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Use of the postbiotic INGUBAL Swine® to reduce antibiotic use and improve health parameters in swine.
Empleo de Ingubal Swine® para la reducción del uso de antibióticos y mejora de parámetros de salud en porcino

The use of fermented supplements in animal nutrition has become one of the best alternatives to antibiotics as growth promoters. This type of product improves production rates, prevents the appearance of diseases and respects animal welfare, in addition to reducing the use of antibiotics on farms.

The two experiments presented in this article investigate the effects of the fermented supplement INGUBAL Swine® on Iberian and white pigs. On the one hand, Iberian piglets showed an improvement in productive parameters and health indicators, especially in average daily gain and hematological parameters, especially the red series. In addition, a change in the intestinal microbiota was observed. On the other hand, in the white pig farm, a reduction in the use of antibiotics was achieved, as well as a reduction in mortality after the appearance of a digestive pathological process.

INTRODUCTION

Until 2006, the use of antibiotics as growth promoters was a very common practice in animal husbandry. This strategy for pathogen control was especially widespread in species with more intensive farming conditions, such as swine, broilers and dairy cattle.

The widespread increase in the use of antibiotics led to the development and emergence of multi-resistant pathogenic bacterial strains whose impact on human health was unknown. There was also increased concern about residual contamination of the food chain with these antibiotics. All this triggered the adoption of new safety measures and a gradual withdrawal of antibiotics as growth promoters. Specifically, in January 2006 its use was finally banned in the European Union (EC Regulation 1831/2003).

This ban has sparked intensive research focused on developing alternative strategies to maintain animal health and performance with the goal of reducing antibiotic use.

Currently, the use of functional additives in animal nutrition is the main option to improve production rates, prevent the occurrence of diseases and respect animal welfare (Bajagai, 2016).

In this sense, we understand as functional additives all those ingredients that incorporated in the diet of animals can improve their welfare and productivity beyond what could be expected or explained by their simple nutritional potential (Ve- lasco, 2006). In principle, this improvement is attributed to the regulatory action of these additives on the intestinal immune response, the functionality and integrity of the mucosa and on the microbiota (Pagnini, 2010).

Recent studies have pointed out that it is common in some production systems for animals to perform up to 30-40% below their genetic potential, mainly due to poor digestion and the appearance of infectious processes. The use of functional ingredients in feed and the development of formulations to optimize intestinal health can help us achieve healthier and more robust animals and, therefore, capable of fully expressing their genetic potential. This will ensure that raw materials are used more efficiently, thereby reducing waste and environmental pollution.

INGUBAL Swine® is a fermented feed supplement produced from yeasts and different strains of lactic acid bacteria with postbiotic activity.

 

OBJECTIVE

The objective we have pursued with our trials has been to evaluate the effectiveness of INGUBAL Swine® versus a group of control animals, in the reduction of antibiotic use, improvement of health parameters and productive indexes in two experiments in Iberian and white piglets.

 

Experience 1: Effect of INGUBAL Swine® on the use of antibiotics in cerdo blanco pigs.

 

DESCRIPTION OF THE TEST

The study was carried out in a pig farm with a weaning room with a capacity of 7,000 places. The installation consists of 6 rows of polyester cubicles, each with two feeders and two drinkers, plus a hand feeder to administer the lactoinitiator in the first days after weaning and a covered outdoor pen in front with capacity for 30 to 40 animals.

The animals were either Danbreed or PIC crosses, and all came from the same dam.

The groups included in the study were as follows:

Control group: Piglets (n=800) fed with pre-starter and starter feed without supplementation.

INGUBAL Swine® Group: Formed by piglets (n=6,190) that have received the same type of feed as the previous group, but in this case supplemented with 2 kg of INGUBAL Swine® per MT of feed in pre-starter and 1.25 kg/tm in starter.

 

Clinical picture

The clinical picture presented by the animals coincides with the usual problems of pig farms after weaning. Approximately 2 or 3 days after starting with the starter feed, digestive problems began to appear in all the houses, with losses in both groups.

 

Sampling

Necropsy of the dead animals was performed as recently as possible, since after several hours, autolysis of the intestine occurs rapidly.

The digestive packets are sent to the laboratory for complete analysis, both fresh for the microbiological study and preserved in 4% formalin for the histopathological study, in both cases refrigerated.

Samples were taken with swabs with transport medium from animals with diarrhea, obtained directly from the rectum.

 

Lip tests

a) Clinical and histopathological study

The clinical signs of the animals affected by the digestive symptoms were recorded and all the animals on the farm were checked for the presence of any symptoms.

In addition, macroscopic study of the lesions observed during necropsies was performed, as well as microscopic study by conventional histopathology techniques.

b) Microbiological study

Rectal swabs and lesion samples from the affected organs were seeded on conventional culture media (Blood Agar in aerobiosis and anaerobiosis, non-selective, and MacConkey agar, selective for enterobacteria) at 37 oC for 24 h.

DNA was extracted from bacteria isolated from pure cultures of each of the samples on the aforementioned solid medium. Bacteria were identified by PCR and detection of pathogenicity factors such as fimbriae or toxins (Stx2e, F41, K88, P987, F18, LT, K99, Sta, Stb, East) was performed.

Finally, antibiograms were performed using the Kirby-Bauer agar disc diffusion method from pure cultures against conventional antibiotics (amoxicillin, tetracycline, gentamicin, neomycin, sulfa-trimethoprim, enrofloxacin, chloramphenicol, cefazolin). The antibiogram will allow us to propose an antibiotic treatment for animals in the early stages of the disease.

 

RESULTS

Clinical signs and anatomopathologic study

The digestive picture was characterized by the presence of a dark, very liquid diarrhea (Image 1).

Dark and liquid diarrhea of piglets.Image 1. Dark, watery diarrhea of piglets.

There were 177 farm-wide casualties related to digestive problems of this type. Mortality in the control group was 3.75% and in the INGUBAL Swine® group was 2.37%.

Among the most relevant macroscopic lesions, a significant congestion of the entire digestive tract stands out, with an empty or completely full stomach and highly reactive mesenteric lymph nodes in all cases (Image 2). In addition, they presented intense congestion of the duodenal mucosa.

Image 2. Reactive and fused mesenteric ganglionic chain.Image 2. Reactive and fused mesenteric lymph node chain.

The kidneys also presented intense congestion in the medullary area, which was edematous.

Image 3. Kidney with congestive and edematous medullary zone.Image 3. Kidney with congestive medullary area and edema.

Histopathological examination showed necrotizing enteritis and atrophy of the intestinal villi (Image 4), without the presence of bacteria adhered to the mucosa, compatible with a viral process, due to rotavirus or coronavirus.

Histopathology of necrotizing enteritis and intestinal atrophy.

Image 4. Histopathology of necrotizing enteritis and intestinal atrophy.

This type of virus produces a severe gastroenteritis with destruction of the intestinal villi, causing profuse and difficult to control diarrhea. In these cases, moreover, the animals are more predisposed to co-infection by other pathogens.

 

Microbiological study

Microbiological culture was performed on all samples sent, both from the control and treated groups, obtaining in all cases pure cultures of bacteria identified as Escherichia coli, compatible with the clinical picture of post-weaning diarrhea (Hevia, 2016).

Table 1 summarizes the pathogenicity factors detected in the different strains belonging to both groups(E. coli C isolated from the control group and E. coli I isolated from the INGUBAL Swine® group).

Table 1. Virulence factors detected in the two E. coli strains by PCR.Table 1. Virulence factors detected in the two E. coli strains by PCR.

Virulence factors detected include the thermostable ST (Sta and Stb) and thermolabile LT enterotoxins, as well as F18 fimbrial adhesins, responsible for post-weaning colibacillosis caused by enterotoxigenic E. coli (ETEC). Toxins act on lose nterocytes producing an alkaline diarrhea by hypersecretion that causes immediate dehydration and acidosis (Hevia, 2016), which in some cases caused the death of piglets.

The two strains isolated have the same profile and also coincide with other strains previously isolated in other similar digestive processes on the farm.

On the other hand, the results obtained in the antibiograms allow us to select the antibiotic that is most effective in each case. These results are summarized in Table 2.

Phenotypic resistance profiles detected in the two E. coli strains by antibiogram.Phenotypic resistance profiles detected in the two E. coli strains by antibiogram.

The two isolated strains showed a high resistance profile, i.e., they are resistant to most of the antibiotics tested, making them difficult to treat.

 

DIAGNOSIS AND TREATMENT

The animals suffered a multifactorial digestive condition of viral origin complicated by a multiresistant enterotoxigenic E. coli infection. The E. coli strains isolated had the same pathogenicity factors in the control and treated groups, with the same profile as those isolated in other similar outbreaks on the same farm.

The only group of antibiotics to which they showed sensitivity were the cephalosporins, so the animals were treated with ceftiofur. The chosen treatment regimen included the administration of parenteral antibiotherapy to the piglets that showed symptomatology, as well as to the rest of the animals in the stalls where there were several affected animals, in a metaphylactic manner.

 

CONCLUSIONS: Impact of INGUBAL Swine®

At the end of the study, the results collected regarding mortality and treatments per block were as follows: The impact of supplementation with INGUBAL Swine®. The results have been very positive, since, on the one hand, there have been a 36.8% reduction in mortality due to digestive processes and, on the other hand, it has achieved a 61.8% reduction in the use of antibiotics for the treatment of post-weaning colibacillosis.

These two important reductions may be due to the many beneficial effects of fermented feeds. There are several mechanisms of action but, fundamentally, in this case it is proposed the modulation of the intestinal microbiota and regulation of colonization by pathogenic microorganisms which, although it does not prevent the entry of pathogens into the farm, prevents their binding to the intestinal epithelium since both types of bacteria share adhesion mechanisms (Bajagai, 2016). Other effects include altering the gene expression of pathogenic microorganisms by producing chemicals that can modify the behavior of bacteria and modulating the innate and acquired immune response (Pagnini, 2010), which are key to stimulating animal defenses when infection occurs.

In conclusion, fermented feeds bring numerous benefits to swine production systems, reducing mortality due to digestive processes and reducing the use of antibiotics, improving production rates and thus making farms more profitable.

Table 3. Total number of animals, number and percentage of animals lost due to digestive processes and percentage of treated stables.Table 3. Number of total animals, number and percentage of animals lost due to digestive processes and percentage of stables treated.

 

EXPERIENCE 2: Effect of INGUBAL Swine® on health and production parameters of the Iberian pig.

 

DESCRIPTION OF THE TEST

This trial took place in a farm located in the province of Cáceres. The study included 14 purebred Iberian dams, all of which were nulliparous and had been covered by natural mating with 100% Iberian boars.

The farrowing took place in an outdoor “camping” type system with individual places (Image 5).

Image 5. Detail of the birthing spacesImage 5. Detail of delivery places

The groups included in the study were as follows:

Control Group: Consisted of 5 breeding females and their piglets (n=35). Throughout lactation, the dams have been fed with lactation feed without any type of supplementation and the piglets from the 2nd week of life with pre-starter feed, without any type of supplementation.

INGUBAL Swine® Group: Consisting of 9 breeding females and their piglets (n=61). They have received the same type of feed as the previous group, but in this case both the ration of the mothers and that of the piglets has been supplemented with 2 kg of INGUBAL Swine® for each MT of feed.

 

Sampling

At 3-5 days after birth, all piglets were identified by a microchip placed on the left side of the neck and weighed individually. The animals have been weighed individually in each action carried out on the farm.

Rectal swabs: samples were taken with swabs with transport medium for total mesophilic aerobic aerobic and lactic acid bacteria counts, as well as the ratio between both.

Feces: collected feces were used for coprological analysis to detect the presence of parasite eggs or larvae.

Blood: Blood samples were collected in dry and EDTA tubes. The sera were used to perform blood biochemistry and the samples with anticoagulant were used to perform hemograms.

In addition, all animals were checked daily to identify possible clinical signs of disease.

 

Laboratory tests

a) Total aerobic and lactic acid bacteria count.

With this test we have performed the counting of representative species in the fecal microbiota. The objective was to investigate the possible beneficial biological responses of the supplements administered on the intestinal microbiota of the animals.

Serial dilutions and plating were performed with the following culture media:

    • Total aerobes: PCA Agar, Standard Method Agar (BD), in-cubation 37 oC in aerobiosis for 24 h.
    • Lactobacilli: MRS Agar (Oxoid) for lactobacilli, incubation in aerobiosis at 37 oC for 48-72 h.

b) Coprologies.

Flotations were performed by the conventional method.

c) Blood tests (Hemogram and Biochemistry).

Complete blood counts (WCB, RCB, HGB. HCT%, MCV, MCH, MCHC and PLT) using a hematology analyzer (Celltac α MEK-6550, Nihon Kohden) and a complete biochemical profile (proteins: total proteins, albumin and globulins; liver profile: ALT, AST and GGT; renal profile: creatinine and urea; nutritional profile: calcium, cholesterol, triglycerides and glucose; general profile: ALP and LDH and others: serotonin), using an automatic clinical chemistry analyzer (Biosystem A15).

d) Analysis of the results.

All analyses were carried out using statistical tests with SPSS 19 (SPSS Inc., Chicago, Illinois, USA) and R software version 3. 0. 3.

 

RESULTS AND DISCUSSION

Production parameters

Mortality at birth

During the development of this experiment, no mortality at birth has been detected, considering up to 3 days after birth.

Lactation mortality

In the lactation phase, there were 2 deaths of animals belonging to the Control group. In one of the animals, death occurred acutely (without symptoms) and in the remaining one after a diarrheic process. The two animals belonged to different litters.

Average daily gain

The average daily gain is a very useful production indicator to assess the animals’ growth rate and provides information about the animals’ performance. A good growth rate would be linked to a “good health” of the digestive system. To assess this parameter, we measured the average daily gain (ADG) at the end of lactation. A 23.8% increase in GMD was observed in the supplemented group with respect to the control group, the differences being statistically significant (Figure 1 and Table 4).

Figure 1 and Table 4. Mean levels of lactate dehydrogenase and alkaline phosphatase enzymes (IU/l) in the study groups.Figure 1 and Table 4. Mean lactate dehydrogenase and alkaline phosphatase enzyme levels (IU/l) in the study groups.

 

Total aerobic and lactic acid bacteria counts: validation parameters

Lactic acid bacteria are known for their beneficial effect on intestinal health (Lama, 2014). A healthy microbial population in production animals corresponds to an increase in animal performance and profitability and a decrease in morbidity and mortality during critical phases of production. In this sense, a higher proportion of this type of bacteria over the total aerobic bacteria is considered as an indicator of good intestinal health, since they present a balanced microbiota that favors a more efficient food conversion by facilitating food digestion. The population of this type of bacteria can be modified through feeding. Therefore, in our study we have analyzed the role that the diets used could have on this parameter.

As reflected in the graphs in Figure 2, higher counts of total mesophilic aerobes (TMA, Figure 2A) and lactic acid bacteria (LAB, Figure 2B) were observed in the supplemented group, although this did not translate into differences in the ratio between the two (Ratio, Figure 2C).

Bacterial counts in colony forming units per milliliter (cfu/mL).  Figure 2. Bacterial counts in colony forming units per milliliter (cfu/mL). A: total mesophilic aerobes (TMA), B: lactic acid bacteria (LAB) and C: LAB/AT ratio.

 

The measurement of this parameter is considered validation, since it allows us to confirm that lactic acid bacteria have settled in the mucosa of the digestive tract.

 

Health indicators: blood tests

Blood and serum samples were collected on the day of weaning for complete hemogram and biochemical profiles.

Tables 5 and 6 show the mean values of the different parameters analyzed for each study group. For both groups, all values were within the normal range, although some statistically significant differences were observed between some parameters.

Table 6. Complete biochemical profiles of both groups, reference values and their units.

Table 5. Complete blood counts of both groups, reference values and their units.

 

Complete biochemical profiles of both groups, reference values and their units.

Table 6. Complete biochemical profiles of both groups, reference values and their units.

 

The treated group shows statistically significant differences with respect to the control group in hemoglobin concentration, mean corpuscular volume (MCV, Figure 3A), mean corpuscular hemoglobin (MCH, Figure 3B) and hematocrit (HTC, Figure 3C). However, no difference was observed in the number of red blood cells. A decrease in white blood cells was also observed in treated animals.

Figure 3. Results of hemogram in both groups.Figure 3. Results of blood counts in both groups. A: Mean corpuscular volume, B: corpuscular hemoglobin and C: hematocrit.

 

The observed differences may be due to better utilization and digestion of nutrients in supplemented animals, including vitamins such as B12 or K and minerals such as iron, which are involved in hematopoiesis regulation processes (Rowland, 2017). Moreover, it is especially noteworthy in the swine species since piglets are born with minimal iron reserves and it is common to administer this mineral at birth, so in this case its utilization would be improved (Perri, 2016).

On the other hand, lower levels of white blood cells rule out the presence of subclinical infectious processes. This parameter, together with the results of the previous health indicators, is consistent with the effect on the immune system of lactic acid bacteria, which are capable of modulating the innate and adaptive immune response, preventing colonization by pathogens and reinforcing defense mechanisms in the event of infection (Pagnini, 2010).

No tissue damage was observed in relation to renal (urea and creatinine), general (LDH), or hepatic parameters. The higher levels of alkaline phosphatase (ALP) found in the supplemented group could be related to the presence of a higher GMD, since in these animals the growth rate is higher, as previously mentioned (Figure 4).

Alkaline phosphatase levels in both groups

Figure 4. Alkaline phosphatase levels in both groups.

 

CONCLUSIONS

The use of INGUBAL Swine® added to the feed led to an improvement in production parameters and health indicators, especially in GMD and red series. In addition, a change in the intestinal microbiota was observed. It is remarkable that the farm parameters have been improved without using antibiotic medicated feed. Feed enriched with fermented supplements is one of the new alternatives to antibiotic growth promoters. They are projected as an affordable strategy for the optimization of swine production, in terms of profitability, health and food safety.

 

ACKNOWLEDGMENTS

The research line for the development of the INGUBAL® range of postbiotic products has been awarded the Seal of Excellence by the European Commission (Ref.H2020-SMEInst-867298 LESSANTIBIOTICS). This work has been co-funded by ICEX/Invest in Spain (Exp. 201702890) and by the Ministry of Science, Innovation and Universities/Industrial Doctorate (Ref. DI-17-09603).

 

REFERENCES

  • Bajagai YS, Klieve AV, Dart PJ, Bryden WL. Probiotics in animal nutrition: production, impact and regulation. FAO. 2016.
  • Miranda R, Gómez M, Costillas S, Carvajal A, Rubio P. Colibacillosis in lactation, transition and fattening. porciNews Magazine. March 2016.
  • Lama JM. Role of probiotics in the porcine intestinal microbiota and its impact on reproductive performance. Breeding and Health 2014; 22, 60-66.
  • Pagnini C, Saeed R, Bamias G, Arseneau KO et al. Probiotics promote gut health through stimulation of epithelial innate immunity. Proc Natl Acad Sci USA 2010; 107(1), 454-459.
  • Perri, AM, Friendship, RM, Harding, J. C., & O’Sullivan, TL. An investigation of iron deficiency and anemia in piglets and the effect of iron status at weaning on posweaning performance. Journal of Swine Health and Production 2016; 24(1), 10-20.
  • Rowland, I, Gibson, G, Heinken, A, Scott, K, et al. Gut microbiota functions: metabolism of nutrients and other food components. European journal of nutrition 2017; 57(1), 1-24.
  • Velasco, J. L. F., Moreno, E. E. C., Ramirez, M. C., & Vara, I. A. D. Functional feeds for pigs at weaning. Veterinaria México 2006; 37(1), 117-136.

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This study was conducted by INGULADOS in collaboration with SALBO SCIENTIFIC, PENTABIOL and AGROPOR. It is published in the magazine Animal Production, 315-Jul-Aug, pp. 54-63.

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