1 TITLE : An alternative method for cultivation of

23 An alternative method for cultivation of Lawsonia intracellularis, an obligate intracellular 24 bacterium and causative agent of proliferative enteropathy, was described using Original Space 25 Bag® inflated with a mixture of gas containing 10% hydrogen, 10% carbon dioxide, and 80% 26 nitrogen. The flexibility of this protocol allows for the testing of various environmental 27 conditions for static cultivation of this bacterium and the development of diagnostic techniques. 28 29 TEXT 30 Lawsonia intracellularis is a fastidious and obligate intracellular bacterium and the causative 31 agent of proliferative enteropathy or ileitis. The disease has been reported in a variety of animal 32 species including nonhuman primates, but it has been best described in hamsters, pigs and horses 33 (2). Dividing eukaryotic cells in culture and strict environmental conditions are required for 34 isolation and cultivation of L. intracellularis in vitro (6). The conventional method for isolation 35 and cultivation of this bacterium in monolayer is well established using various methods of 36 supplying hydrogen for infection followed by incubation in a Tri-gas incubators with 83.2% 37 nitrogen, 8.8% carbon dioxide and 8% oxygen at 37C (3, 8). The cost of these requirements has 38 limited the maintenance of this microorganism in vitro to only a few research institutes. 39 Furthermore, since this disease was first reported (1), there have been only a dozen or so cultured 40 L. intracellularis isolates worldwide so far. This study describes an alternative method for 41 cultivation of L. intracellularis in cell monolayers providing necessary atmospheric conditions 42 for growth without Tri-gas incubators. This alternative protocol presents new opportunities for 43 testing different environmental conditions for isolation and cultivation of this organism. 44 Additionally, it also allows the development of diagnostic approaches including 45 on Jauary 6, 2018 by gest ht://jcm .sm .rg/ D ow nladed fom immmunoperoxidase monolayer assay, indirect immunofluorescence and minimum inhibitory 46 concentration. 47 48 The L. intracellularis isolate PHE/MN1-00 (ATCC PTA-3457) previously isolated from a pig 49 with the hemorrhagic form of proliferative enteropathy was used for evaluating its growth and in 50 vitro infection under two different environmental conditions (conventional and alternative). This 51 isolate was grown in murine fibroblast-like McCoy cells (ATCC CRL 1696), maintained in a cell 52 culture system and stored at -72°C until use, as described previously (3). Frozen bacteria were 53 thawed and grown in cell culture for three continuous passages in order to allow the bacteria to 54 recover from the frozen stage. In these three passages, the bacteria was grown using both the 55 conventional and alternative method (Figure 1), as described below. The infection was monitored 56 during every passage using immunoperoxidase staining with polyclonal antibody specific for L. 57 intracellularis (3). After three passages, 16-well, glass-bottom tissue culture plates containing 58 one-day-old McCoy (30% confluence) cells were infected (Day 0) with bacterial suspensions 59 containing approximately 10 L. intracellularis organisms/well. Murine fibroblast-like McCoy 60 cells were grown in Dulbecco’s Modified Eagles Medium (DMEM; Gibco Invitrogen 61 Corporation) with 1% Lglutamine (Gibco Invitrogen Corporation), 7% fetal bovine serum (FBS; 62 Sigma Chemical) and 0.5% amphotericin B (Cellgro; Mediatech), without antibiotics or medium 63 replacements throughout the study (3). The infected cells were incubated using two different 64 methods. For the conventional method, the cells were placed in the Tri-gas incubator with 83.2% 65 nitrogen gas, 8.8% carbon dioxide and 8% oxygen gas and a temperature of 37C (6). Tissue 66 culture plates were removed and flushed with hydrogen gas daily. For the alternative method, the 67 plates were placed in an Original Space Bag® (Storage Packs, San Diego, CA, USA, 68 on Jauary 6, 2018 by gest ht://jcm .sm .rg/ D ow nladed fom [http://www.spacebag.com]) measuring 54 cm x 85 cm which was hermetically closed. The air 69 inside the bag was then removed by vacuum pump to a pressure of 100 mm Hg. Afterward, the 70 bag was inflated through a cuff containing a 0.22 μm filter connected to a gas cylinder 71 containing 10% hydrogen, 10% carbon dioxide, and 80% nitrogen gas. Finally, the bag was 72 incubated at 37C for eight days (Figure 1). The atmosphere inside the bag was replaced as 73 described above every 24 hours. Carbon dioxide and oxygen gas percentages were monitored in 74 both protocols using CO2 and O2 indicators (FYRITE® Gas Analyzer) at the initiation of 75 incubation and every 24 hours during the eight days. The CO2 and O2 levels were demonstrated 76 to be stable in the bag when they were measured daily after inflating and before replacing the gas 77 mixture. The CyQuant® cell proliferation assay, a fluorescence-based approach for determining 78 numbers of cultured cells (5), was used to monitor the cell growth of infected and non-infected 79 cells for both incubation methods. In addition, the population doubling time (days) of the McCoy 80 cells was calculated using the algorithm provided by http://www.doubling-time.com (11), based 81 on the intensity of the fluorescence demonstrated in the CyQuant® assay. Infection and growth 82 monitoring of L. intracelluaris were performed by direct counting of heavily infected cells 83 (HIC), identified by immunocytochemistry staining with polyclonal antibody specific for L. 84 intracelluaris (3), and by quantitative PCR (qPCR), as previously described (10). The level of 85 infection was also monitored by calculating the estimated population doublings of the HIC, 86 which were previously counted. The number of IHC and the number of L. intracelluaris 87 organisms were quantified using four replicates (wells) in 16-well tissue culture plates. The 88 average from four replicates was used in the statistical analysis. Wilcoxon signed-rank test was 89 performed using SAS software (9.1) to assess differences between both incubation methods. A 90 value of p<0.05 was considered significant. 91 on Jauary 6, 2018 by gest ht://jcm .sm .rg/ D ow nladed fom During the eight days of incubation, the carbon dioxide and oxygen gas were constant (8.8% 92 CO2; 8.0% O2) in the Tri-gas incubator (conventional method). In the alternative method, CO2 93 and O2 levels ranged between 7.0-8.0% and 5.5-6.5%, respectively (Table 1). The CyQuant® cell 94 proliferation assay was used to measure cellular DNA via fluorescent dye binding in non95 infected and infected cells every 24 hours for eight days of incubation. Non-infected cells had 96 similar growth rates and no significant difference on average of estimated population doubling 97 (calculated by day) between the conventional protocol at 5% CO2/37oC (1.83+0.18), the Tri-gas 98 incubator (1.79+0.06) and the plastic bag (1.76+0.08). These results showed that non-infected 99 McCoy cells are able to be grown in these bags and able to support the cultivation of L. 100 intracelluaris. The bag described previously in this study was able to support up to 12 T25 101 (Figure 1) or six T175 tissue culture flasks (Corning®). However, we have observed that larger 102 bags from the same manufacturer support higher number of flasks. 103 104 Enterocyte proliferation is the primary lesion associated with L. intracelluaris infection in vivo 105 (2). Although previous experiments have not reported cellular proliferation in vitro (7, 9) to date, 106 there is no information regarding the cell growth during in vitro infection. Using the CyQuant® 107 cell proliferation assay, the growth curves of infected and non-infected cells were compared for 108 the conventional and alternative methods (Figure 2). The results did not show statistical 109 differences (p<0.05) between infected and non-infected in either incubation method. Oh et al 110 (2010) described up-regulation of cell cycle genes in infected McCoy cells; however, cell growth 111 was not measured in this experiment. Furthermore, epithelial growth factors and their 112 interactions with the lamina propria during in vivo infections can play a critical role in the 113 pathogenesis of the disease, which is still poorly understood. 114 on Jauary 6, 2018 by gest ht://jcm .sm .rg/ D ow nladed fom The number of heavily infected cells increased progressively and reached the peak on day seven 115 post-infection in both the conventional and alternative incubation methods. There was no 116 significant difference (p<0.05) in the number HIC throughout the days of incubation (Figure 3). 117 No significant difference was found in the estimated population doubling of HIC using the 118 conventional (2.8+0.2) and the alternative (3.1+0.3) protocols. Similar to the 119 immunocytochemistry results, the greatest number of L. intracellularis organisms per well was 120 observed on day seven post-infection by quantitative PCR (Figure 3). In addition, the 121 quantitative PCR showed no significant difference between the conventional and alternative 122 methods of incubation. There was no positive reaction for the non-infected cell cultures (negative 123 control) in the qPCR. Previous studies have described L. intracellularis cultivation in five to 124 seven days using a Tri-gas incubator, including experiments to validate diagnostic approaches 125 (4) and to investigate the pathogenesis of proliferative enteropathy (9). However, these studies 126 failed to quantify the numbers of L. intracellularis organisms or the number of HIC in the 127 infected cultures. 128 129 Similar to the conventional method, incubation in the bag provided environmental conditions 130 that enable L. intracellularis to infect and multiply in the cells. Based on these results, we 131 believe this approach can be used for the static cultivation and, potentially, isolation of this 132 bacterium without requiring a Tri-gas incubator. In addition, our experience has shown no 133 difference in the cell growth or level of the infection when the gas inside the bag is replaced at 134 least two times (on second and fifth days post-infection) throughout the period of incubation. 135 This alternative method has been also successfully reproduced (J. S. V. Oliveira and R. M. C. 136 on Jauary 6, 2018 by gest ht://jcm .sm .rg/ D ow nladed fom Guedes, unpublished data) in an independent trial. This fact has confirmed the usefulness and 137 feasibility of the present method. 138 The flexibility of this methodology allows for the testing of various environmental conditions for 139 L. intracelluaris cultivation and production of antigens for the development of diagnostic 140 techniques. Additionally, this affordable technology gives to research institutes an opportunity to141explore this bacterial proliferative disease, which has intriguing and unique properties among142bacterial pathogens.143144Acknowledgement145F. A. Vannucci was supported by the Brazilian government sponsoring agency “Conselho146 Nacional de Desenvolvimento Científico e Tecnológico” (CNPq).147148REFERENCES1491501. Biester, H. E. and L.H. Schwarte. 1931. Intestinal adenoma in swine. Am. J. Pathol.1517:175-185.1521532. Gebhart, C. J. and R. M.C. Guedes. 2010. Lawsonia intracellularis, p. 503-512. In C.154L. Gyles, J. F. Prescott, J. G. Songer and C. O. Thoen (ed.), Pathogenesis of bacterial155 infections in animals, 4th ed. Blackwell Publishing, Ames, Iowa.156157onJauary6,2018bygestht://jcm.sm.rg/Downladedfom 3. Guedes, R. M. C. and C. J. Gebhart. 2003. Preparation and characterization of158polyclonal and monoclonal antibodies against Lawsonia intracellularis. J. Vet. 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Carbon dioxide and oxygen gas levels during eight days of incubation in a conventional193Tri-gas incubator and the Original Space Bag®.194