPHPAD SOFTWARE, SAN DIEGO, CA, USA) and SAS software (version 9.4, SAS Institute Inc., Cary, NC). Viable counts of the gizzard, ileum and caecum contents were subjected to logarithmic conversion (Log 10 ) before statis- Table 10. Primer sequences used for RT-qPCR. ACTB beta-actin, HIF1A hypoxia inducible factor 1 alpha, PTGER2 prostaglandin E receptor 2, TSG-6 tumor necrosis factor-(TNF) stimulated gene-6, F forward, R reverse.  GCA GTT CCT CAT GCA AT  215  R-AAA TGC TGC TAG CCC TTC CC   PTGER2  NM_001083365.1  F-TTG CAC GTC ACC TTC TCG TT  235  R-TGA TGG TCA TGA TGG CGA GG   TSG-6  DQ275160.1  F-ATG GAC AGC GGA TTC ACC TC  219  R-TCT GAA ACC CAC CAG CAG TC   ACTB NM_205518 www.nature.com/scientificreports/ tical analysis. All the results were presented as means of two independent experiments, and differences between treatment groups were determined using the Duncan's multiple range test. Probability values less than 0.05 (P < 0.05) was considered as significant.