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U.S. Department of Health and Human Services

Animal & Veterinary

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OFFICE OF RESEARCH 2000 HIGHLIGHTS

FDA Veterinarian Newsletter May/June 2001 Volume XVI, No III

Introduction

The Office of Research (OR) is the laboratory-based research arm of the Center for Veterinary Medicine (CVM), Food and Drug Administration (FDA). 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, the Office of Research 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 below by the three OR Divisions in which they were conducted—DRC, DAR and DAFM.

Division of Residue Chemistry (DRC)

OR’s Division of Residue Chemistry (DRC) has been responsible for developing, validating and 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.

Depletion of OTC in Shrimp

DRC scientists provided the analytical support needed to complete an oxytetracycline (OTC) residue depletion study in collaboration with Dr. Rodney Williams at the University of Arizona. Currently, OTC is not approved for use in shrimp feed. Moreover, since shrimp aquaculture is a relatively small market in the U.S., it is unlikely that drug sponsors would invest resources necessary for a New Animal Drug Application (NADA) permitting OTC use in shrimp aquaculture. A producer consortium has been generating the data necessary for a NADA.

Dr. Philip Kijak analyzed the OTC-medicated shrimp food and found that it contained 4.5 g OTC-HCl/ kg feed. Drs. Mary Carson and Maureen Ngoh analyzed shrimp tissue from the OTC-depletion study, using an HPLC method validated at OR. Shrimp were fed the medicated diet for 14 consecutive days, and then switched to a control diet for OTC-withdrawal. The results indicated that OTC residues in medicated shrimp had fallen below FDA’s tolerance of 2 ppm (for tetracycline residues in human food) by 48 hours post-withdrawal of medication. The results of this study will be used to support INAD 8069 for OTC use in shrimp.

Cow-Calf Model for Mother-Child Studies

DRC has also developed a cow-calf model which predicts drug transfer rates from mother to infant. The model can be used to monitor drug transfer via the placenta and milk. Data from cow-calf model studies will be used to predict similar drug transfers in a human mother and child. This model has been used to demonstrate that normal dosing of ivermectin (a lipid soluble drug) to the cow leads to an unanticipated accumulation of drug in the calf. A similar accumulation of lipid soluble drug might be anticipated in a nursing infant of a drug-treated mother. Data from the cow-calf model studies, supported by an FDA Office of Women’s Health grant, can be used to predict drug accumulations in mother’s milk for human infants. DRC’s animal data will also be critical in the design of human drug transfer studies.

Fluoroquinolones in Eggs of Laying Hens

DRC scientists recently conducted a series of studies to determine if fluoroquinolone (FQ) residues are transferred into eggs of laying hens and, if so, which biomarker, the parent compound or its metabolites, is most suitable for surveys. 14C-sarafloxacin was orally administered to six laying hens for five consecutive days. Eggs were collected for 15 days after initial drug treatment. Egg yolk and egg albumen were separated and assayed for total radioactive residues (TRR). Radioactivity was detected in egg yolk and egg albumen on the second day of dosing and reached a maximum at 24 hours after drug withdrawal. Thereafter, the sarafloxacin TRR levels in egg albumen declined rapidly and were undetectable two days after the last dose while levels in egg yolk declined at a much slower rate and were undetectable seven days after drug withdrawal. In both the egg albumen and egg yolk, HPLC analysis indicated that the parent sarafloxacin was the major component. Thus, the parent compound was used in the nationwide survey for FQ in eggs of laying hens.

DRC’s next challenge was to develop a multiresidue HPLC method for the determination of three FQs: sarafloxacin, enrofloxacin, and ciprofloxacin in table eggs. In the U.S., sarafloxacin and enrofloxacin are approved for use in broiler chickens and in turkeys for the treatment of bacterial infections, but they are not approved for use in laying hens. Ciprofloxacin is a human drug but is prohibited for use in animals; it was included in the method development because it is a metabolite of enrofloxacin.

Finally, in collaboration with CVM statisticians, DRC scientists designed and conducted a national survey for FQs in table eggs. The objective was to identify possible illegal use of FQs thereby helping to ensure that the U.S. egg supply is safe. With the help of FDA’s Office of Regulatory Affairs (ORA), 276 eggs were sampled from 75 egg production or distribution firms throughout the United States. Because the radiotracer study indicated that drug residues stay in the egg yolk for a longer time than in the egg albumen and that egg yolk is a better matrix of choice for monitoring, we conducted our assays on egg yolk only. Of the 276 eggs assayed, none were found to be positive for FQs. Results from this study suggest that illegal use of fluoroquinolones in laying hens is not a widespread phenomenon, and provide support for CVM’s poultry drug NOOH that there is no evidence of FQ misuse in laying hens.

Division of Animal Research (DAR)

The Division of Animal Research (DAR) has been heavily involved in conducting an array of animal food safety studies. Some noteworthy research accomplishments by DAR personnel in recent years are outlined below.

BSE: Method for Detection

In an attempt to prevent the emergence of bovine spongiform encephalopathy (BSE) in U.S. beef cattle, FDA established a ban on most ruminant materials in feed for ruminant animals. However, CVM did not have an analytical method to detect bovine materials in animal feed. Thus, Dr. Michael Myers optimized a polymerase chain reaction (PCR) method for this purpose. DAR scientists then conducted a multi-laboratory method validation trial that showed the PCR method to be specific and reliable in detecting the presence or absence of bovine materials in animal feed. The PCR test is also rapid and will save time thus ensuring that critical Agency resources are focused on possible violations of the feed additive ban.

Drug Bioequivalence Testing

According to current CVM policy, companies introducing a new generic drug must demonstrate bioequivalence to the pioneer product in every target animal species (thus requiring multiple investigations, 20 animals/species, for each new drug). Traditionally it has been thought that product bioequivalence cannot be extrapolated across target animal species. However, in response to a request from ONADE, DAR scientists examined the validity of extrapolating parental product bioequivalence using two injectable formulations of ampicillin in calves, sheep, and swine. Employing products recognized to be bioinequivalent provided an opportunity to detect species differences. Marked interspecies differences were noted clearly defining the need for further research before current CVM bioequivalence policy can be changed.

Preventing Violative Neomycin Residues in Calves

Violative residues of neomycin in cattle tissues, especially calves, have long been a concern for CVM. Thus, DAR scientists developed a program of research to investigate the transfer of neomycin into tissues of young calves. Results from this program demonstrated that neomycin was absorbed by both ruminating and, to a greater extent, in non-ruminating calves. Other research demonstrated that neomycin levels in kidneys depleted to below tolerance levels at 21 days post-withdrawal of a medicated milk replacer. Further OR investigations examined depletion of neomycin in kidneys following oral administration of the approved DESI dosage of neomycin in cattle. These studies provided critical information necessary to preventing violative neomycin residues in calves.

Division of Animal and Food Microbiology (DAFM)

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 these resistant pathogens has raised growing concerns for therapeutic failures in animals and humans. The President’s Food Safety Initiative has provided CVM with funding to support research of antimicrobial resistance. Under the direction of Dr. Robert Walker, DAFM’s key research goals are to characterize and identify ways to 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 antimicrobial drugs in food animals, including aquatic species. However, the bulk of DAFM’s research focuses on studies aimed at detecting and avoiding development of antibiotic resistant bacteria in the human food chain. A listing of some of DAFM’s studies on antimicrobial resistance follows.

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 recently performed comparison studies of two widely-used microbiological testing methods for Campylobacter—the concentration gradient (Etest) and agar dilution method tests. The DAFM team coordinated experiments and compared results from various investigators around the U.S. and showed that the agar dilution test was uniquely reliable for quantifying antimicrobial susceptibility in Campylobacter. Dr. Walker presented these results at a recent NCCLS-VAST subcommittee meeting and the group agreed that the agar dilution method should be the standard for Campylobacter in vitro antimicrobial susceptibility testing. Moreover, the group agreed upon quality control (QC) limits for various antibiotics for the agar dilution method for testing Campylobacter.

PulseNet: Genetic Fingerprinting of Antibiotic-Resistant Bacteria

Another of DAFM’s projects, PulseNet, has already been highly successful in characterizing and reducing salmonellosis outbreaks. PulseNet is a program designed to provide DNA fingerprints of foodborne bacterial pathogens and store them for future reference in outbreaks. To give an example of PulseNet in action, in the fall of 1999, 30 individuals in Canada developed Salmonella infantis. All those affected were dog owners and many had recently given their dogs pig ear dog treats. Shortly thereafter, the FDA issued a nationwide public health warning on salmonellosis related to contact with these dog treats. PulseNet scientist Dr. Shaohua Zhao isolated and serotyped Salmonella bacteria from various brands of dog treats. She also established PFGE profiles of the Salmonella serotypes and later determined the antibiotic susceptibility of the various serotypes found in the dog treats. Following Dr. Zhao’s work, a nationwide FDA survey (involving 16 FDA district offices and 7 regional labs) showed that 49% of dog treats tested carried Salmonella, and that 36% of serotypes were resistant to one or more antimicrobials with 13% being resistant to 4 or more antimicrobials. In a collaboration with the National Antimicrobial Resistance Monitoring System (NARMS), DAFM researchers shared PulseNet data to determine the association between animal and human Salmonella isolates and to assess mechanisms of antibiotic gene transfer. These same highly successful DNA fingerprinting techniques are being used to examine the possible presence of fluoroquinolone-resistant Campylobacter in retail meats (chicken, turkey, beef, and pork).

Aquaculture

Americans are increasingly health conscious and are more aware of the beneficial effects from eating fish. Thus, the supply and consumption of fish within the U.S. has been steadily increasing over the past 20 years. To meet this demand, aquaculture (or the farming of aquatic organisms including fish, mollusks, crustaceans and aquatic plants) is a rapidly growing industry.

As the number of aquaculture facilities increases, so does the need to develop safe and effective drugs for treating fish diseases. It is critical to understand the effect these treatments might have on fish (and consumers), non-target organisms and on the aquatic environment. As a result, CVM has greatly expanded its commitment to aquaculture research. In January 1999, Dr. Renate Reimschuessel joined OR to develop a research program in aquaculture. Dr. Reimschuessel’s key achievements have largely focused on optimizing OR’s aquaculture facility such that it can support diverse studies in multiple fish species.

OR’s research objectives for aquaculture are to provide data to assist the FDA in assuring that fish derived from aquaculture are safe for human consumption. Species for study include tilapia (Oreochromis sp.), rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar), channel catfish (Lctalurus punctatus) large mouth bass (Micropterus salmoides), toadfish (Opsanus tau), and goldfish (Carassius auratus)—with goldfish being an established model for fish infections. All species, except goldfish, are currently raised for food purposes. There also has been a large effort to develop a rationale for crop grouping (grouping species for drug approvals based on similarities in anatomy, physiology, and drug metabolism).

Since much of the U.S. fish supply is currently from countries where aquaculture drug usage is widespread, it is also critical to conduct drug targeting studies and surveys of aquatic food species being marketed in the USA. In addition, of increasing importance, are studies designed to understand the development and transmission of antimicrobial resistance in both pathogenic and environmental microbes. Understanding those mechanisms will help in designing treatment strategies that minimize the development of resistant pathogens.

STEC and Salmonella Antibiotic Resistance in Cattle

DAFM is collaborating with Dr. David Acheson at New England Medical Center 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 Escherichia coli 0157:H7 in cattle. Molecular subtyping of Escherichia coli isolates is also being done to assess possible development of new E. coli strains. Finally, this information will be used in developing prevention scheme strategies for on-farm control of E. coli transmission.

Surveillance

DAFM also performs surveillance studies of retail products for foodborne bacterial pathogens to assess trends of antibiotic resistance in animal foods for humans. 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 (FSI) goal of helping to reduce the incidence of foodborne disease to the greatest extent possible.