Corrigendum: Histopathologic differences in granulomas of Mycobacterium bovis bacille Calmette Guérin (BCG) vaccinated and non-vaccinated cattle with bovine tuberculosis

PAGE \* Arabic \* MERGEFORMAT 3In the published article, there was an error in the legend for Figure 3 as published. The original legend stated incorrect p values, " (*=p value 0.0434, =p value 0.0064, ns=not significant)."The corrected legend appears below.A) Granuloma size by area (µm 2 ) based on vaccination status. The average area of a granuloma was tabulated by dividing the total area occupied by all granulomas in the mediastinal and tracheobronchial lymph nodes of an animal by the total number of granulomas present. (B) Granuloma size by area (µm 2 ) when broken down by grade (high or low). Values are presented as means ± SEM. (*=p value 0.0004, **=p value <0.0001, ns=not significant).In the published article, there was an error in the legend for Figure 4 as published. The original legend stated incorrect p values, " (=p value 0.0029 for A, =p value 0.0080 for B, **=p value <0.0001)."The corrected legend appears below.Values are broken down by vaccination group (A) and by vaccination group and grade (B). Values are presented as means ± SEM. (*=p value 0.0197 for A, *=p value 0.0143 for B, =p value 0.0081 for A, =p value 0.0010 for B, ****=p value <0.0001, ns=not significant).In the published article, there was an error in the legend for Figure 5 as published. The original legend stated incorrect p values, " (ns=not significant)."The corrected legend appears below Values represent the number per 100µm 2 and are broken down by vaccination group (A) and by vaccination group and grade (B). Values are presented as means ± SEM. (*=p value 0.04, ns=not significant).In the published article, there was an error in the legend for Figure 6 as published. The original legend stated incorrect p values, " (*=p value 0.0344, ns=not significant)."The corrected legend appears below.Values are broken down by vaccination group (A) and by vaccination group and grade (B). Values are presented as means ± SEM. (ns=not significant).In the published article, there was an error in the legend for Figure 7 as published. The original legend stated incorrect p values, "(*=p value 0.0009, **=p value <0.0001, ns=not significant)."The corrected legend appears below.Values are broken down by vaccination group (A) and by vaccination group and grade (B). Each dot represents one animal and its mediastinal and tracheobronchial lymph nodes. Values are presented as means ± SEM. (*=p value 0.04, =p value 0.008, *=p value 0.0006, ns=not significant).The original article has been updated.In the published article, there was an error in Figures 2,3,4,5,6,and 7 as published. Due to differences in challenge strains, experimental groups needed to be treated independently. The corrected figures appear below. The corrected legends are listed in the previous section.PAGE \* Arabic \* MERGEFORMAT 3 PAGE \* Arabic \* MERGEFORMAT 3The original article has been updated.In the published article, there was an error. In fact, two different challenge strains were used in the two experiments, not one. This resulted in the need to analyze groups separately and not combine them. This issue has caused multiple sections to be updated to reflect the correct comparisons and statistics.A correction has been made to Abstract, 1. This section previously stated: "BCG vaccinates had fewer granulomas overall and smaller high-grade granulomas with less necrosis than non-vaccinates. The relative numbers of high-and low-grade lesions were similar as were the amounts of mineralization and the density of MNGC. The amount of fibrosis was higher in low-grade granulomas from vaccinates compared to non-vaccinates. Collectively, these findings suggest that BCG vaccination reduces bacterial establishment, resulting in the formation of fewer granulomas. In granulomas that form, BCG has a protective effect by containing their size, reducing the relative amount of necrosis, and increasing fibrosis in low-grade lesions. Vaccination did not affect the amount of mineralization or density of MNGC"The corrected sentences appear below: "In one set of banked samples BCG vaccinates had fewer granulomas overall and lower numbers of multinucleated giant cells. In the other set of samples, lesions of vaccinates were significantly smaller. In both experimental groups vaccinates had less necrosis than non-vaccinates. The relative numbers of high-and low-grade lesions were similar between vaccinates and nonvaccinates of both groups as were the amounts of fibrosis and mineralization. Collectively, these findings demonstrate the variability of protection offered by BCG. It suggests that BCG vaccination may serve to reduce bacterial establishment, resulting in the formation of fewer granulomas and in granulomas that form, that it may have a protective effect by containing their size and reducing the relative amount of necrosis."PAGE \* Arabic \* MERGEFORMAT 3A correction has been made to Materials and Methods, Samples 1. This section previously stated "Briefly, in Experiment 1, 23 castrated Holstein steers of 4-5 months of age were divided into two groups: non-vaccinates (n=11) and BCG vaccinates (n=12). Animals were kept on pasture prior to the start of the study. As environmental bacteria are present in our area, and in order to prevent possible confounding responses to BCG vaccination, animals were confirmed non-reactive to Mycobacterium avium (M. avium) via interferon gamma release assay (IGRA) immediately before the start of the study. Animals in the vaccinated group received a subcutaneous injection 1 ml of 5 X 10 5 CFU of BCG Danish. Three months following vaccination, both vaccinates and non-vaccinates received 5.5 x 10 2 CFU of M. bovis strain 1315 via aerosolization as described elsewhere (Palmer et al., 2002). Strain 1315 is a virulent field strain obtained from a white-tailed deer in Michigan, USA.The corrected sentences appear below: "Briefly, in Experiment 1 (E1), 23 castrated Holstein steers of 4-5 months of age were divided into two groups: non-vaccinates (n=11) and BCG vaccinates (n=12). Animals were kept on pasture prior to the start of the study. As environmental bacteria are present in our area, and to prevent possible confounding responses to BCG vaccination, animals were confirmed non-reactive to Mycobacterium avium (M. avium) via interferon gamma release assay (IGRA) immediately before the start of the study. Animals in the vaccinated group received a subcutaneous injection 1 ml of 5 X 10 5 CFU of BCG Danish. Three months following vaccination, both vaccinates and non-vaccinates received 5.5 x 10 2 CFU of M. bovis strain 10-7428 via aerosolization as described elsewhere (Palmer et al., 2002)."A correction has been made to Materials and Methods, Samples 1. This section previously stated "In Experiment 2, 21 newborn Holstein steers were divided into two groups: non-vaccinates (n=11) and BCG vaccinates (n=10). Animals were bottle-raised on a pasteurized milk product and kept in clean pens to reduce exposure to environmental mycobacteria. Due to the cleanliness of the housing situation the risk of exposure to environmental mycobacteria was considered low and IGRA testing for M. avium was not performed. Animals in the vaccinated group received a subcutaneous injection 1 ml of 1 X 10 6 CFU of BCG Danish at 2 weeks of age. Three months following vaccination, both vaccinates and non-vaccinates received 1 x 10 3 CFU of M. bovis strain 1315 via aerosolization."The corrected sentences appear below: "In Experiment 2 (E2), 21 newborn Holstein steers were divided into two groups: non-vaccinates (n=11) and BCG vaccinates (n=10). Animals were bottle-raised on a pasteurized milk product and kept in clean pens to reduce exposure to environmental mycobacteria. Due to the cleanliness of the housing situation the risk of exposure to environmental mycobacteria was considered low and IGRA testing for M. avium was not performed. Animals in the vaccinated group received a subcutaneous injection 1 ml of 1 X 10 6 CFU of BCG Danish at 2 weeks of age. Three months following vaccination, both vaccinates and nonvaccinates received 1 x 10 3 CFU of M. bovis strain 95-1315 via aerosolization."PAGE \* Arabic \* MERGEFORMAT 3A correction has been made to Materials and Methods, Samples 1. This section previously stated "The two groups used in this study varied in age at the time of vaccination, 2 wks vs 4-5 mos. It was previously reported that neonatally vaccinated animals had at least as robust an immune response as adults (Hope et al., 2005). Nevertheless, to account for differences between experimental groups we evaluated the effect of group within all statistical analyses and found significance only for fibrosis metrics where group was included as a fixed effect (see stats section)."This statement has been removed.A correction has been made to Materials and Methods, Statistical analysis 9. This section previously stated "For all scored granuloma metrics (count, average area, percent necrosis, MNGC frequency, percent mineralization, and percent fibrosis) data were analyzed with a simple linear regression model (lm) in R (version 4.2.1). Each scored metric was evaluated independently, fitting grade, vaccination status, and their interaction as fixed effects. A fixed effect of 'group', denoting the two separate experimental cohorts, was evaluated for model fit for each metric and only deemed appropriate to include for analysis of percent fibrosis. A pairwise comparison of Least Squares means (lsmeans package) was utilized to determine significant differences between specific grade and vaccination group contrasts."The corrected sentences appear below: "All statistical analyses were performed in GraphPad Prism (Version 9.5.1). E1 and E2 were treated as independent experiments. Evaluated metrics include count, average area, percent necrosis, MNGC frequency, percent mineralization, and percent fibrosis for the mediastinal and tracheobronchial lymph nodes for each animal. Mann-Whitney tests were performed for all metrics listed above to compare vaccinates and non-vaccinates across grades. Additionally, for each metric, differences between vaccination group and high-and low-grade granulomas were evaluated using a two-way ANOVA and Tukey's multiple comparison test."A correction has been made to Results, Granuloma number and breakdown 1. This section previously stated "Non-vaccinated animals had higher numbers of granulomas compared to BCG-vaccinates (p< 0.0001) (Figure 2A). The mean number of individual granulomas per animal in BCG-vaccinates was 18.95 (range 0-51) while the mean number of granulomas per animal in non-vaccinates was 61.5 (range 17-178). In total, 1770 granulomas were evaluated. BCG vaccinates accounted for 417 (23.6%) of these granulomas while 1353 (76.4%) granulomas were in lymph nodes of nonvaccinates."The corrected sentences appear below: "BCG vaccinates in E1 and E2 had significantly fewer granulomas than non-vaccinates (p <0.0001 and p = 0.0229, respectively) (Figure 2A). Overall, in E1 there were 82 low-grade and 62 high-grade granulomas in BCG vaccinates and 419 low-grade and 283 high-grade granulomasPAGE \* Arabic \* MERGEFORMAT 3in non-vaccinates. In E2 there were 176 low-grade and 98 high-grade granulomas in BCG vaccinates and 452 low-grade and 199 high-grade granulomas in non-vaccinates. In E1, nonvaccinated animals had higher numbers of high-and low-grade granulomas compared to BCGvaccinates (p= 0.013 and <0.0001 respectively) (Figure 2B). In E2 while non-vaccinates had both more low-and high-grade granulomas, this was not significant (p>0.05). Overall, the mean number of individual granulomas per animal in E1 BCG-vaccinates was 11.9 (range 0-49) while the mean number of granulomas per animal in non-vaccinates was 63.8 (range 17-126). The mean number of individual granulomas per animal in E2 BCG-vaccinates was 27.4 (range 3-64) while the mean number of granulomas per animal in non-vaccinates was 59.2 (range 23-178)."A correction has been made to Results, Granuloma number and breakdown 2. This section previously stated "While the number of granulomas varied markedly between BCG-vaccinates and nonvaccinates, the relative percentages of each grade of granuloma were similar (Fig 2B). There were approximately equal numbers of high-and low-grade granulomas, within each vaccinate group. Of the total granulomas present in BCG vaccinates, 58.52% of them were low grade while 43.14% were high grade. In non-vaccinates these percentages were 57.56% and 42.44% respectively. Overall, these data indicate that there is a trend for fewer high-grade granulomas compared to low grade granulomas independent of vaccination status (p=0.551 and p=0.0627 for non-vaccinates and vaccinates, respectively)."The corrected sentences appear below:"While the number of granulomas varied markedly between BCG-vaccinates and nonvaccinates, the relative percentages of each grade of granuloma in a given animal were not significantly different (Figure 2C). There were similar percentages of high-and low-grade granulomas, within each vaccinate group. Of the total granulomas present in E1 BCG vaccinates, 57% of them were low grade while 43% were high grade. In E1 non-vaccinates these percentages were 60% and 40% respectively. In E2, low-grade and high-grade granulomas accounted for 64% and 36% respectively while non-vaccinates had 69% and 31% each of lowand high-grade lesions.Overall, these data demonstrate BCG vaccination reduces the number of granulomas in animals challenged with virulent M. bovis, however the relative ratios of high-and low-grade granulomas are similar, independent of vaccination status."A correction has been made to Results, Average granuloma size and amount of necrosis 1. This section previously stated "-BCG vaccinates had smaller high-grade granulomas and lesions with less necrosis.There was no significant difference (p=0.0675) in the average size of granulomas between vaccinates and non-vaccinates, however lesions from non-vaccinated animals tended to be smaller (Figure 3A). Additionally, low-grade granulomas did not significantly vary in size between vaccinates and non-vaccinates however high-grade granulomas were larger in nonvaccinates than vaccinates (p=0.0434) (Figure 3B). Within vaccination groups, BCG vaccinates had similarly sized low-and high-grade granulomas (p=0.6296) while non-vaccinates had larger high-grade granulomas compared to low-grade granulomas (p=0.0064)."PAGE \* Arabic \* MERGEFORMAT 3The corrected sentences appear below: "-BCG vaccination reduces the percentage of necrosis within lesions.In E1 there were no significant differences in the average size of granulomas between vaccinates and non-vaccinates, when total granulomas were compared (Figure 3A) or when broken down by grade (Figure 3B). In contrast, when granulomas from E2 were compared, vaccinated animals had significantly smaller granulomas (p=0.0006) as compared to non-vaccinated animals (Figure 3A). This difference was the result of high-grade lesions being significantly smaller in vaccinated animals (p<0.0001) as compared to non-vaccinates. Low-grade granuloma size did not differ between vaccinated and non-vaccinated animals in E2."A correction has been made to Results, Average granuloma size and amount of necrosis 2. This section previously stated "BCG vaccinates had lesions containing significantly (p=0.0029) less tissue destruction as evidenced by a lower percentage of necrosis than non-vaccinates (Figure 4A). As a result of the categorization method, necrosis was expected to be substantially higher in high grade granulomas compared to low grade and this was true for both vaccinates and non-vaccinates (p<0.001 for both). There was no significant difference in the percentage of necrosis within lowgrade granulomas of vaccinates compared to non-vaccinates with values being nearly identical (p=0.9766) (Figure 4B). High-grade granulomas of non-vaccinates contained a significantly (p=0.0080) higher percentage of necrosis than high-grade granulomas from BCG-vaccinates."The corrected sentences appear below: "In both E1 and E2, granulomas from vaccinates had less necrosis than non-vaccinates (p=0.0081 and p= 0.0197 respectively) (Figure 4A). Specifically, in E1 high-grade granulomas of vaccinates had less necrosis than high-grade granulomas of non-vaccinates (p=0.0010) (Figure 4B). As expected, high-grade granulomas had more necrosis than low-grade granulomas regardless of vaccination status or experimental group (p=0.0143 for E1 BCG-vaccinates, p<0.0001 for E1 non-vaccinates, p<0.0001 for both E2 vaccinates and non-vaccinates) (Figure 4B). There were no significant differences in the percentage of necrosis within low-grade granulomas of vaccinates compared to non-vaccinates in either E1 or E2 (Figure 4B)."A correction has been made to Results, Multinucleated giant cell numbers1. This section previously stated "-MNGC numbers were not dependent on vaccination status or lesion severity. No significant differences were found in the density of MNGC between vaccination groups (Figure 5A). When separated by grade, there were no significant differences in the number of MNGC between high-and low-grade granulomas, however there was a trend (p=0.0632) for low grade granulomas of non-vaccinates to contain a higher number of MNGC than high grade granulomas of the same group (Figure 5B)."PAGE \* Arabic \* MERGEFORMAT 3The corrected sentences appear below: "-BCG vaccinates in E1 had fewer MNGC overall however there were no significant differences in E2 or when comparing different granuloma grades.In E1, granulomas from BCG vaccinates had fewer per 100µm2 than granulomas from nonvaccinates (p=0.0357) (Figure 5A). However, in E2, we did not observe any differences (p=0.8633) in the number of MNGCs between granulomas from vaccinates and non-vaccinates (Figure 5A). Interestingly, when evaluated by granuloma grade, all significance was lost; there were no significant differences in the number of MNGC between high-and low-grade granulomas in either E1 or E2 (Figure 5B)."A correction has been made to Results, Fibrosis and Mineralization 1. This section previously stated "-BCG vaccination increased the amount of fibrosis in low grade granulomas. BCG vaccination did not influence the amount of mineralization. Mineralization increased with the severity of the lesion.Fibrosis was the only metric in which experimental group had a significant effect. The primary difference between the two groups of animals used for this study is age, where the Experiment had higher average fibrosis than the animals Experiment not being there were no significant differences in the percentage of fibrosis per granuloma between vaccinates and non-vaccinates for either experimental group (Figure Collectively, when evaluated by grade, low grade granulomas of vaccinates had a significantly higher percentage of fibrosis than low-grade granulomas of nonvaccinates (Figure When mineralization was no significant differences were in the percentage of mineralization between vaccinates and non-vaccinates (Figure Additionally, mineralization was similar within of different vaccination groups (Figure differences between low grade granulomas and their high-grade granulomas with a in vaccinates and p<0.0001 in corrected sentences appear below: "-BCG vaccination did not the of fibrosis or mineralization within granulomas. Mineralization increased with the severity of the were no significant differences in the percentage of fibrosis per granuloma between vaccinates and non-vaccinates for either experimental group (Figure This was true when fibrosis was analyzed by grade (Figure When mineralization was no significant differences were in the percentage of mineralization in granulomas from vaccinates and non-vaccinates (Figure and mineralization was similar between vaccinates and non-vaccinates when the same grade of granuloma (Figure In E1, granulomas from vaccinates and non-vaccinates had significantly more mineralization in the high-grade granulomas compared to the low-grade granulomas (p= for vaccinates and p= for In contrast, in E2 only granulomas from non-vaccinates had granulomas with significantly higher mineralization than low-grade lesions \* Arabic \* MERGEFORMAT 3A correction has been made to 1. This section previously stated study utilized lymph nodes from animals BCG resulting in the of a total of granulomas. Of 417 granulomas were in BCG vaccinates while 1353 were in corrected sentences appear below: study granulomas from BCG-vaccinates from E1 and from and 1353 from non-vaccinates from E1 and from at the size of the amount of necrosis, number of multinucleated giant the amount of fibrosis and the amount of correction has been made to This section previously stated expected, and in with previous BCG the lesion et al., et al., et al., et al., et al., et al., animals had an average of granulomas per tissue section while non-vaccinated animals 62 granulomas. Interestingly, this contrast, the relative breakdown of high-and low-grade granulomas did not significantly with vaccinates and non-vaccinates similar percentages of This suggests that the protective of BCG in by the of granulomas as to their to a higher grade. findings are with and performed in white-tailed deer which all of granulomas in lymph nodes of BCG vaccinates et al., et al., et al., BCG did not prevent high grade granulomas from being it smaller lesions on average with less corrected sentences appear the lesion in both groups, which is in to previous et al., et al., et al., et al., et al., et al., When evaluated as a E1 vaccinated animals had an average of 11.9 granulomas per tissue section while non-vaccinated animals In E2, vaccinated animals 27.4 granulomas per tissue section while non-vaccinates 59.2 Interestingly, the relative breakdown of high-and low-grade granulomas did not significantly with vaccinates and non-vaccinates similar percentages of This suggests that the protective of BCG in by the of granulomas as to their to a higher grade. findings are with and performed in white-tailed deer which all of granulomas in lymph nodes of BCG vaccinates et al., et al., et al., BCG did not prevent high grade granulomas from being in E2 animals it smaller lesions on average more within the high-grade granulomas. In E1 there were no significant differences in average granuloma area, an of the variability within BCG In both E1 and E2, high-grade granulomas larger than low-grade granulomas, of vaccination status. In both E1 and E2, vaccinates had less necrosis within lesions than In granulomas of non-vaccinates had more necrosis than correction has been made to This section previously stated was the metric in which there were statistical differences between experimental cohorts, of which the primary difference between the groups is animals of Experiment 1 and the PAGE \* Arabic \* MERGEFORMAT 3 animals of Experiment 2. animals from Experiment 1 more fibrosis in granulomas compared to animals of Experiment 2 not This was and as it has not been reported A possible for the difference in fibrosis found between the two age groups be of the for fibrosis. However, to a study performed in is lower in animals vs et al., This suggest other are to these It has been that vaccinated at with BCG similar of interferon gamma as months however the the vaccinates had a in et al., This be as by and be for the differences in fibrosis between age groups et al., It is possible that the being for a to the may have into with environmental which their response when to virulent M. bovis et al., when as a vaccinates and non-vaccinates had similar overall percentages of a in to previous et al., et al., broken down by vaccinate the amount of fibrosis was significantly higher in low grade granulomas of vaccinates than non-vaccinates. the time lesions to this significance was have been removed.A correction has been made to This section previously stated in of the of increased fibrosis in the low-grade lesions of vaccinates is and may represent a response as a result of and an at of the corrected sentences appear below: there were no significant differences in the percentage of fibrosis within granulomas between vaccinates or non-vaccinates, regardless of granuloma grade. This is a in to other et al., et al., findings BCG not protection by increasing fibrosis. the amount of fibrosis compared to fibrosis may of for these The original article has been correction has been made to This section previously stated mineralization is a to necrosis and it is not to of mineralization increased in high grade granulomas. Nevertheless, it is to that the percentage of mineralization between high-grade granulomas were similar between vaccination groups and that amounts of mineralization were present in the low-grade \* Arabic \* MERGEFORMAT 3The corrected sentences appear below: mineralization is a to necrosis and it is not to of mineralization increased in high grade granulomas of both E1 and E2, independent of vaccination status. Nevertheless, it is to that the overall percentage of mineralization between vaccinates and non-vaccinates was of is that amounts of mineralization were present in the low-grade correction has been made to This section previously stated "In this study we found no significant differences between groups or grade, however there was a trend for non-vaccinates to have more MNGC in their high-grade granulomas compared to their low-grade granulomas, increased bacterial may have a corrected appears below: in both E1 and E2 we found no significant differences between granuloma regardless of correction has been made to This section previously stated banked samples from two previous that between the two were not The vaccination and challenge varied between experimental groups, however as these were 1 we it was an of Mycobacterium bovis in is to the the to et al., corrected sentences appear below: not the of this the of banked samples from two separate has an of BCG and M. bovis we expected both experimental groups to in there were 3 where only one experimental group had significance between BCG vaccinates and non-vaccinates and MNGC numbers in E1 and average area in groups varied based on age of vaccination, of BCG and strain and of It be to compare each of these to determine each resulting these suggest and animals the same or the same to these correction has been made to This section previously stated this fact, BCG vaccinates have smaller granulomas with less tissue destruction than Fibrosis was higher in low-grade granulomas of vaccinates compared to non-vaccinates this significance was when grade was Mineralization and MNGC density were similar between vaccinates and non-vaccinates, these may be corrected sentences appear below: "BCG may reduce tissue destruction as not only did E2 vaccinate granulomas have a smaller average size, necrosis was in granulomas of both E1 and E2 The of BCG vaccination in