A Novel Strategy to Mitigate Corynebacterium bovis–Associated Hyperkeratosis in Athymic Nude Mice
Nude mice were inoculated with a nonpathogenic Corynebacterium bovis isolate (NPI) or Corynebacterium amycolatum to assess whether either could prevent skin lesions following inoculation with a pathogenic C. bovis isolate (PI). Crl:NU(NCr)-Foxn1nu mice (n = 6/group) were randomized into 6 groups: NPI (108 colony-forming units [CFU]); C. amycolatum (108 CFU); NPI or C. amycolatum followed 2 weeks later by PI (104 CFU); and negative and positive controls receiving sterile media or the PI (104 CFU), respectively. Colonization was assessed biweekly using isolate-specific PCR assays. Skin lesions were scored 0 to 5 daily for 4 or 6 weeks, at which point skin biopsies were collected, evaluated, and scored. No mice inoculated with the NPI and subsequently infected with the PI developed clinical signs, nor was a significant amount of the PI detected by PCR. Mice inoculated with C. amycolatum before the PI developed milder, delayed skin lesions reaching a significantly lower mean peak clinical score (MPCS; 1.2 ± 0.4) as compared with the positive control (MPCS 2.5 ± 0.5). The C. amycolatum–inoculated mice with and without PI had similar total histopathology scores, both of which were significantly higher than those for the mice inoculated with the NPI followed by the PI. These results led to evaluation of a practical exposure strategy in which nude mice (n = 6/group) were housed on NPI seeded bedding (SB) for 3 or 7 days prior to PI administration; mice housed on C. bovis–free bedding served as controls. Only 1 of 12 mice housed on SB receiving the PI developed Corynebacterium-associated hyperkeratosis (peak score of 4), whereas all unvaccinated mice receiving the PI developed Corynebacterium-associated hyperkeratosis (MPCS 2.83 ± 0.69). The PI was not detected in the SB + PI groups until 21 days postinfection with the PI. There was no significant difference in total histopathology scores across groups, but the histopathology scores were lower in mice receiving the SB.
Schematic of Topical Inoculation Study. Groups NPI and C. amycolatum received 108 CFU/mouse of either the NPI or C. amycolatum. Groups NPI + PI and C. amycolatum + PI received NPI or C. amycolatum and were challenged 14 d later with 104 CFU/mouse of the PI. The sterile media group received only sterile media, and the PI group received 104 CFU/mouse of the PI at the same timepoint as groups NPI + PI and C. amycolatum + PI. dpi, days postinoculation; NPI, nonpathogenic C. bovis; PI, pathogenic C. bovis.
Photographs Demonstrating the Clinical Scoring System Used to Assess C. bovis–Associated Skin Disease in Athymic Nude Mice at Necropsy. Mice were scored 0 to 5 based on clinical signs representing mild, moderate, and severe disease. Reprinted with permission from Mendoza et al.1
Photomicrographs Demonstrating the Histologic Scoring System Used for Assessing the Severity of Acanthosis and Hyperkeratosis in Athymic Nude Mice Infected with C. bovis and/or C. amycolatum. Samples were semiquantitatively scored as normal (0), minimal (1), mild (2), moderate (3), or severe (4), based on the intensity of acanthosis and orthokeratotic hyperkeratosis. A0 to A4, acanthosis grade; H0 to H4, hyperkeratosis grade; black bracket represents the location and amount of acanthosis (A) or hyperkeratosis (H). Reprinted with permission from Mendoza et al.1
Schematic of SB Exposure Study. The SB7 + PI group received the NPI SB 7 d prior to the initial inoculation with the PI. The SB3 + PI group received NPI SB 3 d prior to the initial inoculation with the PI. The PI group received sterile bedding while the SB group received the NPI SB at the same time as the SB7 + PI group but was not inoculated with the PI. Groups SB7 + PI, SB3 + PI, and PI were all inoculated with the PI a second time 35 d after the first PI inoculation.
Clinical Score by Experimental Group. Scores reflect the mean ± SD for n = 6 mice/group assessed daily. The NPI + PI and C. amycolatum + PI groups were inoculated with the PI 2 wk after being inoculated with the NPI or C. amycolatum. *, The PI clinical score was significantly (P ≤ 0.05) higher when comparing to the NPI + PI and C. amycolatum + PI groups. **, The PI clinical score was significantly (P ≤ 0.05) higher when comparing the NPI + PI and C. amycolatum + PI groups, and the C. amycolatum + PI clinical score was significantly (P ≤ 0.05) higher than that of NPI + PI group. ***, The C. amycolatum + PI clinical score was significantly (P ≤ 0.05) higher than that of the PI and the NPI + PI groups. For mice receiving sterile media, only NPI or only C. amycolatum did not develop observable lesions and are excluded. dpi, days postinoculation; NPI, nonpathogenic C. bovis isolate; PI, pathogenic isolate.
Clinical Scores for Experimental Groups. (A and B) Clinical score (mean ± SD) for n = 6 mice/experimental group without (A) and with (B) the outlier, respectively. Red arrows indicate inoculation days (0 and 35) with the PI. The mean clinical scores for mice housed on sterile bedding were significantly higher (*, P ≤ 0.05) than the scores of mice housed on SB after exposure to the PI on the indicated days. (C) Individual clinical scores for mice housed on sterile bedding following inoculation with the PI. (D) Individual clinical scores for mice housed on SB for 7 d prior to inoculation with the PI. A single mouse (no. SB7.5) developed observable lesions at 6 dpi resolving by 10 dpi only to recur with greater severity on 30 dpi. PI, pathogenic isolate; SB, seeded bedding; SB3 = received SB 3 d prior to inoculation with the PI; SB7, received SB 7 d prior to inoculation with the PI.
Mean Estimated Copy Numbers of the NPI, C. amycolatum, or PI at Specific Study Time Points. (A) There were no significant differences in NPI copy numbers in mice that received only NPI as compared with mice that were subsequently inoculated with the PI at 0 and 14 dpi. (B) There were no significant differences in copy numbers of C. amycolatum present on the skin of mice that only received C. amycolatum as compared with those that were subsequently inoculated with the PI at 0 and 14 dpi. (C) The copy numbers of PI were significantly lower in mice that had been inoculated with the NPI at 14 and 28 dpi. In contrast, C. amycolatum only impacted the PI copy numbers at 14 dpi in that they were not significantly different in mice not receiving C. amycolatum at 28 dpi. The horizontal bar represents the data mean. dpi, days postinoculation; NPI, nonpathogenic isolate C. bovis; PI, pathogenic isolate. *, P ≤ 0.05.
Estimated Copy Numbers of the NPI Provided in the SB and the PI. (A) NPI provided in the SB. (B) PI. The horizontal bar represents the data mean. The copy number of the outlier is reflected in pink. *, P ≤ 0.05 (statistics computed excluding the outlier) when comparing the reflected groups. dpi, days postinoculation; SB, NPI SB; NPI, nonpathogenic isolate; PI, pathogenic isolate; SB3, received NPI SB 3 d prior to inoculation with the PI; SB7, received NPI SB 7 d prior to inoculation with the PI.
Histopathology Scores (Combined and Individual Criteria; Mean ± SD) for Each Group (n = 6/Group) in the Topical Inoculation Study. *, P ≤ 0.05 and is located over the group that was significantly different when compared with the other representative groups designated with tick marks on the corresponding line. NPI, nonpathogenic isolate C bovis; PI, pathogenic isolate; SM, sterile media.
Histopathology Scores (Combined and Individual Criteria; Mean ± SD) for Each Group (n = 6/Group) in the SB Study. *, P ≤ 0.05. Mouse no. SB7.5 was excluded. PI, pathogenic isolate; SB, NPI SB; SB3, received NPI SB 3 d prior to inoculation with the PI; SB7, received NPI SB 7 d prior to inoculation with the PI.
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