Susceptibility in vitro
To the authors’ knowledge, this study was the first to successfully evaluate the antimicrobial susceptibility of M. avium
in vitro using monensin sodium or tilmicosin phosphate. The MIC of monensin sodium against this isolate of M. avium
was considerably less than that reported for the same medication against M. phlei
). An explanation for that difference was not determined by the results of this study and is the subject of another investigation. The protocol developed for evaluating susceptibility in vitro in this study was based on results of pilot studies (unpublished data) in our (RBS, DRA) laboratory and has been refined so that it can be performed consistently and with confidence. The medium, prepared as described above, contained Tween 80 (13
). The same medium was used throughout the study. The only difference among treatments was the medication added to the medium. Similar to other studies, the effect noted in our study was attributed to the medication (monensin sodium or tilmicosin phosphate) and was not attributed to the Tween 80 (14
Results of this study support the 1st hypothesis because the MIC of monensin sodium and tilmicosin phosphate were determined in vitro using the 30-day duration of incubation and modified medium described. Results further support the conclusion that monensin sodium and tilmicosin phosphate had antimycobacterial activity against the organisms used and can be included among the few medications that have been shown to have such activity in vitro (5
). Studies are underway to further prove the method of testing in vitro and to evaluate the extent of susceptibility to monensin sodium with other isolates.
Infectivity in a murine model
Hepatic granulomas, characterized as foci of macrophages and epithelioid cells surrounded by lymphocytes, were associated with the ability of the organism to proliferate within macrophages (9
). Those granulomas remain the principal response variable for this model. The murine model, as used for this study, involved younger mice, a smaller volume, and fewer organisms injected than those used in the model as originally described and those used in previous studies (9
). The importance of the series of controls included in this study was substantiated by results that confirmed appropriate reproduction of the model and that the response variable was not compromised by those modifications. The young age of these mice created some logistical problems related to handling and recognition of the congenital conditions. Therefore, use of older mice, as in the original model, is recommended for future studies using this model.
Results with the murine model in this study revealed that our 2nd hypothesis was correct. There was no significant effect of length of time of incubation, concentration of monensin sodium, or concentration of tilmicosin phosphate on the number of hepatic granulomas in the mice. However, incubation of the organism with monensin sodium significantly reduced the number of granulomas. Tilmicosin phosphate did not significantly affect the number of hepatic granulomas caused by this organism. This evidence supports the fact that results in vitro do not always predict results in vivo. The controlled study reported here for evaluation of these medications in vivo using a murine model is one step closer to the naturally-occurring disease than are studies in vitro. There is less encouragement to pursue other studies in vivo with tilmicosin phosphate than with monensin sodium against this organism. Although susceptibility of the organism to tilmicosin phosphate in vitro was encouraging, infectivity, as determined with the murine model, was not encouraging because the number of hepatic granulomas/mm2 within the hepatic section from mice that received the organism exposed to tilmicosin phosphate was no different than that for mice in the positive control group.
Results of studies with the murine model and with cattle suggest that the effect of monensin is not a laboratory oddity. Our first study using this murine model provided evidence of a prophylactic effect of monensin sodium against M. avium
). We subsequently showed a beneficial effect of monensin on lesions in cattle with naturally-occurring Johne’s Disease (11
). Susceptibility in vitro of the organism used in this study was quantitated; the MIC was 0.39 μg of monensin/mL. The beneficial effect of monensin, at a concentration below the MIC, was demonstrated in results with the murine model in this study. Exposure to monensin sodium at a concentration of at least 0.1 μg/mL of medium for at least 12 h reduced the infectivity of M. avium
. Concentrations of monensin and tilmicosin phosphate used in this portion of the study encompassed the MIC of each medication determined in this study. Logarithmic differences in concentrations were chosen because the pharmacodynamic response to a medication is normally distributed as a function of the logarithm of the dose or concentration of that medication (16
). Results were then evaluated with statistical methods applicable to normally distributed response variables, without transformation.
The mechanism of antimycobacterial action of monensin was not the subject of this investigation but possible mechanisms, proposed elsewhere (4
), include direct or indirect ionophoric activity that may alter the bacterial intracellular utilization of metals and electrolytes. Proper utilization of those elements is vital to intermediary metabolism of the organisms or may render organisms susceptible to other substances in the medium, such as the vancomycin that was used routinely in selective media, that are otherwise not effective against the organism (4
Further investigations should be performed to evaluate and define the role for monensin sodium in the control and management of bovine paratuberculosis. The ability to diagnose cattle infected with this organism has greatly improved (6
). While, to date, no treatment for cattle with Johne’s Disease is considered practical, medications currently suggested for treatment may be used in an extra-label manner (6
). Efficacy of those compounds has only been evaluated with, and considered acceptable for, individual clinical patients. The role of those compounds for successful, large-scale control of the disease is doubtful. Efforts continue for development of an effective vaccine against bovine paratuberculosis, but to date, vaccination has not been universally effective (20
Clinical use of monensin sodium as an aid in the control of Johne’s Disease has been largely ignored but may be a rational, economical, and valuable tool. Although laws in the United States (US) do not currently allow extra-label use of feed additives, (AMDUCA — 21 CFR Part 530; Docket number 96N — 0081, RIN 0910-AA47: http:/www.fda.gov/cvm/index/amduca/amducafr.htm
) regulations in other countries may permit such use. Monensin is a fermentation product of the organism Streptomyces cinnamonensis
). A large portion of monensin administered orally to cattle is eliminated in feces (23
). Concentrations of monensin that were used in our murine model encompassed the MIC of the drug for the test organism and were within those present in feces (1.3 to 8.5 ppm — depending on the amount fed) of cattle receiving monensin orally (23
). Organisms shed in feces of infected ungulates are considered to be the source of infection for other susceptible animals (6
). Depending on size of particles ingested, the intestinal transit time for cattle is usually longer than 12 h (27
). Exposure of the organism to monensin during intestinal transit may, therefore, be sufficient to reduce the infectivity of the organism by the time it is excreted with the feces. Based on results of this study, it could be hypothesized that fecal concentrations of monensin may be sufficient to reduce the infectivity of M. avium
in that feces if exposed for at least 12 h. It could be further hypothesized that prolonged exposure to monensin in the fecal patty or soil may continue to decrease the infectivity of the organism, rendering it less likely to infect another animal that would ingest it. Monensin decays within a few weeks, depending on conditions in feces and soil, and it presents no known environmental risk (26
). We suggest that studies be performed to evaluate the effect of monensin on recovery of M. avium
shed in feces from infected cattle and the natural infectivity of those organisms in susceptible cattle.
Results of this and previous studies do not suggest that monensin is a dramatically effective therapeutic agent for infected cattle that demonstrate advanced clinical signs of Johne’s Disease (11
). In those animals, monensin may assist to prolong the usefulness of the animal, as has been attributed to other medications (6
). We hypothesize that the most probable roles of monensin in the control of the disease may be to reduce fecal shedding, infectivity or both of the organism shed from infected animals, and as an aid in prevention of infection in susceptible, young replacement animals (replacement brood-stock), or both. Should those hypotheses prove to be true, prolonged, diligent use of monensin, in time, could reduce contamination of the environment with infective organism, thereby reducing exposure and incidence of the disease in susceptible animals such as replacement brood-stock. Appropriate use of monensin, concurrent with the practice of diagnostic testing and culling of infected animals from the herd, could assist other measures for the eventual elimination of the disease from a herd or reduce the influence of the disease (U.S. Voluntary Johne’s Disease Herd Status Program for Cattle; USAHA; 1998 http://www.aphis.usda.gov/vs/nahps/Johnes/vjdhspusaha1.htm