Comparison of Growth and the Cytokines Induced by Pathogenic Yersinia enterocolitica Bio-Serotypes 3/O: 3 and 2/O: 9

Pathogenic Yersinia enterocolitica is widely distributed in China where the primary bio-serotypes are 3/O: 3 and 2/O: 9. Recently, the distribution of 2/O: 9 strains are being gradually replaced by 3/O: 3 strains where presently 3/O: 3 strains are the major pathogenic Y. enterocolitica in China. To identify the growth conditions and cytokines induced by Y. enterocolitica and providing some clues for this shift, we performed competitive growth in vitro and in vivo for these two bio-serotype strains; and we also compared the cytokines induced by them in infected BALB/C mice. We found 2/O: 9 strains grew more in vitro, while 3/O: 3 strains grew more in vivo regardless of using single cultures or mixed cultures. The cytokines induced by the two strains were similar: interleukin-6 (IL-6), IL-9, IL-13, granulocyte colony-stimulating factor (G-CSF), chemokines (KC), monocyte chemotactic protein 1 (MCP-1), macrophage inflammation protein-1α (MIP-1α), tumor necrosis factor-α (TNF-α), and RANTES were statistically up-regulated upon activation of normal T cells compared to the control. The cytokine values were higher in mixed infections than in single infections except for IL-6, G-CSF, and KC. The data illustrated the different growth of pathogenic Y. enterocolitica bio-serotype 3/O: 3 and 2/O: 9 in vitro and in vivo, and the cytokine changes induced by the two strains in infected BALB/C mice. The growth comparisons of two strains maybe reflect the higher pathogenic ability or resistance to host immune response for Y. enterocolitica bio-serotype 3/O: 3 and maybe it as one of the reason for bacteria shift.


INTRODUCTION
Yersinia enterocolitica is widely distributed in the natural world; and being one of the few intestinal bacteria that can grow at low temperatures, the animal hosts of Y. enterocolitica are widely distributed, including livestock, poultry, rodents, reptiles, and aquatic animals. It causes gastrointestinal symptoms and systemic disease, e.g., liver and spleen abscess (Thoerner et al., 2003), reactive arthritis, erythema nodosum, and anemic goiter (Heyma et al., 1986;Stuart and Woodward, 1992;Gaede and Heesemann, 1995;Wang et al., 2012); and can cause septicemia and death (Bottone, 1999). Biotyping and serotyping are common and valuable methods for Y. enterocolitica identification where at present Y. enterocolitica is divided into six biotypes (1A, 1B, 2, 3, 4, and 5) and 60 serotypes. The highly pathogenic Y. enterocolitica bioserotype 1B/O:8 strain was distributed worldwide (Wang et al., 2008(Wang et al., , 2010Liang et al., 2012); while the other bio-serotypes were the lower or non-pathogenic strains and distributed all over the world (Wang et al., 2011). From our previous investigation, 58 serotypes of Y. enterocolitica had been found and no highly 1B/O: 8 strain has been isolated in China (Wang et al., 2008;Gu et al., 2012). The bio-serotype 3/O: 3 and 2/O: 9 strains were the primary types found in China.
Since the 1990s, the isolation numbers of bio-serotype 2/O: 9 strains have decreased, while the 3/O: 3 strains became the dominant bio-serotype in China. From 2009 to 2011 in a national survey for Y. enterocolitica, 2/O: 9 almost disappeared where only six O: 9 strains were identified from 862 Y. enterocolitica (Liang et al., 2012). The mechanisms of this change were not known, and therefore to determine the reason, we performed comparative growth of the two strains in vitro and in vivo, and compared the bacteria produced cytokine changes of the two strains in infected BALB/C mice.

In vitro Cultures
Two colonies of strain A and B were selected either as a single culture, or the strains were mixed as mixed cultures. All the cultures were inoculated in BHI broth and incubated at 25 • C for 48 h, and shaken at 120 rpm. Colony counts were performed every 3 h, and each colony in the mixed bacterial culture was identified using monoclonal antibodies grown in our laboratory; and finally comparative growth curves were drawn. The experimental procedure was performed twice for each strain; and strains C and D were also included.

In vivo Cultures
Six week-old healthy female BALB/C mice were purchased from the Chinese Academy of Food and Drug Testing Laboratory Animal Resource Center. Experiments were performed using the four strains as mentioned above. For each experiment, O: 3 and O: 9 strains were mixed to infect animals using equal number of each bacterium in the experimental groups; O: 3 and O: 9 strains singly infected mice were the two control groups (O: 3 control group and O: 9 control group), and healthy mice were selected as a blank group. Approximately 10 7 cfu/ml of the bacteria were used for intraperitoneal injection, three mice were removed randomly at 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, and 48 h after infection and sub-ocular sinus blood was separated and the serum was stored at −70 • C. Mice were sacrificed by cervical dislocation; the spleens were removed after immersion disinfection. Three spleens were mixed with 3 ml PBS to form a suspension, and diluted 10-fold to 10 −3 ; and the appropriate concentration to coat plates at 3 h was made. The plates were cultured at 25 • C for 24 h.

Statistical Analysis
The amount of bacteria at each time point was bacterial counts per milliliter and each cytokine measured value were expressed as mean ± standard deviation. The statistical differences between each group were analyzed using analysis of variance or the T-test where differences had significance when P ≤ 0.05.

Ethics Statement
The animals were handled according to the national criterion for animal investigation of China (Ethics Review Committee
The growth of different bio-serotype strains in vitro or in vivo was different. Comparing the growth curves of the four strains in vitro, we found that 2/O: 9 strains had statistically significant higher growth than 3/O: 3 strains (Figures 1A,B), either in single culture or mixed cultures. However, the growth curves of four strains in vivo were inverse, 3/O: 3 grew statistically significantly higher than the 2/O: 9 (Figures 1C,D), either in single infection or mixed infection.

DISCUSSION
In our study, the competitive growth of the two bio-serotype strains were totally different, the 3/O: 3 grew greater than 2/O: 9 in vivo; conversely, the 2/O: 9 grew greater than 3/O: 3 in vitro. This suggested the 2/O: 9 grew better without a host; however, the 3/O: 3 had stronger resistance to the defense mechanisms of the hosts. Therefore, the clearance of the 3/O: 3 was slower than the 2/O: 9 in BALB/C mice; and might reflected the different pathogenic abilities. This showed the higher resistant ability of 3/O: 3 to host clearance than the 2/O: 9, and might be a possible explanation for distribution of the two strains in human hosts.
Cytokines participate in cellular immunity, humoral immunity, hematogenesis regulation, cell proliferation, and injury repair; however, excess cytokine secretion leads to immunopathogenic effects. Some research showed excess cytokines induced host death after infections (Hancock et al., 1986;Segura et al., 1999;Smith et al., 2008). Currently, the studies on different serotypes of Y. enterocolitica that cause different secretions of cytokines were shown (McNally et al., 2006). In our study here, the Yersinia highly susceptible BALB/C mice were used instead of cells (Autenrieth and Heesemann, 1992). Cytokines involved in this study, IL-6, IL-8, MCP-1, MIP-1α, and RANTES in a relatively short period of time reach their peaks in both the two control groups and the experimental group. These cytokines were inflammatory cytokines. This was consistent with the reported literature: Yersinia infection of epithelium (HeLa) cells can significantly enhance the levels of transcription and secretion of inflammatory cytokines MCP-1, GM-CSF, and TNF-α. One hour after infection transcript levels of these cytokines began to increase and within 3-4 h reached their peak and then gradually decline (Kampik et al., 2000). These cytokines played important roles in the hosts' immune response against Y. enterocolitica and the cytokine changes for Y. enterocolitica infection were similar using cell infected models or animal infected models.
Previous studies showed the cytokine values of highly pathogenic Y. enterocolitica were higher than the lower pathogenic strains . Brucella and 2/O: 9 bioserotype Y. enterocolitica caused different cytokine changes, involving their different pathogenic ability and life cycles . However, in our study lower pathogenic Y. enterocolitica had similar cytokine changes compared with 3/O: 3 and 2/O: 9 strains. Therefore, the similar cytokine changes were caused by analogical pathogenic ability of the Y. enterocolitica strains. The different pathogenic abilities or life cycles were important for cytokine changes in Y. enterocolitica infections. The cytokines induced by two kinds of strains were similar, while the growth conditions were different; maybe this reflected the different pathogenic ability or defense ability to hosts, and it may as one of the reason for bacteria shift.