Experimental immunology
Effect of experimental immunization of pigs with a suspension of Yersinia enterocolitica selected strains on changes in serum immunoglobulin G levels

Introduction

The pathogenicity of Yersinia spp. is due to their invasive properties, their capability for proliferation in a host’s body and production of toxins [1-4]. A predominating form of yersiniosis are alimentary disorders, although other symptoms are diagnosed in its course as well, including symptoms resembling appendicitis, reactive arthritis, erythema nodosum, micro-abscesses of internal organs, and sepsis [1, 2]. According to data provided in the latest EFSA (European Food Safety Authority) report, yersiniosis is ranked third amongst alimentary zoonoses, after campylobacteriosis and salmonellosis [5]. Yersinia enterocolitica is a bacteria wide-spread in the natural environment, whilst pigs has been acknowledged as its major reservoir and source of infection [1, 6-10]. The effect of infection in pigs includes, mainly, long-term carrier state and excretion of bacteria to the environment, which poses threat to human health. Attempts of creating herds free of Y. enterocolitica have so far been unsuccessful owing to a too wide dissemination and a variety of transmission routes of this pathogen [10, 11].

World-wide investigations into experimentally-induced yersiniosis of animals have been conducted for years, however pathogenesis and immunological response in the course of Y. enterocolitica infection are still inconclusive. Sparse researches are also available regarding the specific immunoprophylaxis of yersiniosis. This has, to a great extent, been attributed to a long-standing conviction that this bacteria is non-pathogenic to experimental animals, e.g. to rabbits, guinea pigs, rats or mice. A lack of a laboratory experimental model has for years been impairing the development of research addressing this issue. Later studies have demonstrated that some strains of Y. enterocolitica are pathogenic to selected breeding lines of laboratory animals, mice in particular [2, 3, 12]. For this reason, most of original papers focused on the specific immunoprophylaxis of yersiniosis are based on experiments conducted on a mouse model. The infection of mouse results in diarrhea, which however is not the only symptom of the disease [2, 3, 13]. The mouse model of Y. enterocolitica infections does not always mirror processes ongoing in organisms which under natural conditions are most frequently infected with this bacteria. For the major reservoir and, thus, the key source of risk posed to human health are pigs, there is a need for extending knowledge on the feasibility of preventing infections with Y. enterocolitica in this animal species. Therefore, the objective of this study was to develop a method for pigs immunization with a suspension of immunogenic strains of Y. enterocolitica and to analyze the impact of this immunization on the course of immunological processes in animals, and thus to search for possibilities of preventing or reducing potentially negative effects of Y. enterocolitica experimental infections in pigs.

Material and methods

Preparation of immunogenic Yersinia enterocolitica suspension



The suspension was prepared from Y. enterocolitica strains selected out of 60 isolates obtained from internal organs of aborted fetuses, fetal membranes, placentas as well as from vaginal and rectal swabs of sows. Detailed information referring to the origin of samples and bacteriological analyses they were subjected to as well as particular characteristics of isolates applied in the immunogenic suspension were provided in earlier papers [4, 14]. The strains were selected based on immunogenicity assessment using RBA/PKA (respiratory burst activity/potential killing activity) test and MTT (Mitogen Transformation Test). The RBA/PKA test was used to evaluate the effect of Y. enterocolitica on the activity of phagocytic cells, whereas the MTT – on the activity of T lymphocytes subjected to the effect of Y. enterocolitica cells expressed by the extent of proliferative response to mitogens.



Respiratory burst activity



Metabolic activity of polymorphonuclear (PMN) and morphonuclear (MN) cells was evaluated using the RBA test after cell stimulation with PMA (Phorbol Myristate Acetate, Sigma) described by Secombes [15] in modification by Siwicki et al. [16]. Whole blood (100 µl) was poured into 96-well microplates (NUNC), afterwards 100 µl of RPMI 1640 with 0.1% FCS (Foetal Calf Serum, Sigma) were added to each well, and the plates were incubated for 24 h, at a temperature of 4°C. After incubation, the non-adhered cells were removed by drawing off the fluid. The fluid was next replaced by the addition of 100 µl/well of a 0.1% solution of nitrotetrazolium blue (NBT) in RPMI or a 0.1% NBT solution in RPMI with PMA – at the amount of 1 µl/ml of a 0.1% solution of NBT. In addition, 20 µl of the suspension of the examined strains of Y. enterocolitica with density of 104 cfu/ml were added to each well. Each sample was analyzed in triplicate. The plates were incubated for 30 min at a temperature of 37°C. After incubation and medium removal, the cells were washed three times with 70% ethyl alcohol. The production of H₂O2 by the cells was measured in a microplate reader MRX 1.1 (Dynex, Great Britain) at a wavelength of 620 nm.



Potential killing activity test



Potential killing activity of blood PMN and MN cells was measured with the spectrophotometric method using the PKA test according to Rook et al. [17] in modification by Siwicki and Anderson [18]. Whole blood (100 µl) was poured into 96-well plates (NUNC), next 100 µl of RPMI 1640 with 0.1% FCS (Sigma) were added to each plate, and the plates were incubated for 24 h at a temperature of 4°C. After incubation, the non-adhered cells were removed by drawing off the fluid, which was next replaced by a 0.1% NBT solution in phosphate buffered saline (PBS) containing 18-h culture of Staphylococcus aureus. Afterwards, 20 µl doses of a suspension of the examined Y. enterocolitica strains with density of 104 cfu/ml were added in three replications, and afterwards incubated for 30 min at a temperature of 37°C. After incubation and medium removal, the cells were washed three times with 70% ethyl alcohol. The plates were dried for 3 min and next 120 µl of 2M KOH and 140 µl of DMSO were added to dissolve formazan. Reading was performed in a microplate reader

MRX 1.1 (Dynex) at a wavelength of 620 nm.



Mitogen transformation test



The proliferative response of lymphocytes stimulated with mitogens was performed with the colorimetric method based on MTT tetrazolium salt according to Mosmann [19] in modification by Siwicki et al. [16]. Samples of peripheral blood were diluted in a ratio of 1 : 1 with RPMI 1640 medium (Sigma). Lymphocytes were isolated in a Histopaque 1077 gradient (Sigma). The isolated lymphocytes (1 – 5 × 106) were suspended in the RPMI 1640 medium with the addition of 10% FCS (Sigma) and poured into 96-well microplates (NUNC) in doses of 100 µl/well.

Afterwards, 100 µl of mitogen were added to each well, i.e. concanavalin A (ConA, Sigma) at the concentration of 64 µg/ml or lipopolysaccharide (LPS, Sigma) obtained from Escherichia coli serotype 0111:B4, at the concentration of 160 µg/ml. Next, 20 µl portions of the suspension of the examined Y. enterocolitica strains with a density of 104 cfu/ml were added to each well, in three replications. The RPMI 1640 medium at the amount of 100 µl/well were used as the control. The plates were incubated for 72 h at a temperature of 37°C (5% CO2). After incubation, 10 µl of an MTT (3-[4,5-dimethylazol-2-yl]-5,2diphenyl tetrazolium bromide, Sigma) solution at the concentration of 5 mg/ml PBS were added to each well, and the plates were again incubated for 4 h at a temperature of 37°C (5% CO2). Further on, the plates were centrifuged for 15 min at 115 × g, the supernatant was removed and 100 µl of dimethylsulfoxide (DMSO, Polskie Odczynniki Chemiczne SA) were added to each well. After 10 min, the absorbance was read in a microplate reader MRX 1.1 (Dynex) at a wavelength of 620 nm.



Selection of strains



In order to classify the isolates to prepare the experimental Y. enterocolitica immunogenic suspension, out of the 60 strains examined that had earlier been divided into 4 groups depending on proliferation level, four strains were selected from each group. Thus selected and divided strains were used to prepare mixtures (24-h culture, 3° in McFarland’s scale), that were again analyzed with RBA/PKA test and MTT in order to determine the reciprocal effect of the investigated strains on the level of non-specific immunity indices. The scheme of selection of the analyzed strains was presented in Table 1.



Determination of the immunogenic Yersinia enterocolitica suspension dose in vivo



Animals



In order to determine the optimal dose of the immunogenic suspension, 15 pigs of a hybrid variety PIC were used, with body weight of ca. 20 kg, serologically-negative in examinations for the presence of anti – Y. enterocolitica antibodies. The animals were divided at random into three groups and placed in separate rooms, isolated from one another.

All activities involved in the experiment were carried out in accordance with the principles for the care and use of research animals and was approved by the Local Ethic Committee for Animal Experiments (No. 24/N).



Experimental immunization



There were prepared two different doses of a cell suspension of immunogenic Y. enterocolitica strains with a density of 2.7 × 109 cfu/cm³ inactivated with formol. One group of animals was administered subcutaneously 2 ml and the other 5 ml of the suspension, twice in a 2-week interval. The third group of pigs served as the control and received PBS in the analogous scheme.

The evaluation in vivo was conducted after per os challenge with a pathogenic Y. enterocolitica O:3 strain, at a dose of 10 ml and a density of 2.7 × 109 cfu/cm³, performed 3 weeks after immunization.

Over the experimental period, the animals were under constant clinical observation (general health status, appetite, internal body temperature, body weight gain) and bacteriological examination.



Antibodies



Serum levels of specific antibodies against Yop (Yersinia outer protein) Y. enterocolitica antigen were analyzed with the ELISA test using a commercial kit PIGTYPE®

YOPSCREEN (Labor Diagnostic, Leipzig, Germany). The test was conducted following producer’s instructions.



Statistical analysis



The statistical analysis of study results was conducted with the Fisher’s NIR test (least significant difference) and Tukey’s RIR test (reasonable significant difference), using statistical software STATISTICA 6.0.

Results

Selections of strains for preparation of experimental Yersinia enterocolitica suspension



The evaluation of Y. enterocolitica effect on the activity of phagocytic cells demonstrated significant differences in the impact of the analyzed Y. enterocolitica strains on the metabolic and phagocytic activity of PMN and MN cells. Simultaneously, significant differences were noted in the effect of the Y. enterocolitica strains examined on the proliferative response of T and B lymphocytes. Based on the proliferative properties of cells in respect of the control group, the strains examined were divided into four groups. The first included strains having a 10-fold stronger effect on lymphocytes proliferation that the control strains, whereas the second and the third group included strains inducing proliferation respectively 3-9 times and 1-2 times stronger than the control strains. The final fourth group included strains that were inducing proliferation once weaker than the control.

The selected and divided into four groups strains of Y. enterocolitica were used to prepare mixtures (24-h culture in 3° McFarland’s scale), that were again analyzed with the RBA/PKA test and MTT in order to determine the reciprocal interactions of the strains (Tables 2 and 3).

The RBA/PKA test demonstrated a lack of the suppressive effect on phagocytic cells of peripheral blood in all analyzed groups of strains, even in group IV constituting a mixture of Y. enterocolitica strains being the weakest in inducing proliferation. All groups of strains exhibited a strong effect on the metabolic and phagocytic activity of PMN and MN cells, yet no significant differences were noted between them.

The MTT demonstrated that groups I and II included strains with the strongest immunogenic properties, very strongly inducing the mitogen-stimulated immunity. In addition, the strains from groups I and II were shown to strongly induce the proliferative response of lymphocytes individually – without the presence of mitogens. For this reason, those strains were found the most appropriate for preparing the experimental immunogenic Y. enterocolitica suspension.

The highly immunogenic Y. enterocolitica strains selected for immunogenic suspension preparation constituted the following serobiotypes: O:3/4 – 1 strain, O:5/1A – 4 strains, O:8/1A – 1 strain, and two serologically not typable strains belonging to 1A biotype. Properties of the strains were described in detail in earlier papers [4, 14].



Experimental immunization



Changes in levels of anti-Y. enterocolitica antibodies in the course of experimental immunization of pigs, measured with the ELISA, were presented in Fig. 1. Results achieved were expressed as a per cent (%OD) of color reaction inhibition.

The immunization of piglets with the experimental suspension of inactivated highly immunogenic Y. enterocolitica strains was inducing the production of anti-Yop Y. enterocolitica antibodies in the first week post infection (wpi), when an insignificant increase in OD value was recorded in both immunized groups. Those levels were increasing until 3 wpi, reaching a slightly higher level in group II, where OD was at the level of cutoff values referred to as positive. After the challenge, the immune response in both immunized groups was enhanced and the levels of antibodies reached a multiplied value in respect to the level induced by immunization. The immune response as a result of per os infection with Y. enterocolitica occurred the earliest and was the strongest in the control group. In 3 wpi, the levels of antibodies were alike in all groups and maintained at a similar, high level until the end of the experiment.

No effect of immunization, both the first and the repeated one, was observe on the clinical picture nor body weight gains of the immunized animals. Results of bacteriological analyses and clinical examinations will be described in detail in another paper.

Discussion

Most of investigations addressing immunological processes induced by immunization with Y. enterocolitica cells have been conducted with laboratory animals [20-24]. The course of yersiniosis in mice or rabbits differs, to some extent, from its course in humans or in pigs which are the main reservoir of Y. enterocolitica. It may, therefore, be speculated that some differences will also be observed upon immunization of laboratory animals.

The pathogenicity of Y. enterocolitica is strictly related to the presence of a virulence plasmid (pYV) responsible for many typical properties of a microorganism in vitro and in vivo, including e.g. calcium dependent growth [2, 13, 25]. In a study by Toyos et al. [23], vaccinations were conducted with inactivated and viable bacterial cells of Y. enterocolitica O:3, both the Ca2+-dependent and Ca2+-independent ones. This research has demonstrated that the killed bacterial cells, both the Ca2+-dependent and Ca2+-independent ones, assured the same level of immunity when administered intraperitoneally or orally, and that they completely protected mice against oral infection.

In our study, the immunogenic suspension contained Y. enterocolitica strains that belong to various biotypes and serotypes, including biotype 1A which is characterized by, among other things, a lack of virulence plasmids [4, 14]. The main criterion of isolates selection was high immunogenicity demonstrated in vitro by means of RBA/PKA test and MTT. No description was found in the available literature regarding earlier investigations with the use of a similar method for the selection of strains to be used for immunization, however based on experimental results of mice immunization it may be presumed that the presence of virulence plasmids is not directly proportional to the immunogenic capability of Y. enterocolitica strains, which is also indicated by results of own in vitro studies.

In order to elucidate the role of humoral and cellular immunity in the prevention of Y. enterocolitica infections Nakajima et al. [22] were immunized mice with live and killed cells of the microorganism. Both the live and inactivated vaccines were stimulating the body to produce antibodies, and the titre of agglutinins reached  1 : 320. Infection through intraperitoneal or intragastric route did not enhance this effect. Alike results were achieved by other research group [25], which also conducted its experiment with the mice model. The applied vaccines, apart from inducing antibodies production, were preventing bacteria excretion with feces once the titre of agglutinins reached  1 : 320.

In our study, the experimental subcutaneous immunization of pigs with Y. enterocolitica cells, likewise in the mice model, induced that production of antibodies. However, the concentration of IgG in pig serum was at a low detection level by ELISA, yet in group II administered the higher dose of the experimental immunogenic suspension, the titres were slightly higher. According to the test’s criteria, not in all pigs from group I was the OD recorded at a level above 20, referred to as the cutoff value of the positive reaction.

In our study, the oral infection of the pigs was conducted 3 weeks after the second immunization. In contrast to findings by Nakajima et al. [22] reported for mice, the immune response was significantly enhanced and the level of antibodies increased 8 fold within 2 weeks. The increase in the levels of antibodies occurred in all groups, however 1-week retardation of the immune response was observed in the immunized animals, compared to the control pigs. In 4 wpi, levels of antibodies in all groups were at a similar, higher level, being insignificantly higher than in the control group.

The results achieved demonstrated that higher levels of antibodies were obtained in the group of animals administered subcutaneously the higher dose of the experimental immunogenic suspension, i.e. 5 ml 2.7 × 109 cfu/cm³ twice in a 2-week interval. Immunization conducted in this way will be applied in future investigations aimed at searching for methods of preventing infections or reducing Y. enterocolitica shedding in pigs.

Assuming that vaccination has been successful as a means of preventing or reducing the severity of infectious disease [26], investigations conducted with laboratory animals have proven the feasibility of producing an effective vaccine against yersiniosis. Infections with Y. enterocolitica may pose risk especially to public health, hence the practical application of research into the possibility of preventing or reducing infections in livestock is, presumably, only a matter of time, and further investigations in this respect seem to be substantiated.

References

 1. Bottone EJ (1999): Yersinia enterocolitica: overview and epidemiologic correlates. Microbes Infect 1: 323-333.

 2. Schiemann DA, Devenish JA (1980): Virulence of Yersinia enterocolitica determined by lethality in mongolian gerbils and the Sereny test. Infect Immun 29: 500-506.

 3. Wiśniewski J, Bielecki J (1996): Mechanizmy wirulencji bakterii z rodzaju Yersinia. Post Mikrobiol 2: 213-241.

 4. Platt-Samoraj A, Ugorski M, Szweda W, et al. (2006): Analysis of the presence of ail, ystA and ystB genes in Yersinia enterocolitica strains isolated from aborting sows and aborted fetuses. J Vet Med B 53: 341-346.

 5. Osek J, Wieczorek K (2011): Food-borne zoonoses and their etiological agents in the EFSA report for 2009. Życie Wet 86: 588-589.

 6. Fenwick S (1997): Domestic animals as potential sources of human Yersinia infection. Surveillance Wellington 24: 2-5.

 7. Fredriksson-Ahomma M, Björkroth J, Hielm S, et al. (2000): Prevalence and characterization of pathogenic Yersinia enterocolitica in pig tonsils from different slaughterhouses. Food Microbiol 17: 93-1001.

 8. Jakubczak A, Platt-Samoraj A, Siemionek J, et al. (1993): Występowanie pałeczek z rodzaju Yersinia u zwierząt hodowlanych, domowych, dzikich oraz ryb pochodzących z województwa olsztyńskiego. Med Weter 49: 301-303.

 9. Thibodeau V, Frost EH, Quessey S (2001): Development of an ELISA procedure to detect swine carriers of pathogenic Yersinia enterocolitica. Vet Microbiol 82: 249-259.

10. Bhaduri S, Weslez I (2006): Isolation and characterization of Yersinia enterocolitica from swine feces recovered during the National Animal Health Monitoring System Swine 2000 Study. J Food Prot 69: 2107-2112.

11. Nesbakken T, Iversen T, Lium B (2007): Pig herds free from human pathogenic Yersinia enterocolitica. Emerg Infect Dis 13: 1860-1864.

12. Staroniewicz Z, Madej JA (1986): Doświadczalne zakażenie myszy pałeczkami Yersinia enterocolitica. Med Weter 42: 12-15.

13. Laird WJ, Cavanaugh DC (1980): Correlation of autoagglutination and virulence of yersiniae. J Clin Microbiol 11: 430-432.

14. Platt-Samoraj A, Szweda W, Procajło Z (2009): The influence of experimental Yersinia enterocolitica infection on the pregnancy course in sows – preliminary studies. I. Bacteriological examination. Pol J Vet Sci 12: 317-322.

15. Secombes CJ (1990): Isolation of salmonid macrophages and analysis of their killing activity. Tech Fish Immunol 1: 137-145.

16. Siwicki AK, Krzyżanowski J, Bartoszcze M, et al. (1998): Adjuvant properties of killed Propionibacterium avidum KP-40 in vaccination of dogs against canine parvovirosis. Dtsch Tierärztl Wschr 105: 186-190.

17. Rook GA, Steele J, Umar S, et al. (1985): A simple method for the solubilization of reduced NBT, and its use as a colorimetric assay for activation of human macrophages by gamma-interferon. J Immunol Methods 82: 161-167.

18. Siwicki AK, Anderson DP (1993): Nonspecific defence mechanisms assay in fish: II. Potential killing activity of neutrophils and macrophages, lysozyme activity in serum and organs. Fish Diseases Diagnosis and Prevention’s Methods. IFI Olsztyn, FAO-Project GCP/INT/526/JPN; 105-112.

19. Mosmann T (1983): Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol 52: 67-74.

20. Genaro MS, Escudero ME, Munoz E, et al. (1998): Intracellular immunization with Yersinia enterocolitica O:8 cellular extract protects against local challenge infection. Microbiol Immunol 42: 781-788.

21. Mc Ghee JR, Mestecky J, Dertzbaugh MT, et al. (1992): The mucosal immune system: from fundamental concepts to vaccine development. Vaccine 10: 75-87.

22. Nakajima R, Kaneko K, Nashimoto N (1984): Protection against the lethal effect and arthritogenic capacity of Yersinia enterocolitica O:3 for mice. Jpn J Vet Sci 46: 721-727.

23. Toyos JR, Diaz R, Hardisson C (1991): Protection of mice against parenteral and oral infection with Yersinia enterocolitica. Microbiol Immunol 76: 289-298.

24. Uchida I, Kaneko K, Hashimoto N (1982): Cross-protection against fecal excretion of Yersinia enterocolitica and Yersinia pseudotuberculosis in mice by oral vaccination of viable cells. Infect Immun 36: 837-840.

25. Carter PB, MacDonald TT, Collins FM (1979): Host responses to infection with Yersinia enterocolitica. Contrib Microbiol Immunol 5: 346-350.

26. McVey DS, Galvin JE, Olson SC, et al. (2003): A review of the effectiveness of vaccine potency control testing. Int J Parasitol 33: 507-516.