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Characterization of late tuberculosis infection in Sigmodon hispidus
Tuberculosis 89 (2009) 183–188
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Tuberculosis journal homepage: http://intl.elsevierhealth.com/journals/tube
Characterization of late tuberculosis infection in Sigmodon hispidus Robert L. Elwood a, *, Michael Rajnik a, Samuel Wilson a, Kevin Yim c, Jorge C.G. Blanco c, Boris Nikonenko b, Val G. Hemming a, c a
Department of Pediatrics, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, USA Sequella, Inc., 9610 Medical Center Drive, Suite 200, Rockville, MD 20850, USA c Virion Systems, Inc., 9610 Medical Center Drive, Suite 100, Rockville, MD 20850, USA b
a r t i c l e i n f o
s u m m a r y
Article history: Received 26 August 2008 Received in revised form 12 January 2009 Accepted 14 January 2009
We previously described primary tuberculosis in Sigmodon hispidus cotton rats up to 6 months following a pulmonary challenge. At that time, we observed fewer animals demonstrating disease as time from exposure progressed. We hypothesized that some cotton rats may control a primary infection to latency in a similar fashion to humans. The current experiment was designed to examine the natural progression of disease in S. hispidus at a later timepoint following a respiratory challenge with Mycobacterium tuberculosis (Mtb). An additional objective was to test whether cotton rats may become latently infected, and to determine whether latent disease might be activated by cyclophosphamide induced immune suppression. Thirty-four percent of the inoculated cotton rats died prior to 9 months following the challenge. However, 50% of immunocompetent animals surviving past 9 months demonstrated positive lung tissue cultures for Mtb without histologic evidence of disease. None of the immunosuppressed animals demonstrated this pattern. These findings are consistent with the development of latent tuberculosis infection in some cotton rats. Furthermore, it appears reactivation of disease occurs with cyclophosphamide induced immunosuppression. Cotton rats may serve as a model for latent as well as active tuberculosis infection. Published by Elsevier Ltd.
Keywords: Mycobacterium tuberculosis Latency LTBI Cotton rats Sigmodon hispidus
1. Introduction Disease due to Mycobacterium tuberculosis (Mtb) is a leading cause of infectious death worldwide.1,2 Studies examining the pathogenesis of Mtb disease have used several animal species including guinea pigs, mice, rabbits, and non-human primates. However, none has proved an ideal model of human Mtb disease as the pathology associated with Mtb in most small animal models does not closely approximate human tuberculosis.3,4 The American cotton rat (Sigmodon hispidus) was briefly tested as a model for Mycobacterium bovis infection more than half a century ago.5,6 We have recently demonstrated that cotton rats develop primary pulmonary infection when given a low dose respiratory challenge of human Mtb. Our initial experiments followed infected cotton rats for 6 months after respiratory challenge with Mtb. We observed over time that some infected cotton rats resolved their infections.7 We could not determine whether these
* Corresponding author. Department of Pediatric Infectious Disease, Wilford Hall Medical Center, 2200 Bergquist, Suite 1, Lackland AFB, TX 78236, USA. Tel.: þ1 202 782 9774; fax: þ1 301 295 3898. E-mail address: [email protected] (R.L. Elwood). 1472-9792/$ – see front matter Published by Elsevier Ltd. doi:10.1016/j.tube.2009.01.003
animals had fully eradicated Mtb or whether they might have latent infection. The current study characterizes the natural history of Mtb disease in S. hispidus up to 10 months after respiratory challenge. We also tested whether these animals controlled Mtb infection to latency and whether latent infection could be reactivated by aggressive immune suppression. In this study we define latency as positive lung and/or spleen culture with no gross or microscopic evidence of granuloma formation or detectable acid-fast organisms. 2. Materials and methods 2.1. Animals Female cotton rats (Sigmodon hispidus), between 6 and 8 weeks old, were obtained from inbred colonies maintained at Virion Systems, Inc. (Rockville, MD). These cotton rats were sib-mate inbred up to 25 generations at the Veterinary Resources Branch, National Institutes of Health (Bethesda, MD). An additional 45 generations of inbreeding was carried out by Virion Systems, Inc. from this stock colony obtained from NIH. Genetic variability in the colony after inbreeding for a total of 70 generations would be insignificant to the outcome of the study. Inbred cotton rats from
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this colony have been made available to two commercial vendors in the US (Harlan and Ace animals). Breeding colony cotton rats maintained at Virion Systems, Inc. are seronegative for adventitious viruses, rodent and murine pathogens. Live animal submissions are sent quarterly and serology is conducted every 6 weeks as part of a sentinel program. All the animals used in this study were in good health and free of other pathogens to the best of our knowledge. They were singly housed in large polycarbonate rat cages with filter tops to prohibit any animal–animal transmission. They were given paper mill and fed a diet of rodent chow and water throughout the experiments. All animals were weighed monthly and examined daily for signs of clinical illness. This experiment was performed under a protocol approved by our Institutional Animal Care and Use Committee in accordance with the accepted guidelines. 2.2. Bacteria Mycobacterium tuberculosis strain H37Rv, obtained from JoAnne Flynn (University of Pittsburgh School of Medicine, Pittsburgh, PA) was used for the inoculations as in our prior experiments. This strain has been previously passaged in mice. Stock inoculum was prepared as previously described and stored in 1.5 mL ampules in a 70 C freezer until needed.7 The titer of the stored ampules was 3.9 106 CFU/mL. All preparations and handling of the bacteria, animal inoculation, sacrifice and dissection were done in a biosafety hood. 2.3. Infection BSL-3 facilities that would accommodate aerosolization of Mtb were not available. Therefore, intranasal inoculations were used in this experiment to introduce Mtb into the lower respiratory tract of the cotton rats. Frozen ampules containing H37Rv were thawed, vortexed and diluted 1:100 in sterile PBS. Next, the suspension was briefly sonicated to disperse aggregated bacteria. Animals were anesthetized with ketamine injected intramuscularly, and a volume of 0.1 mL of inoculum was instilled in the nares (50 mL per nare). Forty-seven animals were inoculated in this manner and recovered. 2.4. Histology Animals were euthanized by CO2 narcosis. Lung tissues used for histologic evaluation were dissected en bloc, inflated through the trachea with 10% buffered formalin to their normal volume, and finally immersed into the formalin solution. Spleens were dissected in half, and one portion was prepared for histology by immersion in a 10% buffered formalin solution. Adrenal glands and liver sections were also collected in the 10% formalin solution. Sections of all formalin-fixed lungs, spleens, livers, and adrenal glands were stained with hematoxylin and eosin (H&E) to evaluate histopathology, and modified acid-fast tissue stain to look for the presence of acid-fast bacilli (AFB). Evaluation of the stained tissue sections was performed prior to identification of individual animals for correlation with clinical parameters and prior to tissue culture results being available. 2.5. Culture of organ tissues Lung and spleen organ tissues were aseptically removed from all cotton rats at the time of sacrifice. The entire lingular lobe of the lung of each animal was removed and homogenized for culture. Spleens were bi-sected with half of the organ being homogenized for culture and the other half prepared for histology. Serial 10-fold dilutions of homogenates prepared from the tissue samples were plated using sterile technique and incubated as we have previously
described.7 Colonies were counted after 18–21 days by an investigator that was blinded to clinical findings and the histological findings associated with any of the animals. Cultures were not obtained from animals that died prior to their planned day of sacrifice. This decision was based on the non-standardized time between death of the animal and necropsy as well as the inability to prepare culture media in a timely fashion on a case by case basis. 2.6. Immunosuppression Nine months after inoculation, all surviving animals were divided into two groups. One group served as infected controls. The second group was immune suppressed with intraperitoneal injections of cyclophosphamide. A calculated dose of 50 mg/kg of cyclophosphamide was injected three times per week. This dose was previously used in a study by Ottolini et al., and was noted to reliably induce leucopenia in cotton rats.8 Cyclophosphamide injections were given for 4 weeks, and then the animals were allowed to recover for 2 weeks. At the end of this period all remaining cyclophosphamide treated and infected control animals were sacrificed. 3. Results Forty-seven S. hispidus cotton rats were anesthetized, challenged with 3.9 103 CFU of human Mtb (H37Rv) by intranasal inoculation, and recovered. Five out of six animals sacrificed at 3 months following inoculation demonstrated histologic evidence of a primary tuberculosis infection with nodular lung lesions and pulmonary granulomas and with visible AFB. The animal without histologic evidence of disease also had a negative lung tissue culture. One animal with histologic evidence of disease had all lung tissue specimens placed in formalin preventing the ability to culture. The mean colony forming units (CFU) recovered from lung tissue culture in the remaining four animals was 1.98 105. Fourteen of 41 (34%) inoculated animals died prior to the 9 month time point at which immunosuppression was begun. Ten of these 14 (71%) demonstrated histologic evidence of active pulmonary tuberculosis infection. Following the group division, five of the 15 animals treated with cyclophosphamide died prior to the planned time of sacrifice, while only one of 12 of the infected control animals died before this time point. Most animals that died prior to their planned time of sacrifice appeared ill and wasted and uniformly had extensive pulmonary disease. Many had almost confluent pulmonary inflammation with few clear alveoli open for gas exchange. Gross examination of other organ systems at the time of necropsy was normal. However, we did not systematically evaluate for all forms of extrapulmonary active disease (including bowel, mesentery, kidney or central nervous system) in these animals. These should be evaluated in future experiments. To repeat, in this study, we defined a latent tuberculosis infection as a positive lung and/or spleen tissue culture together with the absence of histologic evidence of pulmonary or splenic disease. Data obtained from the animals that survived past the time of separation into cyclophosphamide treated and infected control groups are presented in Table 1. Animals that died prior to the planned day of sacrifice did not have tissue culture for Mtb performed. This included five animals in the cyclophosphamide treated group and one animal in the infected control group. Once cyclophosphamide injections began, these animals were counted as not having latent infection if there was histologic evidence (granulomas with AFB) of active Mtb disease. Twelve animals were included in the infected control group. Six of these animals fulfilled our definition for latent Mtb infection. Careful examination revealed no visible areas of inflammation
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Table 1 Results from S. hispidus cotton rats after 9–10 months following respiratory Challenge with M. tuberculosis.
Cyclophosphamide treated (10 animals survived until planned sacrifice)
(5 animals died prior to planned sacrifice)
Infected controls (11 animals survived until planned sacrifice)
(1 animal died prior to planned sacrifice)
Peak Wt/death Wt (g)
% Wt loss
Quantitative Mtb cultures (CFU/mL)* Lung
Spleen
110/90 115/100 110/80 135/135 125/125 145/145 145/145 120/120 145/145 155/155 115/100 135/80 95/90 90/70 110/100 140/130 140/140 135/135 145/145 140/140 145/145 140/140 150/150 140/140 135/135 135/135 130/130
18.18 13.04 27.27 0 0 0 0 0 0 0 13.04 40.74 5.26 26.32 9.08 7.14 0 0 0 0 0 0 0 0 0 0 0
1.1 108 1.0 109 1.0 109 2.0 105 5.3 107 NG NG NG NG NG
2.4 105 4.0 106 1.6 105 1.3 102 6.0 104 NG NG NG NG NG
2.0 107 2.7 107 6.0 106 8.0 102 9.3 102 1.3 102 4.0 102 6.6 102 2.7 102 NG NG
1.0 105 9.6 103 contaminated 6.0 102 NG NG NG NG NG NG NG
Description of pulmonary histologyy
Patchy sml granulomas, few AFB Confluent lg granulomas, abundant AFB Patchy mod granulomas, abundant AFB Patchy mod granulomas, few AFB Patchy sml granulomas, mod AFB No granulomas, no AFB No granulomas, no AFB No granulomas, no AFB No granulomas, no AFB No granulomas, no AFB Confluent lg granulomas, abundant AFB Confluent lg granulomas, abundant AFB Patchy mod granulomas, abundant AFB Patchy mod granulomas, mod AFB No granulomas, no AFB Patchy mod granulomas, mod AFB Patchy sml granulomas, few AFB Patchy mod granulomas, mod AFB No granulomas, no AFB No granulomas, no AFB No granulomas, no AFB No granulomas, no AFB No granulomas, no AFB No granulomas, no AFB No granulomas, no AFB No granulomas, no AFB Patchy sml granulomas, few AFB
* Animals that died prior to the date of planned sacrifice but after immunosuppression had begun were evaluated for histology but did not have cultures performed. NG ¼ No growth. y Lung, spleen, liver and adrenal glands were also evaluated. Of note, no animals demonstrated evidence of Mtb infection in extrapulmonary organs without also demonstrating evidence of Mtb infection within the lungs.
despite the low level positive cultures. Three animals in this group demonstrated active Mtb disease with both positive cultures and histologic evidence of pulmonary disease while one animal had positive histologic evidence of disease but cultures were not obtained. The remaining two animals in this group appeared to be free of infection (negative cultures and histology). Geometric means of the colony forming units (CFU) of Mtb recovered from lung tissue culture of cotton rats were calculated and compared with a one-way ANOVA followed by Tukey’s post-hoc tests. The geometric mean CFU recovered from animals with latent Mtb infection was 4.36 102 (95% CI ¼ 1.98 102–9.59 102). The geometric mean CFU of Mtb recovered from culture in cotton rats from the infected control group with active disease was 1.48 107 (95% CI ¼ 2.05 106–1.07 108). This difference was significant (p < 0.001). Fifteen animals began receiving cyclophosphamide injections at 9 months after inoculation. Nine of these animals had histologic evidence of active pulmonary disease. Most demonstrated severe pulmonary disease including granulomas with central necrosis and abundant AFB. Five animals in this group appeared to be free of disease, and zero animals fulfilled our definition of latent Mtb infection. A single animal in the cyclophosphamide treated group that died prematurely was without histologic evidence of Mtb disease. This animal was excluded from calculations because no culture data was available. The geometric mean CFU of Mtb recovered from lung tissue culture in cotton rats with active disease in the cyclophosphamide treated group was 6.51 107 (95% CI ¼ 8.54 105–4.96 109). The difference in geometric mean CFU between this group of animals and those from the infected control group demonstrating latent infection was significant (p < 0.001). The difference in geometric mean CFU among the animals demonstrating active disease in the cyclophosphamide treated and
infected control group was not significant (p ¼ 0.636). Table 2 provides further statistical analysis of the histopathology and culture data from all animals that survived until the groups were divided at 9 months when immunosuppression began. Figure 1 illustrates comparative gross and pulmonary histopathology results from cotton rats with noted active disease in the immunosuppressed and infected control groups as well as a cotton rat in the infected control group with latent disease. The mean peak weight of cyclophosphamide treated animals with active disease was 115 g while their death weight was 97 g. This represents a mean 16% weight loss (range ¼ 5–41%). The mean peak weight of infected control animals with active disease was 136 g. A single animal in this group demonstrated a 7% weight loss. There was no weight loss in any of the animals that demonstrated latent disease or the absence of infection. In most cases, the weight loss correlated with degree of pulmonary disease as is shown in Table 1. Granulomas with visible AFB were seen in the liver in a single evaluated specimen from an animal that died prior to the initiation of immunosuppression. Histology of liver and adrenal gland specimens were evaluated along with the lungs and spleens in all animals that remained to the day of final sacrifice. Granulomas with abundant AFB were noted in the liver and an adrenal gland in one animal that underwent immunosuppression. None of the infected control animals evaluated at 10 months from the time of inoculation demonstrated evidence of Mtb in the adrenals. Figure 2 illustrates some of the extrapulmonary histopathology seen. 4. Discussion Cotton rats have several advantages over other animals used in Mtb research. Compared to larger animals, cotton rats are
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Table 2 Comparison of immunosuppressed vs. immunocompetent S. hispidus cotton rats at 10 months following challenge with Mycobacterium tuberculosis.
Positive tissue culturesy Lung Spleen Positive histologic evidence of disease Latent infection (positive culture þ negative histology)y
P*
Cyclophosphamide treated
Infected controls
5/10 (50%) 5/10 (50%) 9/15 (60%)
9/11 (82%) 0.18 3/10 (30%) 0.65 4/12 (33%) 0.25
0/14 (0%)
6/12 (50%) 0.004
Statistical difference were compared by 2-sided Fischer’s exact test. Animals that died prior to the date of planned sacrifice but after immunosuppression had begun were evaluated for histology but did not have cultures performed. * y
inexpensive, and easy to handle and maintain in large groups. Also, a wide variety of primers and other reagents are available to study the immune response in cotton rats.9,10 We have previously shown that compared to other rodent models, cotton rats develop a primary Mtb infection that histologically appears more similar to primary Mtb disease in humans following a respiratory exposure. In the first few months following exposure, Mtb infected cotton rats develop granulomas, often with caseating necrosis, and show evidence of spread to lymphoid organs.7 However, little was previously known about the natural progression of the infection after 6 months.
The normal lifespan of a cotton rat is between 12 and 15 months. About a third of the cotton rats died prior to their planned time of sacrifice (at about 11 months of age). Seventy-one percent of these animals demonstrated histologic evidence of active pulmonary Mtb disease. While some degree of attrition not attributable to the infection is expected in an experiment lasting this long, we believe that Mtb contributed to, if not caused, the early death of many of these animals. We have made previous attempts to evaluate for latency and reactivation of Mtb disease in cotton rats. The first attempt is an unreported administration of steroids between 4 and 6 months following challenge. We noted more severe disease in the steroid treated animals. We next used cyclophosphamide to induce immunosuppression in cotton rats between 6 and 10 months after the challenge. Trends of clinically latent Mtb infection were noted, but the number of animals was too small to reach statistical significance.11 It is difficult to study latency in the pathogenesis of Mtb. While it is widely accepted that humans become latently infected with Mtb, there is no agreed upon standard of defining latency. It is usually diagnosed by demonstrating a likely exposure to the Mtb bacillus (usually by an immunologic response to Mtb antigens) along with the absence of active disease. True latent tuberculosis infection (LTBI) can be easily misdiagnosed. It is unknown how lung tissue culture for Mtb in humans correlates with a diagnosis of LTBI. In the cotton rat model, we believe that histological evidence for
Figure 1. Gross lung, H & E, and modified acid-fast stains from cotton rats sacrificed at 10 months following respiratory challenge with Mtb. Level of magnification is labeled on the photomicrographs. A, B, and C demonstrate severe disease with large tubercules, expanding granulomas with mild necrosis and many AFB in a cotton rat that received cyclophosphamide injections for 4 weeks followed by 2 weeks of recovery. D, E, and F demonstrate small tubercules, granulomas with central necrosis and calcification and several AFB in a cotton rat that did not receive cyclophosphamide but had high recoverable CFU of Mtb on lung tissue culture consistent with active disease. G, H, and I demonstrate normal appearing lungs by gross and histologic examination in a cotton rat that did not receive cyclophosphamide and had low recoverable CFU of Mtb on lung tissue culture consistent with latent disease.
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Figure 2. Multiple acid-fast organisms (red) found in: (A) liver, (B) spleen, and (C) adrenal gland of a cotton rat with disseminated disease following 4 weeks of cyclophosphamide injections and 2 weeks of recovery. Magnification levels are indicated.
pulmonary granulomas and visible AFB are better measures for active disease than is culture of tissues because we can examine the entire lung for areas of inflammation that denote a focus where there is an active immune response to the invading organism. However, a positive tissue culture certainly confirms the presence of viable Mtb. Therefore, the finding of a positive lung tissue culture, in the absence of granulomatous inflammation can logically be assessed to be a latent infection as per our definition. While we have defined the diagnosis of latency in the cotton rat differently than the manner it is diagnosed in humans, other recognized animal models of LTBI such as the Cornell mouse model also define LTBI in a different manner.12,13 Although there were no grossly abnormal lesions within the abdomen or elsewhere in cotton rats without evidence of active spleen or lung disease, we concede that we cannot rule out the possibility of extrapulmonary active TB disease (in organ systems that we did not fully evaluate such as the bowel, CNS, skeleton, or lymph nodes) in the cotton rats that met our definition of latent disease. However, latent disease seems more likely in these animals when you consider that the immunosuppressed group demonstrated either obvious pulmonary TB disease or no disease at all. Furthermore, it is possible that animals that appeared to be uninfected (negative histology and negative cultures) might have grown Mtb from tissue cultures were it possible to perform both histologic evaluation and culture of each entire lung specimen at the same time. In this case, our calculations would underestimate the true number or latently infected cotton rats. Two murine models of latent Mtb infection have been previously described. The Cornell mouse model was first reported in the 1950’s, and several variations followed.12,13 In this mouse model, Mtb was inoculated intravenously. Latency was defined as negative Mtb cultures following treatment with isoniazid and pyrazinamide. However, week’s later cultures again became positive with organisms that were still sensitive to treatment with these drugs. Similar to the cotton rat, these mice demonstrated low titers of organisms. However, unlike our model, this state of latency was artificially induced. Other murine models of LTBI describe an equilibrium of chronic disease with higher titers (w3–4 Log10) of cultureable Mtb.14 This is believed to be dissimilar to the number of bacilli present in human LTBI. Other animals that have been evaluated for latency include rabbits and non-human primates. While cynomolgus macaques may closely resemble the variations of Mtb infections seen in humans including the ability to skin test with PPD, they are much more expensive and require more specialized training and facilities to use.15 Recently, New Zealand white rabbits have also been described to demonstrate Mtb disease including latency and spontaneous reactivation that is more similar to humans than the murine models.16 However, rabbit models are restricted by the lack of inbreeding and absence of immunologic reagents such as are available for both mice and cotton rats.
There are two prevailing theories as recently described by Cardona and Ruiz-Manzano for how Mtb maintain latency in lung parenchyma that is dynamic in nature.14 One suggests that the bacteria are constantly disseminating and reactivating at a low level but are kept in check by the host’s immune system. The second purports that bacteria are dormant inside necrotic material of a granuloma, where the movement of macrophages would be limited until it is slowly reabsorbed. Our cotton rats with latent disease had positive lung tissue cultures from lingular lung lobes but were grossly and microscopically normal. Our findings are most consistent with the theory that a low level dissemination resulted in Mtb being hematogenously deposited in this part of the lung. An interesting difference from our previous report is that at later time points, positive lung tissue cultures exceeded positive spleen cultures whereas the opposite is true earlier in the course of disease. Our data show that once infection is controlled to latency low levels of viable organisms remain in the absence of detectable pulmonary or splenic inflammation. It is unclear where these organisms reside. We expect to examine this question in subsequent experiments. Although there was not a discernible pattern of predilection for any part of the lungs in the animals with active disease (such as the upper lobes as in humans), it is important to note that there is concordance between the histopathology and quantitative culture results in an all or nothing pattern. That is, when granulomas are found, there are also positive lung cultures, usually with large numbers of bacteria. When the lungs contain no evident granulomas, counts are either negative, or very low, as in our latent cases. An alternate explanation for the animals that were found to have both negative cultures and histological examinations is that they either never became infected or completely cleared their infection. Similarly humans do not always develop active or latent infection following a single exposure. We acknowledge that the intranasal instillation of organisms is less ideal than aerosolization to deliver the inoculum to lungs. Also, personal communications with other researchers working with cotton rats from the same colony but with different strains of Mtb indicate a higher level of resistance to infection than what we have noted. Because of this, we believe that the virulence of the bacterial strain of Mtb is an important variable in this model. Approximately one-third of the cotton rats that developed a primary Mtb infection eventually succumbed to the disease between 4 and 10 months following challenge. Many of these animals demonstrated severe pulmonary disease. However, half of the remaining animals that were allowed to progress naturally demonstrated a latent infection that is characterized by positive lung tissue culture (with a low recoverable CFU of Mtb) but the absence of any histological evidence of active infection. Furthermore, because cyclophosphamide treated cotton rats demonstrated either the absence of any infection or active pulmonary infection,
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we conclude that it is possible to reactivate the Mtb infection with immune suppression. Immune suppressed cotton rats develop more severe Mtb disease. This is clinically demonstrated by increased weight loss and a wasted appearance. AFB were seen in an adrenal gland of one immune suppressed animal. Addison’s disease is described in patients with adrenal tuberculosis, and the immune suppressed cotton rat model may present an opportunity to study adrenal function in tuberculous adrenalitis, but further characterization and development of this model would be needed. Limitations of this model of latent tuberculosis infection include a high degree of attrition and different progression of disease in populations of cotton rats (some with active infection, some with latent infection, and some without infection). The latter limitation would make it important to discover a way to differentiate the groups prior to sacrifice in order to better utilize the model for future study. We have previously reported that tuberculosis skin testing was unreliable in these animals.7 Earlier experiments evaluated skin testing of cotton rats in the footpads (an area of thick skin). While no overt reactions were evident, histologic evaluations demonstrated lymphocytic infiltrates in the area. However, this required amputation of the limb at necropsy to evaluate. Animals reported in our previous paper were tested in a shaved area on the back. We noted three animals with overtly positive reactions, but other animals with confirmed TB disease had no reaction. The skin in this area is very thin, and it is difficult to ensure that PPD is placed intradermally and not subcutaneously. Therefore, we determined it not worthwhile to pursue further skin testing by this method. Future studies should evaluate the utility of quantitative interferon-gamma release assays to confirm infection and radiography to evaluate for active disease. Our results, while preliminary, now justify the expense and logistical difficulty associated with these types of studies. The way in which cotton rats control Mtb infection to latency and may have reactivation of disease induced is unique among small animal models of tuberculosis disease. This model may need further development, and many new questions now arise. For example, it would be interesting to see the effects of newer immune modulators such as TNF inhibitors on the progression or reactivation of disease in the cotton rat. However, it is clear that cotton rats combine many of the advantages individually found in other animal models. This makes cotton rats a useful tool to study many aspects of tuberculosis disease including mycobacterial vaccinology. Our follow up studies will evaluate the effect of Bacille Calmette–Gue´rin on the natural history of tuberculosis in cotton rats. Acknowledgment We would like to thank David Porter MD, of the Department of Pathology and Laboratory Medicine at the University of California at Los Angeles, for assisting in histopathologic interpretation, and Cara Olsen at the Biostatistics Consulting Center, Uniformed Services University, for help with statistical analysis. Funding: None of the authors associated with this project have a commercial or other association that might pose a conflict of interest.
This study was supported in part by Uniformed Services University of the Health Sciences research grant GS86FC and corporate funds from Virion Systems, Incorporated. Competing interests: Experiments reported here were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals published by the Institute of Laboratory Animal Resources, National Research Council, National Academy Press, 1996. The opinions and assertions presented here are the private ones of the authors and are not to be construed as official or as reflecting the view of the Uniformed Services University of the Health Sciences or the Department of Defense. Ethical approval: Not required. References 1. Cegielski J, Chin DP, Espinal MA, Frieden TR, Rodriguez CR, Talbot DE, et al. The global tuberculosis situation. Progress and problems in the 20th century, prospects for the 21st century. Infect Dis Clin North Am 2002;16:1–158. 2. Grange JM, Zumla A. Paradox of the global emergency of tuberculosis. Lancet 1999;353:996. 3. Gupta UD, Katoch VM. Animal models of tuberculosis. Tuberculosis 2005;85: 277–93. 4. Dannenberg Jr AM, Collins FM. Progressive pulmonary tuberculosis is not due to increasing numbers of viable bacilli in rabbits, mice and guinea pigs, but is due to a continuous host response to mycobacterial products. Tuberculosis (Edinb) 2001;81:229–42. 5. Steinbach MM, Duca CJ. Experimental tuberculosis in the cotton rat. Proc Soc Exp Biol and Med 1940;44:288–90. 6. Daigeler A. Die baumwollratte (Sigmodon hispidus hispidus) als Versuchstier bei der tuberkulosediagnostik. Zeitschrift fu¨r Hygiene und Infektionskrankheiten; Medizinische Mikrobiologie. Immunol Virol 1952;135:588–91. 7. Elwood RL, Wilson S, Blanco JCG, Yim K, Pletneva L, Ninkonenko B, et al. The American cotton rat: a novel model for pulmonary tuberculosis. Tuberculosis 2007;87:145–54. 8. Ottolini MG, Curtis SR, Mathews A, Ottolini SR, Prince GA. Palivizumab is highly effective in suppressing respiratory syncytial virus in an immunosuppressed animal model. Bone Marrow Transplant 2002;29:117–20. 9. Blanco JCG, Pletneva L, Boukhvalova M, Richardson JY, Harris KA, Prince GA. The cotton rat: an underutilized animal model for human infectious diseases can now be exploited using specific reagents to cytokines, chemokines, and interferons. J Interferon Cytokine Res 2004;24(1):21–8. 10. Blanco JCG, Richardson JY, Darnell ME, Rowzee A, Pletneva L, Porter DD, et al. Cytokine and chemokine gene expression after primary and secondary respiratory syncytial virus infection in cotton rats. J Infect Dis 2002;185(12): 1780–5. 11. Elwood RL, Hemming VG. Characterization of pulmonary tuberculosis in cotton rats over 12 months [abstract 8428.8]. In: Programs and abstracts of the 2007 annual meeting of the Pediatric Academic Societies (Toronto, Canada) May 5–8, 2007. 12. McCune RM, Tompsett R. Fate of Mycobacterium tuberculosis in mouse tissues as determined by the microbial enumeration technique. I. The persistence of drug-susceptible tubercle bacilli in the tissues despite prolonged antimicrobial therapy. J Exp Med 1957;104:737–62. 13. McCune RM, Tompsett R, McDermott W. The fate of Mycobacterium tuberculosis in mouse tissues as determined by the microbial enumeration technique. II. The conversion of tuberculous infection to the latent state by the administration of pyrazinamide and a companion drug. J Exp Med 1957;104:763–802. 14. Cardona PJ, Ruiz-Manzano J. On the nature of Mycobacterium tuberculosis-latent bacilli. Eur Respir J 2004;24:1044–51. 15. Capuano III SV, Croix DA, Pawar S, Zinovik A, Myers A, Lin PL, et al. Experimental Mycobacterium tuberculosis infection of cynomolgus macaques closely resembles the various manifestations of human M. tuberculosis infection. Infect Immun 2003;71(10):5831–44. 16. Manabe YC, Kesavan AK, Lopez-Molina J, Hatem CL, Brooks M, Fujiwara R, et al. The aerosol rabbit model of TB latency, reactivation and immune reconstitution inflammatory syndrome. Tuberculosis 2008;88:187–96.
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Tuberculosis journal homepage: http://intl.elsevierhealth.com/journals/tube
Characterization of late tuberculosis infection in Sigmodon hispidus Robert L. Elwood a, *, Michael Rajnik a, Samuel Wilson a, Kevin Yim c, Jorge C.G. Blanco c, Boris Nikonenko b, Val G. Hemming a, c a
Department of Pediatrics, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, USA Sequella, Inc., 9610 Medical Center Drive, Suite 200, Rockville, MD 20850, USA c Virion Systems, Inc., 9610 Medical Center Drive, Suite 100, Rockville, MD 20850, USA b
a r t i c l e i n f o
s u m m a r y
Article history: Received 26 August 2008 Received in revised form 12 January 2009 Accepted 14 January 2009
We previously described primary tuberculosis in Sigmodon hispidus cotton rats up to 6 months following a pulmonary challenge. At that time, we observed fewer animals demonstrating disease as time from exposure progressed. We hypothesized that some cotton rats may control a primary infection to latency in a similar fashion to humans. The current experiment was designed to examine the natural progression of disease in S. hispidus at a later timepoint following a respiratory challenge with Mycobacterium tuberculosis (Mtb). An additional objective was to test whether cotton rats may become latently infected, and to determine whether latent disease might be activated by cyclophosphamide induced immune suppression. Thirty-four percent of the inoculated cotton rats died prior to 9 months following the challenge. However, 50% of immunocompetent animals surviving past 9 months demonstrated positive lung tissue cultures for Mtb without histologic evidence of disease. None of the immunosuppressed animals demonstrated this pattern. These findings are consistent with the development of latent tuberculosis infection in some cotton rats. Furthermore, it appears reactivation of disease occurs with cyclophosphamide induced immunosuppression. Cotton rats may serve as a model for latent as well as active tuberculosis infection. Published by Elsevier Ltd.
Keywords: Mycobacterium tuberculosis Latency LTBI Cotton rats Sigmodon hispidus
1. Introduction Disease due to Mycobacterium tuberculosis (Mtb) is a leading cause of infectious death worldwide.1,2 Studies examining the pathogenesis of Mtb disease have used several animal species including guinea pigs, mice, rabbits, and non-human primates. However, none has proved an ideal model of human Mtb disease as the pathology associated with Mtb in most small animal models does not closely approximate human tuberculosis.3,4 The American cotton rat (Sigmodon hispidus) was briefly tested as a model for Mycobacterium bovis infection more than half a century ago.5,6 We have recently demonstrated that cotton rats develop primary pulmonary infection when given a low dose respiratory challenge of human Mtb. Our initial experiments followed infected cotton rats for 6 months after respiratory challenge with Mtb. We observed over time that some infected cotton rats resolved their infections.7 We could not determine whether these
* Corresponding author. Department of Pediatric Infectious Disease, Wilford Hall Medical Center, 2200 Bergquist, Suite 1, Lackland AFB, TX 78236, USA. Tel.: þ1 202 782 9774; fax: þ1 301 295 3898. E-mail address: [email protected] (R.L. Elwood). 1472-9792/$ – see front matter Published by Elsevier Ltd. doi:10.1016/j.tube.2009.01.003
animals had fully eradicated Mtb or whether they might have latent infection. The current study characterizes the natural history of Mtb disease in S. hispidus up to 10 months after respiratory challenge. We also tested whether these animals controlled Mtb infection to latency and whether latent infection could be reactivated by aggressive immune suppression. In this study we define latency as positive lung and/or spleen culture with no gross or microscopic evidence of granuloma formation or detectable acid-fast organisms. 2. Materials and methods 2.1. Animals Female cotton rats (Sigmodon hispidus), between 6 and 8 weeks old, were obtained from inbred colonies maintained at Virion Systems, Inc. (Rockville, MD). These cotton rats were sib-mate inbred up to 25 generations at the Veterinary Resources Branch, National Institutes of Health (Bethesda, MD). An additional 45 generations of inbreeding was carried out by Virion Systems, Inc. from this stock colony obtained from NIH. Genetic variability in the colony after inbreeding for a total of 70 generations would be insignificant to the outcome of the study. Inbred cotton rats from
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this colony have been made available to two commercial vendors in the US (Harlan and Ace animals). Breeding colony cotton rats maintained at Virion Systems, Inc. are seronegative for adventitious viruses, rodent and murine pathogens. Live animal submissions are sent quarterly and serology is conducted every 6 weeks as part of a sentinel program. All the animals used in this study were in good health and free of other pathogens to the best of our knowledge. They were singly housed in large polycarbonate rat cages with filter tops to prohibit any animal–animal transmission. They were given paper mill and fed a diet of rodent chow and water throughout the experiments. All animals were weighed monthly and examined daily for signs of clinical illness. This experiment was performed under a protocol approved by our Institutional Animal Care and Use Committee in accordance with the accepted guidelines. 2.2. Bacteria Mycobacterium tuberculosis strain H37Rv, obtained from JoAnne Flynn (University of Pittsburgh School of Medicine, Pittsburgh, PA) was used for the inoculations as in our prior experiments. This strain has been previously passaged in mice. Stock inoculum was prepared as previously described and stored in 1.5 mL ampules in a 70 C freezer until needed.7 The titer of the stored ampules was 3.9 106 CFU/mL. All preparations and handling of the bacteria, animal inoculation, sacrifice and dissection were done in a biosafety hood. 2.3. Infection BSL-3 facilities that would accommodate aerosolization of Mtb were not available. Therefore, intranasal inoculations were used in this experiment to introduce Mtb into the lower respiratory tract of the cotton rats. Frozen ampules containing H37Rv were thawed, vortexed and diluted 1:100 in sterile PBS. Next, the suspension was briefly sonicated to disperse aggregated bacteria. Animals were anesthetized with ketamine injected intramuscularly, and a volume of 0.1 mL of inoculum was instilled in the nares (50 mL per nare). Forty-seven animals were inoculated in this manner and recovered. 2.4. Histology Animals were euthanized by CO2 narcosis. Lung tissues used for histologic evaluation were dissected en bloc, inflated through the trachea with 10% buffered formalin to their normal volume, and finally immersed into the formalin solution. Spleens were dissected in half, and one portion was prepared for histology by immersion in a 10% buffered formalin solution. Adrenal glands and liver sections were also collected in the 10% formalin solution. Sections of all formalin-fixed lungs, spleens, livers, and adrenal glands were stained with hematoxylin and eosin (H&E) to evaluate histopathology, and modified acid-fast tissue stain to look for the presence of acid-fast bacilli (AFB). Evaluation of the stained tissue sections was performed prior to identification of individual animals for correlation with clinical parameters and prior to tissue culture results being available. 2.5. Culture of organ tissues Lung and spleen organ tissues were aseptically removed from all cotton rats at the time of sacrifice. The entire lingular lobe of the lung of each animal was removed and homogenized for culture. Spleens were bi-sected with half of the organ being homogenized for culture and the other half prepared for histology. Serial 10-fold dilutions of homogenates prepared from the tissue samples were plated using sterile technique and incubated as we have previously
described.7 Colonies were counted after 18–21 days by an investigator that was blinded to clinical findings and the histological findings associated with any of the animals. Cultures were not obtained from animals that died prior to their planned day of sacrifice. This decision was based on the non-standardized time between death of the animal and necropsy as well as the inability to prepare culture media in a timely fashion on a case by case basis. 2.6. Immunosuppression Nine months after inoculation, all surviving animals were divided into two groups. One group served as infected controls. The second group was immune suppressed with intraperitoneal injections of cyclophosphamide. A calculated dose of 50 mg/kg of cyclophosphamide was injected three times per week. This dose was previously used in a study by Ottolini et al., and was noted to reliably induce leucopenia in cotton rats.8 Cyclophosphamide injections were given for 4 weeks, and then the animals were allowed to recover for 2 weeks. At the end of this period all remaining cyclophosphamide treated and infected control animals were sacrificed. 3. Results Forty-seven S. hispidus cotton rats were anesthetized, challenged with 3.9 103 CFU of human Mtb (H37Rv) by intranasal inoculation, and recovered. Five out of six animals sacrificed at 3 months following inoculation demonstrated histologic evidence of a primary tuberculosis infection with nodular lung lesions and pulmonary granulomas and with visible AFB. The animal without histologic evidence of disease also had a negative lung tissue culture. One animal with histologic evidence of disease had all lung tissue specimens placed in formalin preventing the ability to culture. The mean colony forming units (CFU) recovered from lung tissue culture in the remaining four animals was 1.98 105. Fourteen of 41 (34%) inoculated animals died prior to the 9 month time point at which immunosuppression was begun. Ten of these 14 (71%) demonstrated histologic evidence of active pulmonary tuberculosis infection. Following the group division, five of the 15 animals treated with cyclophosphamide died prior to the planned time of sacrifice, while only one of 12 of the infected control animals died before this time point. Most animals that died prior to their planned time of sacrifice appeared ill and wasted and uniformly had extensive pulmonary disease. Many had almost confluent pulmonary inflammation with few clear alveoli open for gas exchange. Gross examination of other organ systems at the time of necropsy was normal. However, we did not systematically evaluate for all forms of extrapulmonary active disease (including bowel, mesentery, kidney or central nervous system) in these animals. These should be evaluated in future experiments. To repeat, in this study, we defined a latent tuberculosis infection as a positive lung and/or spleen tissue culture together with the absence of histologic evidence of pulmonary or splenic disease. Data obtained from the animals that survived past the time of separation into cyclophosphamide treated and infected control groups are presented in Table 1. Animals that died prior to the planned day of sacrifice did not have tissue culture for Mtb performed. This included five animals in the cyclophosphamide treated group and one animal in the infected control group. Once cyclophosphamide injections began, these animals were counted as not having latent infection if there was histologic evidence (granulomas with AFB) of active Mtb disease. Twelve animals were included in the infected control group. Six of these animals fulfilled our definition for latent Mtb infection. Careful examination revealed no visible areas of inflammation
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Table 1 Results from S. hispidus cotton rats after 9–10 months following respiratory Challenge with M. tuberculosis.
Cyclophosphamide treated (10 animals survived until planned sacrifice)
(5 animals died prior to planned sacrifice)
Infected controls (11 animals survived until planned sacrifice)
(1 animal died prior to planned sacrifice)
Peak Wt/death Wt (g)
% Wt loss
Quantitative Mtb cultures (CFU/mL)* Lung
Spleen
110/90 115/100 110/80 135/135 125/125 145/145 145/145 120/120 145/145 155/155 115/100 135/80 95/90 90/70 110/100 140/130 140/140 135/135 145/145 140/140 145/145 140/140 150/150 140/140 135/135 135/135 130/130
18.18 13.04 27.27 0 0 0 0 0 0 0 13.04 40.74 5.26 26.32 9.08 7.14 0 0 0 0 0 0 0 0 0 0 0
1.1 108 1.0 109 1.0 109 2.0 105 5.3 107 NG NG NG NG NG
2.4 105 4.0 106 1.6 105 1.3 102 6.0 104 NG NG NG NG NG
2.0 107 2.7 107 6.0 106 8.0 102 9.3 102 1.3 102 4.0 102 6.6 102 2.7 102 NG NG
1.0 105 9.6 103 contaminated 6.0 102 NG NG NG NG NG NG NG
Description of pulmonary histologyy
Patchy sml granulomas, few AFB Confluent lg granulomas, abundant AFB Patchy mod granulomas, abundant AFB Patchy mod granulomas, few AFB Patchy sml granulomas, mod AFB No granulomas, no AFB No granulomas, no AFB No granulomas, no AFB No granulomas, no AFB No granulomas, no AFB Confluent lg granulomas, abundant AFB Confluent lg granulomas, abundant AFB Patchy mod granulomas, abundant AFB Patchy mod granulomas, mod AFB No granulomas, no AFB Patchy mod granulomas, mod AFB Patchy sml granulomas, few AFB Patchy mod granulomas, mod AFB No granulomas, no AFB No granulomas, no AFB No granulomas, no AFB No granulomas, no AFB No granulomas, no AFB No granulomas, no AFB No granulomas, no AFB No granulomas, no AFB Patchy sml granulomas, few AFB
* Animals that died prior to the date of planned sacrifice but after immunosuppression had begun were evaluated for histology but did not have cultures performed. NG ¼ No growth. y Lung, spleen, liver and adrenal glands were also evaluated. Of note, no animals demonstrated evidence of Mtb infection in extrapulmonary organs without also demonstrating evidence of Mtb infection within the lungs.
despite the low level positive cultures. Three animals in this group demonstrated active Mtb disease with both positive cultures and histologic evidence of pulmonary disease while one animal had positive histologic evidence of disease but cultures were not obtained. The remaining two animals in this group appeared to be free of infection (negative cultures and histology). Geometric means of the colony forming units (CFU) of Mtb recovered from lung tissue culture of cotton rats were calculated and compared with a one-way ANOVA followed by Tukey’s post-hoc tests. The geometric mean CFU recovered from animals with latent Mtb infection was 4.36 102 (95% CI ¼ 1.98 102–9.59 102). The geometric mean CFU of Mtb recovered from culture in cotton rats from the infected control group with active disease was 1.48 107 (95% CI ¼ 2.05 106–1.07 108). This difference was significant (p < 0.001). Fifteen animals began receiving cyclophosphamide injections at 9 months after inoculation. Nine of these animals had histologic evidence of active pulmonary disease. Most demonstrated severe pulmonary disease including granulomas with central necrosis and abundant AFB. Five animals in this group appeared to be free of disease, and zero animals fulfilled our definition of latent Mtb infection. A single animal in the cyclophosphamide treated group that died prematurely was without histologic evidence of Mtb disease. This animal was excluded from calculations because no culture data was available. The geometric mean CFU of Mtb recovered from lung tissue culture in cotton rats with active disease in the cyclophosphamide treated group was 6.51 107 (95% CI ¼ 8.54 105–4.96 109). The difference in geometric mean CFU between this group of animals and those from the infected control group demonstrating latent infection was significant (p < 0.001). The difference in geometric mean CFU among the animals demonstrating active disease in the cyclophosphamide treated and
infected control group was not significant (p ¼ 0.636). Table 2 provides further statistical analysis of the histopathology and culture data from all animals that survived until the groups were divided at 9 months when immunosuppression began. Figure 1 illustrates comparative gross and pulmonary histopathology results from cotton rats with noted active disease in the immunosuppressed and infected control groups as well as a cotton rat in the infected control group with latent disease. The mean peak weight of cyclophosphamide treated animals with active disease was 115 g while their death weight was 97 g. This represents a mean 16% weight loss (range ¼ 5–41%). The mean peak weight of infected control animals with active disease was 136 g. A single animal in this group demonstrated a 7% weight loss. There was no weight loss in any of the animals that demonstrated latent disease or the absence of infection. In most cases, the weight loss correlated with degree of pulmonary disease as is shown in Table 1. Granulomas with visible AFB were seen in the liver in a single evaluated specimen from an animal that died prior to the initiation of immunosuppression. Histology of liver and adrenal gland specimens were evaluated along with the lungs and spleens in all animals that remained to the day of final sacrifice. Granulomas with abundant AFB were noted in the liver and an adrenal gland in one animal that underwent immunosuppression. None of the infected control animals evaluated at 10 months from the time of inoculation demonstrated evidence of Mtb in the adrenals. Figure 2 illustrates some of the extrapulmonary histopathology seen. 4. Discussion Cotton rats have several advantages over other animals used in Mtb research. Compared to larger animals, cotton rats are
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Table 2 Comparison of immunosuppressed vs. immunocompetent S. hispidus cotton rats at 10 months following challenge with Mycobacterium tuberculosis.
Positive tissue culturesy Lung Spleen Positive histologic evidence of disease Latent infection (positive culture þ negative histology)y
P*
Cyclophosphamide treated
Infected controls
5/10 (50%) 5/10 (50%) 9/15 (60%)
9/11 (82%) 0.18 3/10 (30%) 0.65 4/12 (33%) 0.25
0/14 (0%)
6/12 (50%) 0.004
Statistical difference were compared by 2-sided Fischer’s exact test. Animals that died prior to the date of planned sacrifice but after immunosuppression had begun were evaluated for histology but did not have cultures performed. * y
inexpensive, and easy to handle and maintain in large groups. Also, a wide variety of primers and other reagents are available to study the immune response in cotton rats.9,10 We have previously shown that compared to other rodent models, cotton rats develop a primary Mtb infection that histologically appears more similar to primary Mtb disease in humans following a respiratory exposure. In the first few months following exposure, Mtb infected cotton rats develop granulomas, often with caseating necrosis, and show evidence of spread to lymphoid organs.7 However, little was previously known about the natural progression of the infection after 6 months.
The normal lifespan of a cotton rat is between 12 and 15 months. About a third of the cotton rats died prior to their planned time of sacrifice (at about 11 months of age). Seventy-one percent of these animals demonstrated histologic evidence of active pulmonary Mtb disease. While some degree of attrition not attributable to the infection is expected in an experiment lasting this long, we believe that Mtb contributed to, if not caused, the early death of many of these animals. We have made previous attempts to evaluate for latency and reactivation of Mtb disease in cotton rats. The first attempt is an unreported administration of steroids between 4 and 6 months following challenge. We noted more severe disease in the steroid treated animals. We next used cyclophosphamide to induce immunosuppression in cotton rats between 6 and 10 months after the challenge. Trends of clinically latent Mtb infection were noted, but the number of animals was too small to reach statistical significance.11 It is difficult to study latency in the pathogenesis of Mtb. While it is widely accepted that humans become latently infected with Mtb, there is no agreed upon standard of defining latency. It is usually diagnosed by demonstrating a likely exposure to the Mtb bacillus (usually by an immunologic response to Mtb antigens) along with the absence of active disease. True latent tuberculosis infection (LTBI) can be easily misdiagnosed. It is unknown how lung tissue culture for Mtb in humans correlates with a diagnosis of LTBI. In the cotton rat model, we believe that histological evidence for
Figure 1. Gross lung, H & E, and modified acid-fast stains from cotton rats sacrificed at 10 months following respiratory challenge with Mtb. Level of magnification is labeled on the photomicrographs. A, B, and C demonstrate severe disease with large tubercules, expanding granulomas with mild necrosis and many AFB in a cotton rat that received cyclophosphamide injections for 4 weeks followed by 2 weeks of recovery. D, E, and F demonstrate small tubercules, granulomas with central necrosis and calcification and several AFB in a cotton rat that did not receive cyclophosphamide but had high recoverable CFU of Mtb on lung tissue culture consistent with active disease. G, H, and I demonstrate normal appearing lungs by gross and histologic examination in a cotton rat that did not receive cyclophosphamide and had low recoverable CFU of Mtb on lung tissue culture consistent with latent disease.
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Figure 2. Multiple acid-fast organisms (red) found in: (A) liver, (B) spleen, and (C) adrenal gland of a cotton rat with disseminated disease following 4 weeks of cyclophosphamide injections and 2 weeks of recovery. Magnification levels are indicated.
pulmonary granulomas and visible AFB are better measures for active disease than is culture of tissues because we can examine the entire lung for areas of inflammation that denote a focus where there is an active immune response to the invading organism. However, a positive tissue culture certainly confirms the presence of viable Mtb. Therefore, the finding of a positive lung tissue culture, in the absence of granulomatous inflammation can logically be assessed to be a latent infection as per our definition. While we have defined the diagnosis of latency in the cotton rat differently than the manner it is diagnosed in humans, other recognized animal models of LTBI such as the Cornell mouse model also define LTBI in a different manner.12,13 Although there were no grossly abnormal lesions within the abdomen or elsewhere in cotton rats without evidence of active spleen or lung disease, we concede that we cannot rule out the possibility of extrapulmonary active TB disease (in organ systems that we did not fully evaluate such as the bowel, CNS, skeleton, or lymph nodes) in the cotton rats that met our definition of latent disease. However, latent disease seems more likely in these animals when you consider that the immunosuppressed group demonstrated either obvious pulmonary TB disease or no disease at all. Furthermore, it is possible that animals that appeared to be uninfected (negative histology and negative cultures) might have grown Mtb from tissue cultures were it possible to perform both histologic evaluation and culture of each entire lung specimen at the same time. In this case, our calculations would underestimate the true number or latently infected cotton rats. Two murine models of latent Mtb infection have been previously described. The Cornell mouse model was first reported in the 1950’s, and several variations followed.12,13 In this mouse model, Mtb was inoculated intravenously. Latency was defined as negative Mtb cultures following treatment with isoniazid and pyrazinamide. However, week’s later cultures again became positive with organisms that were still sensitive to treatment with these drugs. Similar to the cotton rat, these mice demonstrated low titers of organisms. However, unlike our model, this state of latency was artificially induced. Other murine models of LTBI describe an equilibrium of chronic disease with higher titers (w3–4 Log10) of cultureable Mtb.14 This is believed to be dissimilar to the number of bacilli present in human LTBI. Other animals that have been evaluated for latency include rabbits and non-human primates. While cynomolgus macaques may closely resemble the variations of Mtb infections seen in humans including the ability to skin test with PPD, they are much more expensive and require more specialized training and facilities to use.15 Recently, New Zealand white rabbits have also been described to demonstrate Mtb disease including latency and spontaneous reactivation that is more similar to humans than the murine models.16 However, rabbit models are restricted by the lack of inbreeding and absence of immunologic reagents such as are available for both mice and cotton rats.
There are two prevailing theories as recently described by Cardona and Ruiz-Manzano for how Mtb maintain latency in lung parenchyma that is dynamic in nature.14 One suggests that the bacteria are constantly disseminating and reactivating at a low level but are kept in check by the host’s immune system. The second purports that bacteria are dormant inside necrotic material of a granuloma, where the movement of macrophages would be limited until it is slowly reabsorbed. Our cotton rats with latent disease had positive lung tissue cultures from lingular lung lobes but were grossly and microscopically normal. Our findings are most consistent with the theory that a low level dissemination resulted in Mtb being hematogenously deposited in this part of the lung. An interesting difference from our previous report is that at later time points, positive lung tissue cultures exceeded positive spleen cultures whereas the opposite is true earlier in the course of disease. Our data show that once infection is controlled to latency low levels of viable organisms remain in the absence of detectable pulmonary or splenic inflammation. It is unclear where these organisms reside. We expect to examine this question in subsequent experiments. Although there was not a discernible pattern of predilection for any part of the lungs in the animals with active disease (such as the upper lobes as in humans), it is important to note that there is concordance between the histopathology and quantitative culture results in an all or nothing pattern. That is, when granulomas are found, there are also positive lung cultures, usually with large numbers of bacteria. When the lungs contain no evident granulomas, counts are either negative, or very low, as in our latent cases. An alternate explanation for the animals that were found to have both negative cultures and histological examinations is that they either never became infected or completely cleared their infection. Similarly humans do not always develop active or latent infection following a single exposure. We acknowledge that the intranasal instillation of organisms is less ideal than aerosolization to deliver the inoculum to lungs. Also, personal communications with other researchers working with cotton rats from the same colony but with different strains of Mtb indicate a higher level of resistance to infection than what we have noted. Because of this, we believe that the virulence of the bacterial strain of Mtb is an important variable in this model. Approximately one-third of the cotton rats that developed a primary Mtb infection eventually succumbed to the disease between 4 and 10 months following challenge. Many of these animals demonstrated severe pulmonary disease. However, half of the remaining animals that were allowed to progress naturally demonstrated a latent infection that is characterized by positive lung tissue culture (with a low recoverable CFU of Mtb) but the absence of any histological evidence of active infection. Furthermore, because cyclophosphamide treated cotton rats demonstrated either the absence of any infection or active pulmonary infection,
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we conclude that it is possible to reactivate the Mtb infection with immune suppression. Immune suppressed cotton rats develop more severe Mtb disease. This is clinically demonstrated by increased weight loss and a wasted appearance. AFB were seen in an adrenal gland of one immune suppressed animal. Addison’s disease is described in patients with adrenal tuberculosis, and the immune suppressed cotton rat model may present an opportunity to study adrenal function in tuberculous adrenalitis, but further characterization and development of this model would be needed. Limitations of this model of latent tuberculosis infection include a high degree of attrition and different progression of disease in populations of cotton rats (some with active infection, some with latent infection, and some without infection). The latter limitation would make it important to discover a way to differentiate the groups prior to sacrifice in order to better utilize the model for future study. We have previously reported that tuberculosis skin testing was unreliable in these animals.7 Earlier experiments evaluated skin testing of cotton rats in the footpads (an area of thick skin). While no overt reactions were evident, histologic evaluations demonstrated lymphocytic infiltrates in the area. However, this required amputation of the limb at necropsy to evaluate. Animals reported in our previous paper were tested in a shaved area on the back. We noted three animals with overtly positive reactions, but other animals with confirmed TB disease had no reaction. The skin in this area is very thin, and it is difficult to ensure that PPD is placed intradermally and not subcutaneously. Therefore, we determined it not worthwhile to pursue further skin testing by this method. Future studies should evaluate the utility of quantitative interferon-gamma release assays to confirm infection and radiography to evaluate for active disease. Our results, while preliminary, now justify the expense and logistical difficulty associated with these types of studies. The way in which cotton rats control Mtb infection to latency and may have reactivation of disease induced is unique among small animal models of tuberculosis disease. This model may need further development, and many new questions now arise. For example, it would be interesting to see the effects of newer immune modulators such as TNF inhibitors on the progression or reactivation of disease in the cotton rat. However, it is clear that cotton rats combine many of the advantages individually found in other animal models. This makes cotton rats a useful tool to study many aspects of tuberculosis disease including mycobacterial vaccinology. Our follow up studies will evaluate the effect of Bacille Calmette–Gue´rin on the natural history of tuberculosis in cotton rats. Acknowledgment We would like to thank David Porter MD, of the Department of Pathology and Laboratory Medicine at the University of California at Los Angeles, for assisting in histopathologic interpretation, and Cara Olsen at the Biostatistics Consulting Center, Uniformed Services University, for help with statistical analysis. Funding: None of the authors associated with this project have a commercial or other association that might pose a conflict of interest.
This study was supported in part by Uniformed Services University of the Health Sciences research grant GS86FC and corporate funds from Virion Systems, Incorporated. Competing interests: Experiments reported here were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals published by the Institute of Laboratory Animal Resources, National Research Council, National Academy Press, 1996. The opinions and assertions presented here are the private ones of the authors and are not to be construed as official or as reflecting the view of the Uniformed Services University of the Health Sciences or the Department of Defense. Ethical approval: Not required. References 1. Cegielski J, Chin DP, Espinal MA, Frieden TR, Rodriguez CR, Talbot DE, et al. The global tuberculosis situation. Progress and problems in the 20th century, prospects for the 21st century. Infect Dis Clin North Am 2002;16:1–158. 2. Grange JM, Zumla A. Paradox of the global emergency of tuberculosis. Lancet 1999;353:996. 3. Gupta UD, Katoch VM. Animal models of tuberculosis. Tuberculosis 2005;85: 277–93. 4. Dannenberg Jr AM, Collins FM. Progressive pulmonary tuberculosis is not due to increasing numbers of viable bacilli in rabbits, mice and guinea pigs, but is due to a continuous host response to mycobacterial products. Tuberculosis (Edinb) 2001;81:229–42. 5. Steinbach MM, Duca CJ. Experimental tuberculosis in the cotton rat. Proc Soc Exp Biol and Med 1940;44:288–90. 6. Daigeler A. Die baumwollratte (Sigmodon hispidus hispidus) als Versuchstier bei der tuberkulosediagnostik. Zeitschrift fu¨r Hygiene und Infektionskrankheiten; Medizinische Mikrobiologie. Immunol Virol 1952;135:588–91. 7. Elwood RL, Wilson S, Blanco JCG, Yim K, Pletneva L, Ninkonenko B, et al. The American cotton rat: a novel model for pulmonary tuberculosis. Tuberculosis 2007;87:145–54. 8. Ottolini MG, Curtis SR, Mathews A, Ottolini SR, Prince GA. Palivizumab is highly effective in suppressing respiratory syncytial virus in an immunosuppressed animal model. Bone Marrow Transplant 2002;29:117–20. 9. Blanco JCG, Pletneva L, Boukhvalova M, Richardson JY, Harris KA, Prince GA. The cotton rat: an underutilized animal model for human infectious diseases can now be exploited using specific reagents to cytokines, chemokines, and interferons. J Interferon Cytokine Res 2004;24(1):21–8. 10. Blanco JCG, Richardson JY, Darnell ME, Rowzee A, Pletneva L, Porter DD, et al. Cytokine and chemokine gene expression after primary and secondary respiratory syncytial virus infection in cotton rats. J Infect Dis 2002;185(12): 1780–5. 11. Elwood RL, Hemming VG. Characterization of pulmonary tuberculosis in cotton rats over 12 months [abstract 8428.8]. In: Programs and abstracts of the 2007 annual meeting of the Pediatric Academic Societies (Toronto, Canada) May 5–8, 2007. 12. McCune RM, Tompsett R. Fate of Mycobacterium tuberculosis in mouse tissues as determined by the microbial enumeration technique. I. The persistence of drug-susceptible tubercle bacilli in the tissues despite prolonged antimicrobial therapy. J Exp Med 1957;104:737–62. 13. McCune RM, Tompsett R, McDermott W. The fate of Mycobacterium tuberculosis in mouse tissues as determined by the microbial enumeration technique. II. The conversion of tuberculous infection to the latent state by the administration of pyrazinamide and a companion drug. J Exp Med 1957;104:763–802. 14. Cardona PJ, Ruiz-Manzano J. On the nature of Mycobacterium tuberculosis-latent bacilli. Eur Respir J 2004;24:1044–51. 15. Capuano III SV, Croix DA, Pawar S, Zinovik A, Myers A, Lin PL, et al. Experimental Mycobacterium tuberculosis infection of cynomolgus macaques closely resembles the various manifestations of human M. tuberculosis infection. Infect Immun 2003;71(10):5831–44. 16. Manabe YC, Kesavan AK, Lopez-Molina J, Hatem CL, Brooks M, Fujiwara R, et al. The aerosol rabbit model of TB latency, reactivation and immune reconstitution inflammatory syndrome. Tuberculosis 2008;88:187–96.