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Ciência Rural

Print version ISSN 0103-8478On-line version ISSN 1678-4596

Cienc. Rural vol.46 no.8 Santa Maria Aug. 2016

https://doi.org/10.1590/0103-8478cr20151269 

MICROBIOLOGY

Virulence factors of Escherichia coli in relation to the importance of vaccination in pigs

Fatores de virulência da Escherichia coli associados à importância da vacinação nos suínos

Daniele Araujo Pereira1 

Marilda Carlos Vidotto2 

Karla Alvarenga Nascimento1 

Anne Caroline Ramos dos Santos1 

Marina Lopes Mechler3 

Luís Guilherme de Oliveira1  * 

1Departamento de Clínica e Cirurgia Veterinária, Universidade Estadual Paulista "Júlio de Mesquita Filho" (UNESP), Via de Acesso Prof. Paulo Donato Castellane, s/n., 14884-900, Jaboticabal, SP, Brasil.

2Departamento de Medicina Veterinária Preventiva, Universidade Estadual de Londrina (UEL), Campus Universitário Centro de Ciências Agrárias, Londrina, PR, Brasil.

3Departamento de Medicina Veterinária, Universidade Federal de Lavras (UFLA), Lavras, MG, Brasil.


ABSTRACT:

Enterotoxigenic Escherichia coli (ETEC) is the major cause of diarrhea in newborn and weaned pigs. Bacteria adhesion to the host cell is considered a specific phenomenon among fimbrial and non-fimbrial adhesins with their respective receptors on enterocytes. Enteric disorders are related with the fimbriae F4 (K88), F5 (K99), F6 (987P), F41, and F18. In addition to ETEC, another category of E. coli , porcine pathogenic E. coli (PEPEC),can cause diarrhea in pigs; it produces the porcine attaching and effacing-associated (Paa) adhesin in, which is capable to cause a typical lesion known as an attaching and effacing (A/E) lesion. Immunization of sows with adhesin is important to stimulate the production of antibodies and their subsequent transfer to piglets through colostrum. The aim of this paper is to illustrate the main impacts of enteric diseases caused by E. coli in swine production and to highlight the importance of continuing research on this bacterium to improve disease prevention through vaccination.

Key words: colibacillosis; diarrhea; vaccines; swine; colostrum.

RESUMO:

Escherichia coli

( E. coli ) enterotoxigênica (ETEC) é considerada importante causa de diarreia em suínos neonatos e desmamados. A adesão da bactéria à célula do hospedeiro é considerada um fenômeno específico entre as adesinas fimbriais e não fimbriais com seus respectivos receptores nos enterócitos. Os distúrbios entéricos estão relacionados com as fímbrias F4 (K88), F5 (K99), F6 (987P), F41 e F18. Além da ETEC, outra categoria de E. coli pode causar diarreia nos suínos, denominada de PEPEC (porcine pathogenic E. coli ), a qual produz a adesina Paa (Porcine attaching adherence), capaz de provocar uma lesão típica denominada A/E (attaching and effacing). A imunização das matrizes com adesinas é importante para estimular a produção de anticorpos e a consequente transferência através do colostro aos leitões. O objetivo deste trabalho foi mostrar os principais impactos das doenças entéricas causadas por Escherichia coli na produção de suínos, e mostrar a importância de atualizar o estudo dessa bactéria para prevenir a doença através da vacinação.

Palavras-chave:  colibacilose; diarreia; vacinas; suínos; colostro.

INTRODUCTION:

Enteric diseases of bacterial etiology, particularly those caused by Escherichia coli , are the major concern in pig farming and have a heavy impact on pork industry ( HYPERLINK "#mkp_ref_029" ). Therefore, to avoid losses in pig farming, care during the initial disease phase is essential through a quick and accurate enteric diagnosis associated with a full history, clinical signs, and pathology. Recent advances in diagnostic methods have enabled a better understanding of diseases and have helped to reduce the effects in swine ( HYPERLINK "#mkp_ref_047" ).

The main cause of economic losses in young pigs is related to enteritis because it causes diarrhea, decreased weight loss in food conversion, culling, and sudden death. The diarrheal syndrome is responsible for 6% of piglets mortality on farms ( HYPERLINK "#mkp_ref_008" ). Among the agents that cause diarrhea in suckling piglets are Isospora suis , coronavirus, rotavirus, and Clostridium perfringens type C, although enterotoxigenic E. coli (ETEC) can be considered the most important agent capable of causing illness and death in newborn and weaned piglets ( HYPERLINK "#mkp_ref_002" ). Colibacillosis may have a mortality rate of approximately 25% if measures such as prevention and treatment are not adopted ( HYPERLINK "#mkp_ref_033" ).

Etiology

E. coli is a facultative anaerobic bacterium belonging to the family Enterobacteriaceae ; it is a Gram-negative rod, non-sporulating, and either non-mobile or moved by peritrichous flagella ( HYPERLINK "#mkp_ref_017" ). These bacteria have the ability to reduce nitrate to nitrite and to ferment glucose. They also are oxidase- negative and metabolize a wide variety of substances such as carbohydrates, proteins, amino acids, lipids, and organic acids. They produce catalase and glucose and use ammonia as the only source of carbon and nitrogen ( HYPERLINK "#mkp_ref_043" ).

Although E. coli bacteria are part of the normal flora of human and other animal species intestinal tracts, a few strains are pathogenic due to specific features. These can be classified into six groups: enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), entero aggregative E. coli (EAEC), entero invasive E. coli (EIEC), and diffusely adherent E. coli (DAEC) ( HYPERLINK "#mkp_ref_044" ). Among these, the bacteria belonging to the ETEC group are primarily responsible for colibacillosis in both newborn and weaned pigs ( HYPERLINK "#mkp_ref_028" ).

Serological classification of E. coli is based on the four surface antigens: O (somatic antigen), k (capsular antigen), H (flagellar antigen), and F (fimbrial antigen) ( HYPERLINK "#mkp_ref_038" ). ETEC strains that cause diarrhea in pigs have two fundamental factors of virulence, the adhesins and enterotoxins (MOON & BUNN, 1993). These may be expressed by several genes located on different segments of chromosomes and/or plasmids, and they are differentiated by their hemagglutinating, serological, and morphological properties. The majority of the protein subunits of the fimbriae are attached to the erythrocytes' integrated sugars, causing hemagglutination in the presence of D-mannose ( HYPERLINK "#mkp_ref_007" ).

The ETEC found in pigs have shown several adhesin in types, defined as fimbriae F4, F5, F6, F18, and F41 ( HYPERLINK "#mkp_ref_045" ; HYPERLINK "#mkp_ref_026" ). The expression of these fimbriae is stimulated in vitro by growth conditions such as pH, rearing temperature, aeration, and osmolarity of the medium, with a specific medium for each fimbria ( HYPERLINK "#mkp_ref_014" ).

Pathogenesis associated with the bacterial virulence factors

Colibacillosis begins when there is binding of the bacteria to specific receptors of enterocytes through the fimbrial adhesins. Toxins are then released to activate the production of the enzymes adenylate cyclase and guanylyl cyclase, which increases the intracellular concentration of cyclic adenosine monophosphate (cAMP), generating a greater transfer of sodium bicarbonate and water from the gut lumen cells ( HYPERLINK "#mkp_ref_033" ). Colibacillosis, when caused by ETEC toxins, causes dehydration leading to animal death by secretory diarrhea, with fluid excretion higher than the absorption capacity generating a dysfunction of the physiological processes ( HYPERLINK "#mkp_ref_049" ).

The enterotoxins are classified into thermo stable (STa and STb) and thermo labile (LT-1 and LT-2); these are responsible for causing imbalances in the absorption and secretion of electrolytes and water by the enterocytes, leading to diarrhea, dehydration, and death of animals ( HYPERLINK "#mkp_ref_033" ). ETEC is able to produce several protein toxins that differ in weight, molecular structure, and biological and immunological activity ( HYPERLINK "#mkp_ref_015" ).

The K88 fimbria, also currently known as F4, was the first studied after it was discovered in 1961 by serological testing from pigs with edema disease and enteritis ( HYPERLINK "#mkp_ref_037" ); its antigenic variants were differentiated into K88ab, K88ac, and K88ad ( HYPERLINK "#mkp_ref_012" ), with the K88ac variant being most commonly reported in piglets with diarrhea. This fimbria is characterized by a diameter of 2.1nm and molecular weight of 27.5 kilodalton (kDa). The specific culture medium for expression of F4 is agar glucose, and this fimbria enables E. coli to hemagglutinate guinea pig erythrocytes in the presence of mannose ( HYPERLINK "#mkp_ref_032" ).

HYPERLINK "#mkp_ref_041" ) tested the first F4 vaccine in sows to protect the newborn piglets with passive immunity via colostrum. An F4 vaccine was able to protect piglets that received antibodies through colostrum during weaning ( HYPERLINK "#mkp_ref_036" ). A study conducted with samples from a diarrhea outbreak in the Paraná state of Brazil found a 52% prevalence of F4 in the analyzed samples ( HYPERLINK "#mkp_ref_042" ).

The K99 fimbria, currently known as F5, has a molecular weight of 18.5kDa and a diameter of 4.8nm. F5 can be found in ETEC samples in calves and swine ( HYPERLINK "#mkp_ref_030" ). Selective medium Minca is used to induce expression of the fimbriae.

Purification and characterization of F41 was reported by De GRAAF & ROODA (1982); this fimbria has a molecular weight of 29.5kDa and a diameter of 3.2nm. Its production can be compared with the production of F5. Use of F41 as an antigen in vaccines is less well defined when compared with the fimbriae F4, F5, and F6 (MOON, 1990). This fimbria is usually reported in bovines and swine ( HYPERLINK "#mkp_ref_014" ). F5 and F41 can hemagglutinate human, guinea pig, and sheep erythrocytes in the presence of mannose ( HYPERLINK "#mkp_ref_035" ).

The strains most commonly found in newborn piglets have F5 (K99) and F6 (987P) fimbriae. During the first week of life, the receptors for F5 are found in enterocytes of piglets and then decrease significantly ( HYPERLINK "#mkp_ref_001" ). The 987P fimbria was analyzed by electron microscopy, showing a diameter of 7nm, molecular weight of 18.9kDa, and pH of 3.7 ( HYPERLINK "#mkp_ref_034" ). It is usually found in newborn piglets ( HYPERLINK "#mkp_ref_014" ); however, the receptors in pigs for this fimbria are also present in young and adult animals (ALFIERI et al., 2010). Blood agar culture medium is more selective for inducing this fimbria ( HYPERLINK "#mkp_ref_013" ). Fimbria 987P does not have hemagglutinating properties ( HYPERLINK "#mkp_ref_020" ), but attaches to glycoprotein receptors on swine enterocytes.

The F18 fimbria was expressed only in special laboratory culture conditions ( HYPERLINK "#mkp_ref_048" ). This fimbria has two variants, F18ab and F18ac, and their identification was analyzed by serology and polymerase chain reaction (PCR) ( HYPERLINK "#mkp_ref_019" ). According to HYPERLINK "#mkp_ref_009" ), F18 has the ability to colonize the intestinal epithelium, adhere to specific receptors, and cause edema disease or diarrhea after weaning.

In pig farming, fimbriae F4, F5, F6, and F41 are linked to enteric disorders, and the F18 fimbria is linked with edema disease ( HYPERLINK "#mkp_ref_011" ). Edema disease is an illness that affects weaned piglets, and it is related to E. coli strains linked to different virulence factors such as fimbriae and toxins ( HYPERLINK "#mkp_ref_015" ). HYPERLINK "#mkp_ref_031" ) reported that the impact of the fimbriae in the diagnosis and vaccination depends on the number and prevalence of antibody types, favoring intestinal colonization of occurring species.

In addition to ETEC, another class of E. coli that causes diarrhea in newborn pigs is porcine pathogenic E. coli (PEPEC), which is similar to the E. coli that causes enteric infections in humans and animals through the lesion type A/E (attaching and effacing) ( HYPERLINK "#mkp_ref_003" ).These lesions are characterized by close adherence to the membranes of the enterocytes and activation of a variety of chromosomal gene products that interact with components of the host cells, leading to the destruction of enterocytes and cytoskeletons edges ( HYPERLINK "#mkp_ref_024" ).

Phenotypic and genotypic characterizations of PEPEC showed that they lack the virulence factors of ETEC but present porcine attaching and effacing-associated (paa) nucleotide sequences, associated with the ability to produce injury type A/E in piglets and pig ileum sections ( HYPERLINK "#mkp_ref_023" ). The E. coli A/E lesion is caused by an outer membrane protein, known as intimin, which is encoded by the eae gene and facilitates bacterial adhesion ( HYPERLINK "#mkp_ref_027" ).

Histopathological changes caused by A/E injuries are indistinguishable from those produced in humans by EPEC. A/E injuries are also caused by other bacteria (including EHEC), by agents of hemorrhagic colitis, and by uremic syndrome in humans ( HYPERLINK "#mkp_ref_022" ); EPEC also causes diarrhea specifically in rabbits ( HYPERLINK "#mkp_ref_032" ). All genes involved in A/E injuries are located on the locus of enterocyte effacement (LEE) chromosome.

The gene identified in paa PEPEC was cloned, and it showed strong correlation with the eae gene of EHEC O157:H7 and with isolates of dogs, rabbits, and pigs ( HYPERLINK "#mkp_ref_004" ). Characterization of e paa gene suggests its contribution to the early stages of PEPEC virulence ( HYPERLINK "#mkp_ref_006" ) and its rPaa protein weights ( HYPERLINK "#mkp_ref_040" ). In Paraná, 300 stool samples of 100 pigs from different farms were analyzed using PCR for the paa gene and its link to the enterotoxins Sta, Stb, and LT. Results were 22% positive for the paa gene, which is associated with the presence of genes for other adhesins and toxins (F4, F18, F41, Paa, Sta, Stb, and LT). This demonstrates the importance of the paa gene as a virulence factor ( HYPERLINK "#mkp_ref_046" ). The characteristics described above are listed in table 1.

Table 1 Summary of E. coli groups and their relation to different species. 

Diagnosis

It is difficult to diagnose diarrhea caused by ETEC using clinical signs and necropsy lesions, which are considered non-specific. Because E. coli are commonly present in the intestinal flora of animals, their discovery is not a conclusive diagnosis. A phenotypic or genotypic description of the isolated strain is required to recognize bacterial fimbriae and characterize the genes encoding the fimbrial proteins and enterotoxins ( HYPERLINK "#mkp_ref_001" ).

Control measures

A number of factors influence the presence of diarrhea within pig production. One of the main problems is improper disinfection of the stall and poor sanitation among the sows. A lack of adequate temperature control for the piglets and sows (the ideal temperature is 33°C), and poor air circulation also play a role. Another factor is low attention by the handler to hygiene aspects, such as the use of dirty boots, which serve as a source of infection for the piglets. Additionally, diets low in digestibility can cause the substrate to remain longer in the small intestine of piglets, favoring the multiplication and prevalence of E. coli . Finally, environmental conditions and the immune state of sows also influence the appearance of diarrhea ( HYPERLINK "#mkp_ref_033" ).

There are several ways to minimize the damage caused by ETEC, some of which include sow vaccination before labor, autogenous vaccines in piglets, use of prophylactic antibiotics, and selection of animals lacking genes encoding receptors for fimbriae. Other methods to reduce losses due to ETEC are active immunization (intramuscular immunization with fimbriae or oral immunization with attenuated or non-virulent strains which contain fimbriae), passive immunization (oral administration of yolk immunoglobulin from eggs (IgY) against fimbriae), use of powdered plasma proteins ( HYPERLINK "#mkp_ref_012" ), and intranasal vaccines in piglets to prevent diarrhea ( HYPERLINK "#mkp_ref_025" ). Measures of control and prevention are listed in table 2.

Table 2 Measures of control and prevention in piglets, gilts, and sows. 

Vaccination schedules proposed by companies that produce vaccines against colibacillosis recommend vaccination of swine matrices during pregnancy in two ways: at four and two weeks before labor in gilts, and a revaccination two weeks before labor in sows. The first contact of the mother with the vaccine should be preconized in two doses to stimulate a higher immune response. As weaned piglets are often affected by ETEC, they must be able to produce immunity through antibodies received in colostrum ( HYPERLINK "#mkp_ref_005" ). The colostrum can be absorbed by the enterocytes in piglets up to the first 24 hours after birth, as this passive immunization is sufficient for protection by the immunoglobulin (IgG) to prevent diarrhea ( HYPERLINK "#mkp_ref_021" ). The vaccination programs are listed in table 3. HYPERLINK "#mkp_ref_039" ) evaluated the humoral immune response of immunized sows with recombinant proteins of ETEC (F4, F5, F6, F18, and F41), and the response was compared with a group that received a commercial vaccine containing ETEC bacterins and a control group. There was a statistically significant increase in the levels of fimbriae F4, F5, F6, and F18 antibodies in sows vaccinated with recombinant proteins compared to the control group. Furthermore, all piglets showed significantly elevated antibody levels to all fimbriae, demonstrating the transfer of antibodies via colostrum.

Table 3 Vaccination programs according to each manufacturer. 

Studies show that the swine matrices immunized with colibacillosis vaccines are able to better protect their piglets as compared to unvaccinated ones ( HYPERLINK "#mkp_ref_018" ). The vaccine is capable of inducing the production of specific antibodies, which decrease bacterial adhesion receptors on the intestinal cells and neutralize enterotoxins. The commercial vaccines contain inactivated bacterins or purified vaccine fimbrial subunits, and some already contain enterotoxin LT; they are given via a parenteral route ( HYPERLINK "#mkp_ref_001" ). The rPaa protein could be included in the development of a vaccine against colibacillosis ( HYPERLINK "#mkp_ref_040" ).

Commercial vaccines are not completely efficient, owing the dependence on maternal antibodies, however, the commercial vaccines administered parenterally to pregnant sows are effective against newborn diarrhea if the vaccine has all fimbrial antigens that occur on the farm, if there is sufficient intake of colostrum per animal, and if there is a low pressure of infection ( HYPERLINK "#mkp_ref_016" ).

CONCLUSION:

Immunization of sows with adhesins is essential for stimulating the production of antibodies and conferring these to piglets through colostrum. Vaccination may be considered an excellent strategy and quite effective when used properly, and it is considered one of the best ways to prevent diseases within a production system where the pressure of infection is high. At the same time, risk factors also need to be avoided through control measures; this is fundamental to ensure a healthy environment free of disease. Although, the risk of death in swine after a single infection with ETEC is not high, complex infections with ETEC, rotavirus, and coccidia are often found in piglets at both newborn and post-weaning stages, leading to high economic losses for pig producers.

ACKNOWLEDGEMENTS

Research supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

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1CR-2015-1269.R2

Received: September 07, 2015; Accepted: February 15, 2016; Revised: May 04, 2016

*Corresponding author: Luís Guilherme de Oliveira, email: HYPERLINK "luis.guilherme@fcav.unesp.br"

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