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U.S. Department of Health and Human Services

Animal & Veterinary

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CVM'S Office of Research 2001 Highlights

FDA Veterinarian Newsletter 2002 Volume XVII, No II

The Office of Research (OR) is the laboratory-based research arm of FDA's Center for Veterinary Medicine (CVM). OR's research priorities are ever changing, being driven by the needs of other CVM offices-i.e., the Office of New Animal Drug Evaluation (ONADE) and the Office of Surveillance and Compliance (OSC) and by FDA-wide requirements to thoroughly assess the latest food safety concerns. To meet these needs, OR is staffed by researchers with diverse scientific backgrounds-microbiology, biochemistry, toxicology, analytical chemistry, pharmacology, etc.-as well as scientists with specialist training-e.g., aquatic science specialists and antimicrobial resistance geneticists.

To give the reader an idea of the broad array of research studies conducted by OR scientists, this section briefly describes some of OR's recent studies. These studies are organized by the three OR Divisions in which they were conducted-Division of Residue Chemistry (DRC), Division of Animal Research (DAR) and Division of Animal and Food Microbiology (DAFM).

DIVISION OF RESIDUE CHEMISTRY

OR's Division of Residue Chemistry has been responsible for developing, and validating monitoring methods used in FDA's highly effective milk safety programs. More recently, DRC has been focused on developing methods to measure antibiotic residues in various tissues. They also have been conducting surveys to examine possible misuse of antibiotics, particularly fluoroquinolones, to prevent the emergence of antibiotic-resistant bacteria.

AQUACULTURE CROP GROUPING

There is a vital need to expand the repertoire of therapeutic drugs for veterinary use in aquaculture. Compared with traditional farm species, few drugs are currently approved by FDA for use in aquaculture species. In order to facilitate the drug approval process for multiple cultured fish species, it is desirable to establish crop (species) grouping based on similar drug metabolic profiles and drug residue patterns between species. Similarities (and dissimilarities) between metabolic profiles and species residue depletion will determine the likelihood of deriving species groupings.

DRC scientists in collaboration with DAFM scientists conducted studies to determine metabolic profiles and depletion pattern of albendazole in muscle tissue of cold water (rainbow trout) and warm water (tilapia) fish. The preliminary results indicate that albendazole is metabolized by both species and also a similar depletion pattern was observed for its pharmacologically active metabolite albendazole sulfoxide in fish, rainbow trout and tilapia. This suggests there is a potential for crop (species) grouping of cold and warm water fish for this model compound.

DRC scientists have developed and are completing the validation of a multiresidue confirmatory method for aminoglycoside antibiotics in tissues. This study builds on previous DRC work on confirmation of aminoglycosides in milk. It utilizes liquid chromatography-ion trap mass spectrometry. This approach yields highly specific residue identification. The method is capable of confirming nine different aminocyclitol and aminoglycoside antibiotics at or below their U.S. tolerances: spectinomycin, streptomycin, dihydrostreptomycin, hygromycin, amikacin, kanamycin, apramycin, gentamicin, and neomycin. It has been demonstrated to work in a variety of tissues: bovine kidney, liver and muscle; swine kidney, liver, and muscle; chicken kidney and muscle; rabbit and horse kidney. CVM's method is intended to be used by USDA's Food Safety and Inspection Service (FSIS) in their tissue residue monitoring programs. Currently, FSIS is unable to take action with some potentially adulterated carcasses because their screening and microbiological assays will not distinguish individual drugs within a class. As part of the validation of DRC's mass spectrometry method, FSIS sent DRC numerous bovine kidney samples that had screened presumptive positive for either streptomycin, gentamicin, or neomycin. DRC scientists were able to confirm the identity of aminoglycoside in nearly all of these tissues. In several of the tissues, the presence of two or more distinct drug residues was confirmed.

DRUG-DOSE REGIMEN REQUIREMENTS IN BOVINE PRODUCTION CLASSES

A comparative pharmacokinetic study of four different drugs in both steers and lactating dairy cows indicate a significant difference in the elimination half-life of the extensively metabolized drugs enrofloxacin and fendbendazole and no difference in elimination half-life phenylbutazone and ivermectin which are not metabolized. The data provide support for extensive pharmacokinetic review and drug dose regimen development in steers and dairy cows for drugs that are metabolized and the minimization of this requirement to one production class for drugs that are eliminated without extensive metabolization.

DIVISION OF ANIMAL RESEARCH

The Division of Animal Research has been heavily involved in investigating the safety of animal feeds. These investigations included developing methodology for detecting prohibited substances in ruminant animal feeds as part of the Center's bovine spongiform encephalopathy (BSE) prevention program.

BSE - METHODS FOR DETECTING PROHIBITED SUBSTANCES

In an attempt to prevent the emergence of BSE in U.S. beef cattle, FDA established a ban on ruminant materials in feed for ruminant animals. However, CVM did not have an analytical method to detect the prohibited substances in animal feeds. Previous efforts at OR established the validity of bovine specific PCR primers to detect bovine-derived materials in complete feed. Current efforts have established that a set of "universal" PCR primers is capable of detecting DNA from cattle, sheep, goats, deer, elk, horse and swine. Other than horse and swine, these primers do not detect any other exempt species. We have established sets of primers for swine, poultry and dog that detect only each individual species. We are currently focusing on developing sets of PCR primers that detect only horse and only cat. Once these are completed, CVM will conduct a method validation trial using these new primers. After validation, the Center will be able to analyze a feed sample using PCR methodology with the universal set of primers and the swine and horse primers to determine if the feed contains material from a prohibited species.

The second approach to the problem is the development of an ELISA test capable of discriminating between prohibited and exempt materials. The initial efforts have centered on bovine-derived materials. CVM researchers have separated and are in the process of identifying four heat stable proteins found in bovine meat and bone meal (BMBM) that are not present in blood and milk. (The gelatin which is always present in BMBM is removed by ammonium sulfate precipitation.) Efforts are underway to purify enough of these proteins to permit preparation of polyclonal and monoclonal antibodies for development of an ELISA test. In addition, CVM will attempt to identify and isolate unique heat-stable proteins from other prohibited species to include in our ELISA work.

DIVISION OF ANIMAL AND FOOD MICROBIOLOGY

Currently, one of the FDA's most important tasks is to ensure the safety of foods from microbial hazards, particularly from antibiotic-resistant bacteria. The increase in the incidence of human infections caused by resistant foodborne bacterial pathogens has raised concerns about the increased possibility of therapeutic failures in animals and humans. Under the direction of Dr. Robert Walker, DAFM's research goals are to characterize and reduce microbial hazards associated with all phases of animal food production and to address the effects of therapeutic and non-therapeutic antimicrobials used in food-producing animals on commensal bacteria and foodborne bacterial pathogens.

ANTIMICROBIAL RESISTANCE

To achieve these public health goals, DAFM collaborates on and has initiated a number of research studies, both internally and externally funded, aimed at developing approaches to support the safe use of antimicrobials in food animals, including aquatic species. This research focuses on strategies designed to provide greater understanding of the mechanisms of antibiotic resistance in order to reduce the prevalence of antibiotic resistant bacteria in the human food chain.

IMPROVING RELIABILITY OF MICROBIOLOGICAL TESTS

Currently, many microbiological tests suffer from poor reproducibility, poor comparability, and lack of agreement among microbiologists as to which tests are reliable and should be used by all investigators. Recognizing these shortcomings, Dr. Walker and his team coordinated an experiment involving investigators from the U.S., Canada, and Europe, and were able to demonstrate that when performing antimicrobial susceptibility testing on Campylobacter species the agar dilution testing method provided the intra-and inter-laboratory reproducibility required by the NCCLS to be accepted as an NCCLS standardized testing method. The DAFM team recently performed comparison studies of two widely used antimicrobial susceptibility testing methods for Campylobacter-the concentration gradient (Etest) and agar dilution testing methods.

PULSENET-GENETIC FINGERPRINTING OF ANTIBIOTIC-RESISTANT BACTERIA

PulseNet, a national computer network of DNA fingerprinting database for foodborne pathogens, was established in 1996 through a collaborative effort of CDC, FDA, USDA, and State Health Departments. The program uses pulsed-field gel electrophoresis (PFGE) as the DNA fingerprinting method to pinpoint an exact source of foodborne illness outbreak. PulseNet has already been highly successful in preventing and reducing foodborne outbreaks. In the past, PulseNet was focused on foodborne pathogens isolated from patients and foods because foodborne pathogen isolates from animals are limited. In a collaboration with the National Antimicrobial Resistance Monitoring System (NARMS), DAFM researchers obtain approximately 600 Salmonella isolates each year from NARMS, which are isolated from a variety of animals, including cattle, swine, chicken, turkey, equine, cat, and dog. All isolates are subtyped by PFGE and the DNA fingerprinting patterns are submitted to PulseNet. The patterns are compared to human clinical isolates through PulseNet. The study will reveal if there is a clonal spread of resistant isolates between animals and humans or widespread dissemination of unrelated strains. Also, Salmonella isolates are screened for the presence of class1 antibiotic resistance integron. Integrons are antibiotic resistance gene "cassettes" and play an important role in the dissemination of resistance genes. This study will help us to understand the genetic diversity of Salmonella, the link of Salmonella strain between animals and humans, and how antibiotic usage in animal husbandry can influence antimicrobial resistance in foodborne pathogens as well as the mechanism of resistance gene transfer between animal and human bacterial pathogens. In addition to Salmonella, the DNA fingerprinting database of E. coli 0157:H7 and Campylobacter are also established at CVM/OR.

STEC AND SALMONELLA ANTIBIOTIC RESISTANCE IN CATTLE

DAFM is collaborating with Dr. David Acheson at University of Maryland, Baltimore, MD to examine Shiga-toxin producing E. coli (STEC), e.g., E. coli 0157:H7, and Salmonella in cattle. These studies will determine the epidemiology of antimicrobial resistance phenotypically and genotypically in Salmonella and STEC as the organisms move longitudinally from feed into animals.

PREVENTION OF WATERBORNE E. COLI TRANSMISSION

DAFM collaborates with Dr. Charles Kaspar at the University of Wisconsin - Madison, on a study examining the waterborne transmission of E. coli 0157:H7 in cattle. Molecular subtyping of E. coli isolates will also be done to assess possible development of new E. coli strains. This information will be used in developing prevention scheme strategies for on-farm control of E. coli transmission.

SURVEILLANCE

Assessment of the dissemination of antibiotic resistant bacteria in food and within the food animal environment has been an important component of the DAFM mission. Preliminary data were developed in conjunction with NARMS on the prevalence of resistant enterococci, Salmonella, Campylobacter, and E. coli associated with animal-derived food. This work was preliminary to the development of an annual national survey, which will be initiated in FY02 and will be a collaboration between CVM and the Centers for Disease Control and Prevention.

The survey of animal feed commodities was also initiated as a pilot project during FY01 and will be expanded in FY02 in conjunction with FDA's Office of Regional Operations. This work forms part of CVM's ongoing risk assessment activities and is being conducted in concert with NARMS. The rationale is based on the potential for feed commodities to serve as vectors for the dissemination and maintenance of antibiotic resistant determinants within animal production environments.

During FY01, CVM was in the midst of conducting a formal risk assessment on the potential human health impact of Enterococci serving as a vector for transferring synercid resistance to humans through the food supply. This potential risk arises from the long-term use of virginiamycin, a Synercid analog, as a growth promoter in animal production. DAFM has been significantly involved in this risk assessment activity by providing microbiological analytical support through data collection and analysis.

DEVELOPMENT OF FLUOROQUINOLONE RESISTANCE IN CAMPYLOBACTER JEJUNI

Historically, antimicrobials approved for use in food animals have been evaluated based on their efficacy against the animal pathogen. There is increasing concern about their effect on resistance development within non-target, foodborne bacteria that are pathogenic to humans. These studies examined the effect of the veterinary-specific fluoroquinolones against the foodborne pathogen, Campylobacter jejuni. A series of experiments were conducted in broiler chickens using sarafloxacin and enrofloxacin. The results demonstrated that the use of fluoroquinolones in chickens, under label indications, generates a rapid increase in the fluoroquinolone minimum inhibitory concentrations (MICs) of resident C. jejuni (from 0.250 to 32 µg/mL), appearing within the treatment time frame and persisting long after treatment is stopped. No resistant isolates were detected in the non-treated control groups. In the case of C. jejuni from animals treated with sarafloxacin, an increased proportion of susceptible isolates appeared beginning at day 12. At day 26 (3 weeks after ending treatment), 72% of the isolates tested displayed MICs >32 µg/mL. In contrast, 100% of isolates analyzed from the enrofloxacin-treated animals displayed ciprofloxacin MICs of 32 µg/mL throughout the experiment, which ended 16 days after ending treatment. These results highlight the potential of inducing resistance in non-target bacterial species.

The above represent just a few of the ongoing highly successful research studies being conducted by DAFM scientists. These studies are achieving The President's Food Safety Initiative goal of helping to reduce the incidence of foodborne disease to the greatest extent possible.