Introduction

Although the knowledge base of zoo and wildlife medicine continues to grow rapidly, basic conundrums continue to stymie the clinician seeking to treat nondomestic species. Often, simply determining whether an animal is actually ill, let alone affected with a treatable infectious organism, can be remarkably difficult. Nondomestic species, both wild and captive, tend to conceal disease extremely well. Frequently, sudden death is the only indication that an animal harbored significant disease, although necropsy may indicate a long‐standing infection.

Nevertheless, even if an antemortem diagnosis of infection is made in a wild animal species, evidence‐based decisions for antimicrobial use may be extremely challenging. Pharmacological studies relevant to zoo and wildlife species continue to be scant. This is due in part to the technical difficulty of performing drug studies in wild animals as well as the perceived risks of such research in rare or endangered species. Additionally, the unusual physiology of nonmammalian species such as reptiles, amphibians, avians, and fish can add yet another layer of complexity to drug studies. Even nondomestic mammalian taxa may metabolize drugs in surprising ways compared to domestic counterparts.

Lacking hard data on pharmacokinetics, safety, and efficacy of most antimicrobials, clinicians caring for zoo and wildlife species must extrapolate drugs and doses from unrelated species, a less than ideal situation. Finally, the technical aspects of providing a course of antimicrobial therapy to wild animals, most of which are uncooperative and potentially dangerous even if sick, provide a final level of complexity.

Clinical Breakpoint Interpretation in Zoological Medicine

Global events and perceptions regarding the use of antimicrobial agents in animals have placed even more importance on the essential role of antimicrobial susceptibility testing of bacteria isolates from animals. However, little information is available on microorganism–antimicrobial–host interactions with zoo species. Currently, veterinary‐specific breakpoints have been compiled and published in the Clinical Laboratory Standards Institute’s (CLSI) VET01S (CLSI, 2021). This also indicates the criteria for several antimicrobials that are commonly used in veterinary medicine yet do not have veterinary species‐specific breakpoints. The number of drugs in the latter category gets smaller with each revision, but even the veterinary‐specific criteria must be interpreted with caution when applied to zoological species for treatment considerations and therapy. This is emphasized in VET09 from CLSI (CLSI, 2019a).

The first reason for this caution is how the breakpoints are determined by the Veterinary Antimicrobial Susceptibility Testing subcommittee. The path for breakpoint determination is outlined in the CLSI VET02 document “Development of Quality Control Ranges, Breakpoints, and Interpretive Categories for Antimicrobial Agents Used in Veterinary Medicine” (CLSI, 2019b). This document outlines the pathway for antimicrobial agents for the setting and recommendation of veterinary‐specific clinical breakpoints. “S” is the susceptible interpretive test category implying that the infection, due to the isolate, may be effectively treated with the normal dosage regimen of an antimicrobial recommended for that type of infection and causative bacterial species. This is the fundamental piece of information that is often not understood by the attending zoo veterinarian because of limited information about pharmacokinetics in the species to be treated and the lack of accurate prediction of pharmacokinetics across species (Hunter and Isaza, 2008). The recorded results indicate that the isolate is S or I or R for the culture submitted when tested against the diagnostic lab’s standard array of antimicrobial agents. The S/I/R are reported using the information provided in VET01S previously mentioned. The reporting institution does not know, in many cases, the host species, route of administration, or pharmacokinetics of the antimicrobial agent in the treated species being evaluated and reported. They generally only have two pieces of the puzzle: microorganism and class representative antimicrobial agent tested.

Most veterinarians then assume that if the microorganism is reported as S, they simply treat it with that agent and positive results will follow. Antimicrobial susceptibility testing of bacteria of animal origin is ultimately intended for the selection of antimicrobial agents for better clinical outcomes. The premise of veterinary antimicrobial susceptibility testing (VAST) is that in vitro test results can be used to guide the veterinarian in antimicrobial drug therapeutic decision making when the testing is performed in a standardized and reproducible manner. Just as important, clinicians must understand that antimicrobial resistance is not necessarily an inherent or absolute characteristic of bacteria, but rather that resistance indicates the crossing of a threshold.

Although “S” and “R” are usually considered binary characteristics, in fact resistance can only be identified if a clinical breakpoint or threshold of antimicrobial concentration is predetermined and agreed upon by regulatory agencies and/or standard‐setting organizations such as the CLSI. The threshold (“interpretive criterion”) cannot be arbitrarily determined (e.g., by saying that all bacteria with a zone of inhibition of less than “X” mm are resistant) but must be validated with the appropriate data, including knowledge of concentrations of antimicrobial drug that can be achieved in an animal (pharmacokinetics), the best presentation of the drug to the bacteria in the host (pharmacodynamics), range of concentrations of antimicrobial drug required to inhibit the growth of populations of wild‐type bacterial pathogens, and clinical outcome of treatment of the pathogen with approved or commonly accepted doses of an antimicrobial drug. It goes without saying that the determination of S is a complex process for the indication(s) on the product label. Attempting to determine this value for each and every “bug/drug/species” combination in zoological medicine would be an astronomical undertaking.

To avoid misinterpretation, CLSI VET01S and VET09 recommend that diagnostic laboratories only test and report breakpoints for antimicrobials appropriate for therapeutic or control use. Antimicrobials could be added based on specific therapeutic needs (such as for specific zoological species where a specific agent and formulation are commonly used). Given the limited number of antimicrobial agents approved for use in some animal species, “extra‐label” use of antimicrobial agents is common. The US Congress, in the Animal Medicinal Drug Use Clarification Act (AMDUCA), has defined extra‐label use as the “actual use or intended use of a drug in an animal in a manner that is not in accordance with the approved labeling.” This includes, but is not limited to, use in species or for indications (disease or conditions) not listed in the labeling; use at a dosage level higher than that stated in the label; and use of routes of administration other than those stated in the labeling. This type of use has regulatory acceptance in many countries (e.g., extra‐label use permitted under the AMDUCA regulations). While laboratory personnel should be familiar with the extra‐label use of antimicrobial agents in animals, the laboratory client is responsible for using the compound appropriately in the animal.

The laboratory client is also responsible for using the agent appropriately for the various animal types or categories (e.g., calves, lactating dairy cattle). The laboratory client assumes all responsibility for efficacy, safety, and residue avoidance with extra‐label uses of antimicrobials. The laboratory should be prepared to offer advice to the veterinarian to enable appropriate decision making. Although the laboratory may choose to modify the list of antimicrobials it tests and reports, on the basis of public health concerns, it needs to be done in consultation with appropriate experts, based on good clinical judgment, and in accordance with recognized principles of judicious use. Veterinarians working with minor or zoological species should make themselves aware of the tables provided in VET01S and VET09 so as to understand what the breakpoints are and what bug/drug/species indications they are based on.

Numerous antimicrobials are approved for use in different animal species by the US FDA‐CVM or comparable regulatory authorities in other countries. Factors such as microbiological activity, clinical efficacy, and pharmacology should be considered for therapy, indications, and restrictions. CLSI document VET01S lists compounds in groups in which drugs are approved for use in the indicated animal species by the US FDA‐CVM (Groups A, B, C, and D). It is most appropriate to report those antimicrobials that have veterinary‐specific interpretive criteria over those using human interpretive criteria (Group A). These antimicrobials have demonstrated an acceptable correlation between in vitro susceptibility test results and clinical criteria outlined in VET01S. While antimicrobials evaluated using human interpretive guidelines (Group B) may perform adequately in diseased animals, the interpretive relationship for veterinary applications has not been determined. Some antimicrobials are FDA‐CVM approved for use in a specific animal species but have neither veterinary‐specific nor human‐specific interpretive criteria (Group C) and reporting interpretive criteria from one animal species to another (extra‐label use, Group D) is not recommended due to various differences in dosages and pharmacokinetics.

Intra‐ and Interspecies Dose Extrapolation

Species differences in drug absorption, distribution, metabolism, and excretion (ADME) for numerous antimicrobials have been well documented for domestic species; however, there is limited information concerning the ADME of drugs in nondomestic species (Hunter, 2017). Lack of approved drugs and/or pharmacokinetic data in the literature for zoo species is a major issue for veterinarians attempting to treat these animals. Zoological medicine practitioners take approved antimicrobials (veterinary or human) and extrapolate their use to nonapproved species. The range of animals a zoo veterinarian cares for varies from very small invertebrates (e.g., honeybees) to megavertebrates such as elephants and whales. The decision on the dose, duration, and treatment interval is often made with limited species‐specific pharmacokinetic and/or efficacy information. Because of the monetary value of these animals or their status as endangered species, the method of “trial and error” for antimicrobial dosage selection is inappropriate.

In zoological medicine, various methods have been used in an attempt to extrapolate or predict safe and effective dosage regimens (Hunter and Isaza, 2008). The simplest and most typical method of extrapolating a dosage to a nondomestic species is to use a mg/kg dose established for another domestic species or humans. However, this calculation results in a linear increase in the amount of drug administered as body weight increases. Although common, this method tends to overdose large animals and underdose small animals. A second method is similar except that it takes the approved dose in a specific species and makes an additional assumption that links the dosage to a physiological function or anatomical feature. Examples are the use of basal metabolic rate or body surface area as the basis for dosage extrapolation. Allometric scaling of pharmacokinetic parameters is the final method of dosage extrapolation between species. This is commonly used in the pharmaceutical industry to establish the first dosage in human drug investigations. Adaptation of this method for zoological medicine is believed to enhance the ability to estimate therapeutic dosages for nondomestic species. This tool, when used appropriately, can provide an estimate for designing dosage regimens. The example of differences in ketoprofen inversion across species emphasizes the need to understand and be aware of the assumptions when designing treatment regimens based on allometric scaling data (Hunter et al., 2003).

Just as mammals can range from a few grams to thousands of kilograms, reptiles and birds can also vary in body weight across a wide range. It has been suggested that it is impossible to derive a single equation correlating body mass to metabolic rate for all 6000 species of reptiles (Funk, 2000). Without knowledge of the extent and route of elimination of an administered antimicrobial, extrapolation of dosage regimens from one class to another is difficult, if not impossible, with any certainty. Some reports question the practical use of this approach (Hunter et al., 2008; Mahmood et al., 2006; Martinez et al., 2006).

Before extrapolation of any drug dose, the veterinarian should appreciate the mathematical and physiological assumptions involved and the limitations that are associated with allometry. Careful consideration of the available literature to understand the route of elimination and the extent of metabolism of antimicrobials will greatly assist in determining allometric relationships of pharmacokinetic parameters. There is a continuing need to consider and apply methods for reducing the size and risk of extrapolation error, as this can affect both target animal safety and therapeutic response. Data from at least one large animal (nonhuman and a body weight >70 kg) should be included to reduce potential error (Mahmood et al., 2006).

A Practical Example of Allometry and Breakpoints

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