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Antibiotic residue surveillance at the Veterinary Services Laboratory

H. C. Hall, V. S. St. John, R. S. Watson, L. J. Padmore, and S. M. Parris, Ministry of Agriculture & Rural Development, Veterinary Services Laboratory, The Pine, St. Michael, Barbados

Surveillance for antibiotic residues in foods of animal origin has been ongoing at the Veterinary Services Laboratory since 1996 with approximately 4,200 tests performed on meat, milk and eggs between January 1996 and April 15th 2003. Approximately 3% of these tests yielded positive results. When milk testing commenced, 6% of the samples tested positive, however, by the year 2002 this figure had fallen to 1%. The tests are performed with a Charm II 7600 analyser and samples of meat, milk and eggs are checked for the residues of four antibiotic classes: beta-lactams, aminoglycosides, sulphonamides and tetracyclines. Chloramphenicol was recently included for meat only. The majority of meat tested has been chicken and tetracyclines have been the most common residues detected. Only one of the 730 tests which were run on chicken in 2002 recorded a positive result.

___________________________________ Key words: antibiotic residues, beta-lactams, aminoglycosides, sulphonamides, tetracyclines, chloramphenicol, Charm II 7600 analyser

The wide variety of medical and health programs on television as well as medical features in newspapers and magazines have made the general public more aware of food safety issues. Bacterial and chemical contamination of foods may happen at any stage of food production and may have lethal consequences for consumers caught unawares. The advent of globalisation has introduced major changes in business. An exporter will be more likely to trade if he can prove that his product is wholesome. On the other had, a trader can suffer great financial losses or may be put out of business if his products are found to be contaminated. Antibiotic residues in foods of animal

origin are one of the sources of concern among the public and medical health professionals (WHO/73, Bren). Many of the antibiotics used to treat bacterial infections in humans also have veterinary applications; they are used to treat infections in sick and injured animals and as prophylactics and growth promoters. In the latter two cases, the antibiotics are used at concentrations lower than those used for treatment; a potentially dangerous practice since it can encourage the production of antibiotic resistant strains of bacteria (Khachatourians, 1998 and Simonsen et al 1998). One example is the emergence of fluoroquinoloneresistant Campylobacter, one of several bacterial species that cause food poisoning in humans (Khachatourians, 1998 and Enberg et al.).

Antibiotic residues may produce allergic or anaphylactic reactions in susceptible individuals (with the sulphonamides and penicillins being of particular local importance). Some antibiotics are directly toxic, e.g. chloramphenicol which destroys blood-forming tissue. Allergic reactions and toxic side effects may have fatal consequences. Most countries have established tolerance or safe levels (T/SL) or maximum residue levels (MRL) of drugs, below which it is considered that the drug may be safely used without harming the consumer. The Codex Alimentarius Commission is the accepted international agency responsible for food safety issues and has established T/SL and MRL listings for many antibiotics (MacNeil, 1998). A country may set its own levels or may accept those set by Codex Alimentarius.

lactam kit will detect all penicillins, whether man-made (synthetic, such as Amoxycillin) or from natural sources (e.g. Penicillin G). Ceftiofur as well as all other cephalosporins are also detected since their structure is very similar to that of penicillins. In some cases a kit exists for a specific drug, e.g. Cloxacillin. Although the tests may also be performed on liver, kidney, urine or serum (Charm Sciences Protocols), lean muscle tissue is the preferred specimen type.

General Test Principle

The tests utilize microbial or antibody receptor assay technology. The sample is incubated with a binding agent (microbial cells with specific receptor sites or with specific antibodies attached) and a tracer (the radio-labelled version of the antibiotic to be detected). The amount of tracer on the binding agent is measured using a scintillation counter and is compared to a pre-determined cut-off or control point. If contaminating antibiotic is present, it will prevent the binding of the tracer by occupying the receptors on the binding agent (Figure 1). The less labelled tracer detected, the more contaminating antibiotic there is present in the sample. Charm II tests are very sensitive and have been compared favourably to high performance liquid chromatography (HPLC) (Anderson et al.) The tests are capable of detecting antibiotic residues at or below the MRLs and tolerance levels established by some countries (Table 1). VSL currently conducts surveillance for residues of four (4) antibiotic classes: beta-lactams, aminoglycosides (gentamycin/neomycin type and streptomycins), sulphonamides and tetracyclines. Testing for chloramphenicol was recently included as part of an International Atomic Energy Agency (IAEA) project: The Development of Strategies for the Effective Monitoring of Veterinary Drug Residues in Livestock and Livestock Products in Developing Countries. 2

A residue testing system was purchased from Charm Sciences Incorporated (Malden, Massachusetts USA) by the Barbados Egg and Poultry Producers Association for the Veterinary Services Laboratory (VSL) in 1996. The system purchased by the VSL is the same standard type used at six U. S. Food and Drug Administration (FDA) laboratories, 55 state laboratories and seven US Army facilities, as well as by industry and regulatory agencies for international trade (Charm Sciences). The detection capabilities can be adjusted to meet specific regulations of importing/exporting countries (e.g. Australia, New Zealand). Many of the Charm II tests for milk have been approved as adequate screening tests by the FDA and NCIMS (National Conference of Interstate Shipments) (Charm Sciences). Antibiotics are placed into categories or families based on the compound from which they are derived. Charm II test kits are designed to detect all antibiotics of a specific family. For example, the beta-


Sample containing antibiotic




Binding Agent

Most sites filled by tracer CPM high

Figure 1. Charm test principle

Many sites filled by unlabelled antibiotic PM low

Table 1. Antibiotic detection limits and drugs used


Beta-lactams Cloxacillin Gentamycin/Neomycin Streptomycin Sulphonamides Tetracyclines

20 Penicillin G 250 Gentamycin 500 Streptomycin 10 Sulfamethazine 100 Chlortetracycline 0.15 Chloramphenicol

4.5 Penicillin G 10 Cloxacillin 24 Gentamycin 25Gentamycin 10 Sulfamethazine 28 Chlortetracyclin e -

500 Streptomycin 50 Sulphamethazine 200 Chlortetracycline -


Reproduced from Charm Sciences Literature International Atomic Energy Agency (IAEA) project: The Development of Strategies for the Effective Monitoring of Veterinary Drug Residues in Livestock and Livestock Products in Developing Countries.

VSL currently conducts surveillance for residues of four antibiotic classes: betalactams, aminoglycosides (gentamycin/neomycin type and streptomycins), sulphonamides and tetracyclines. Testing for chloramphenicol was recently included as part of an

Materials and Methods

Sample Collection Meat, milk and egg samples were submitted by Animal Health Assistants (AHAs) of the Ministry of Agriculture and Rural Development, Environmental Health Officers (EHOs) of the Ministry of Health, private farmers, companies and veterinarians. Samples were collected at meat processing plants, farms, shipping containers inspected by AHAs and occasionally at post mortem by the Veterinary Pathologist. Lean muscle tissue of table quality was submitted fresh, cool (at 2 - 6C) or frozen. At least 60g of chicken, pork, beef and lamb are required for a test profile. Milk was submitted cool (at 2 - 6C) or frozen in plastic disposable 50ml centrifuge tubes or in 100ml WhirlPak bags. Fresh table eggs were submitted in egg crates at room temperature. Reagents, Supplies and Equipment Test kits comprise of tablet reagents for an antibiotic family, positive control standards, zero or negative control standards and buffer solutions in the case of meat and eggs. Also needed are scintillation fluid, distilled and deionised water and sodium hydroxide.

tube of homogenate was cooled in an ice water bath for 10 minutes then centrifuged at high speed for 10 minutes. The resulting supernatant was decanted into another centrifuge tube for use in testing and the meat pellet was discarded. Milk Sample Processing using Charm Sciences Protocols 9-14 Frozen milk was thawed completely and thoroughly mixed. Both fresh and thawed milk were centrifuged at high speed for 5 minutes to bring the butterfat to the surface. The samples were cooled in a freezer for 45 minutes before the fat layer was removed. The defatted milk was kept at 2 - 6C until ready for testing. Egg Sample Processing using Charm Sciences Protocols1-2 Eggs were broken into individual plastic disposable 150ml cups. Yolk and white of each egg were blended together using a spatula. 10ml of the mixture were poured into individually labelled plastic disposable centrifuge tubes, which were placed in a boiling water bath for 6 minutes. The heated egg was allowed to cool slightly then transferred to a mini food processor. 30ml of MSU buffer solution were added and the whole blended for 30 seconds. The suspension was returned to the centrifuge tube and then centrifuged at high speed for 10 minutes. The resulting supernatant was decanted into another centrifuge tube for use in testing and the egg solids were discarded.

Sample Processing
Meat Sample Processing using Charm Sciences Protocols 3-8 Frozen meat was thawed completely. The tissue (fresh or thawed) was trimmed of excess fat and approximately 10g (20g for new chloramphenicol method) added to 30ml (20ml for chloramphenicol) MSU buffer in a plastic disposable centrifuge tube. The meat was homogenised in the buffer using a mini food processor and returned to the tube. This homogenate was placed in an 80C dry bath for 30 or 45 minutes, depending on which test was being conducted. After incubation, the

Test Procedure
The basic test procedure is similar for all the drugs and for meat, milk and eggs. Procedures are either sequential or competitive. In sequential procedures, the sample is first incubated with the binding agent then re-incubated when the tracer has been added. In competitive procedures sample and tracer are added together and there is one incubation period. Other differences between tests are the volume of extract used, length of

time extracts are incubated as well as incubation temperatures. Tests were performed following the protocols given in the Charm Operator Manuals received with each test kit. 1-14

residue surveillance program until April 2003, a total of 4,264 tests were performed, 151 of which yielded a positive result (Figure 2). 1722 of these were done on meat, 2335 on milk and 195 on eggs. Figure 3 also shows the type of meat being tested

From the beginning of the antibiotic

5000 4000 3000 2000 1000 0


Figure 2. Total tests and positives, November 1996 to April 2003

2500 2000



1500 1000 500

50 87 14 195









Figure 3. Tests done by sample type. January 1996 April 15th 2003

The number of tests performed for each antibiotic type and for each type of sample are shown in Table 2.

The details of the positive tests number of positives for antibiotic type and sample type are given in Table 3.

Table 2. The number of tests performed for each antibiotic type and for each type of sample. TESTS Antibiotic Milk Chicken Pork Beef Mutton Turkey Eggs TOTAL

Beta-lactams Cloxacillin

405 377

313 86 50

15 2


749 377 88

Enrofloxacin Gentamycin / Neomycin Gentamycin / Streptomycin Macrolides Novobiocin Sulphonamides Tetracyclines TOTAL 367 396 4 4 405 377 2335

4 11 15 2 21 21 87 3 3 14 3 3 12 4 2 2 6 10 0 0 10 10 50 50 50 41 54 195

54 610 790 54 4 750 784

224 313 2 267 316 1571


Table 3. Number of positives recorded according to antibiotic and sample type Antibiotic Milk Chicken Pork Beef Turkey Eggs TOTAL

Beta-lactams Cloxacillin Gentamycin / Neomycin Gentamycin / Streptomycin Sulphonamides Tetracyclines TOTAL

26 30 30 2 4 17 109

1 7 0 1 12 21

0 3 0 2 4 9

2 0 0 0 1 3

0 0 0 2 1 3

0 0 6 6

29 30 40 2 9 41 151

Initially, the only residues being monitored were sulphonamides and tetracyclines. A wider range of tests was introduced in 1997. Macrolide testing was done briefly but was stopped after a list was compiled of the antibiotics most often used by veterinarians in Barbados (Table 4).

Tetracyclines were the most commonly detected residues in meat - 18 positives total, 12 in chicken, 4 in pork and 1 each in beef and turkey (Figure 4). Chicken was the meat sample type most frequently tested. A total of 1,571 tests were performed on chicken over the period of discussion. The per capita

consumption of chicken in Barbados is the second highest in the world after India (White, A Travellers Tale Barbados), and most of the monitoring was

performed on this tissue (Figure 5). Other types of meat were also tested pork, turkey, beef and mutton, in descending order of quantity

Table 4. Tests performed by antibiotic and tissue type Chicken Pork Mutton Beef Turkey

Beta-lactams Enrofloxacin Gentamycin / Neomycin Gentamycin / Streptomycin Macrolides Tetracyclines TOTAL

313 50 224 313 2 316 1571

15 2

10 4

Chloramphenicol 86

11 15 2 21 21 87

2 2 3 3 12 4 3 3 14

6 10 10 10 50

Sulphonamides 267

25 20 15 10 5 0
7 12


Beta-lactams Gentamycin / Neomycin

9 4 2 2 1

3 3

3 1 1

Tetracyclines TOTAL

2 1





Figure 4. Residues detected in meat according to sample type

The number of chicken meat samples received for residue testing increased in 1999 with the start of the VSLs food safety project: A Monitoring Programme for Veterinary Drug Residues and

Pathogenic Bacteria in Foods of Animal Origin. The number of positives detected in chicken has declined from approximately 3% at the beginning of the monitoring project to less than 1% in 2003

1600 1400 1200 1000 800 600 400 200 0 1996 1997 19971998 19981999 19992000 20002001 20012002 20022003 9 4 12 1 283 9 202 3 7 1 331 730



Figure 5. Number of tests and positives for chicken, November 1996 to April15th 2003

A total of 2,335 tests were conducted on bulk tank and individual cow samples between 1997 and 2003 (Figure 6). At the start of the residue surveillance program, approximately 6% of samples tested positive for various residues. By

the end of 2002, the figure had declined to approximately 1%. Figure 7 shows tests performed for the various antibiotic classes. Four tests each were performed for Novbiocin and Macrolides. Tests for these two antibiotics ceased after the compilation of a list of drugs most often used to treat dairy cattle

2500 2000 1500 1000 500 0

1997- 1998 1998- 1999 1999-2000 2000- 2001 2001- 2002 2002-2003


616 235 15 38

524 30

444 17

492 5 24 4 109

Figure 6. Antibiotic residue tests in milk, 1997 April 2003



Beta-lactams Cloxacillin



Gentamycin/Streptomycin Macrolides
405 377 367 396 405 377


Novobiocin Sulphonamides Tetracyclines

500 4 0

Figure 7. Antibiotic tests by drug class, 1997 2003

Most of the positive results recorded were for beta-lactams (combined total beta-lactams and cloxacillin, 70), followed by tetracyclines (40) and gentamycin/neomycin-type aminoglycosides (40) (Figure 8).

A total of 195 tests were performed on eggs. Of these, six yielded a positive result (Figure 9).

However, these results are biased since most of the tests were not done on random samples, but rather on eggs from layers after treatment during the moult. Samples are submitted to certify that all antibiotics (namely tetracyclines) have left the birds systems and are no longer contaminating the eggs When the screening of eggs began, it was decided that four categories of antibiotics would be monitored. These were macrolides, streptomycin, sulphonamides and tetracyclines (Figure 10).



Beta-lactams Cloxacillin Gentamycin/Neomycin Gentamycin/Streptomycin

6% 1% 26%


Sulphonamides Tetracyclines

Figure 8. Antibiotic residues detected in milk 1997 2003 by percentage

Tetracyclines 27%

Gentam ycin / Streptom ycin 26%

Sulphonam ides 21%

M acrolides 26%

Figure 9. Residue tests in eggs, 1999 - 2003

200 150 104 100 54 50 0 27 6 1999-2000 2000-2001 2001-2002 10 2002-2003





Figure 10. Antibiotic classes tested in eggs, 1999 2003

The incidence of contamination in both chicken and milk has declined since the monitoring system came into effect. More comprehensive testing of other meat types and eggs will be needed before any conclusions can be drawn

about these animal derived foods. Very few samples from imported meats have been tested and therefore their residue status remains unknown. Smaller poultry producers whose samples were submitted by the EHOs from the Ministry of Health were investigated and counselled when positives were reported from their farms (personal communication). It


should be noted that most of these samples were collected from small poultry farmers in the St. Thomas area. Antibiotics are only routinely added to animal starter feeds manufactured in Barbados because young animals are susceptible to diseases which can result in serious intestinal problems such as scouring (diarrhoea) (Pinnacle Feeds, 2002). The Pine Hill Dairy (PHD) routinely screens bulk tank milk for beta-lactams and tetracycline residues using different screening tests from those employed at VSL (Idexx SNAPTests, Idexx Laboratories Inc., Maine USA). Two or three dairy farms are screened at random on a daily basis. If the milk tests positive, it is rejected on the spot. Bulk tank milk is tested daily as it is brought into the PHD. If a tank tests positive, two more tests are conducted. Should the milk still register as positive, milk from the individual farms is tested to identify the violator. Once the positive farm is known, milk from this source is rejected and screening is conducted daily until residue test results are negative for three consecutive days (Alkins, personal communication). If there is some question as to the veracity of a result, a farmer will on occasion submit a sample for independent testing at the VSL. The status of antibiotic residues in eggs is unclear except from those farms tested by the VSL. Antibiotics are administered prophylactically when the layers are forced to moult to prevent ovarian infections to which the birds are more susceptible to at this time. The submission of samples from other sources would give a clearer picture of the use of antibiotics on other farms. Antibiotic residues can be prevented from entering the food chain at the producer level if farmers and processors are educated about the potential hazards associated with antibiotic residues in foods

of animal origin. Producers and processors should also be made aware of the financial losses they may incur as a direct result of having product dumped, or not being able to trade as a result of contamination, which has happened to farmers in Europe and the USA (FDA 2002). There are now several programs in the USA and Europe which make use of HACCP principles in quality management systems for farms, e.g. Milk & Dairy Beef Quality Assurance Program Milk and Dairy Beef Residue Prevention Protocol. In this program, the farmer works closely with a veterinarian and follows a 10-step process to minimise the risk of antibiotic residues entering the food chain. The Pine Hill Dairy recently held a series of seminars the theme of which was the above quality assurance program. At present, Barbados has no legislation governing the sale or use of veterinary drugs (Gary, 2001), and there are no set safe/tolerance levels or maximum residue limits for veterinary drugs in foods of animal origin; such legislation needs to be enacted. Medication for use in animals may be purchased over the counter. Restriction of drug sales to a licensed veterinarian could greatly reduce the indiscriminate and sometimes incorrect use of antibiotics.

Antibiotic residue testing should be increased for eggs and meat samples other than chicken. Imported meat and meat products also need to be tested.

The authors would like to thank the following for their assistance and communications: Mr. Bryan Alkins, Farm Liason Manager,


Pine Hill Dairy, Mr. Geoffrey Goddard, Chickmont Foods, Mr. Steven Holmes, Technical Assistance Director, Charm Sciences, Mr. Brad Johnson, Technical Assistant, Charm Sciences, Ms. Jennifer Melanson, Technical Assistant, Charm Sciences, Ms. Andrea Osbourne, Environmental Health Officer, Ministry of Health, Mr. Bryan Sanford, Senior Animal Health Assistant, Ministry of Agriculture, Mr. Adrian Yarde Extension Officer, Roberts Manufacturing Company, Farmers, veterinarians and others who made contributions in any way.

8. Charm II Test for Tetracyclines in Tissue, Serum and Urine. Operators Manual TETRA10.DOC (JAN 2003) 9. Charm II Beta-lactam Test for Amoxycillin, Ampicillin, Ceftiofur, Cephairin and Penicillin G (Competitive Assay). Operators Manual Validated for Raw, Commingled Bovine Milk. Potocol ID BLCOM01 (JULY 2001) 10. Charm II Test for Cloxacillin (Competitive Assay). Operators Manual Validated for Raw, Commingled Bovine Milk. Protocol ID CLXCOM6 (DEC 2000) 11. Charm II Aminoglycoside Test for Gentamicin and Neomycin at U.S. Tolerance/Safe Levels (T/SL) or Maximum Residue Limits (MRL) (Competitive Assay) Operators Manual for Milk. Protocol ID Gent comp 06.doc (May 2001) 12. Charm II Aminoglycoside Test for Gentamicin, Streptomycin and Dihydrostreptomycin (Competitive Assay). Operators Manual for Raw, Commingled Bovine Milk. Protocol ID STCOM06 (10/31/00) 13. Charm II Sulfa Drug Test for Sulfadiazine, Sulfadimethoxine, Sulfamethazine and Sulfathiazole (Competitive Assay) Operators Manual Validated for Raw, Commingled Bovine Milk . Protocol ID fdasulf2 (DEC 2000) 14. Charm II Tetracycline Test for Chlortetracycline, Oxytetracycline and Tetracycline (Competitive Assay). Operators Manual for Raw, Commingled Bovine Milk. Protocol ID TETCOMDIL02 (JULY 2001)

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Charm II 7600/6600 System


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Appendix A
Table 1. Charm Food Safety Tests: Charm II Testing Range*
Muscle Urine Serum Egg Feed
Product Contact Surface Clear Juice, Water, Beer & Wine




Farmed Fish

Fruits & Vegetables

Ground Cooked Meat & Seafood Nuts

Sulphonamides Beta-lactams Tetracyclines Macrolides Amphenicols Aminoglycosides: Streptomycin type Gentamycin type Spectinomycin Novobiocin Aflatoxins Pasteurisation Efficiency1 Pesticides ATP/Hygiene Microbial Quality Charm Heat Efficiency Test 2












X Measures alkaline phosphatase activity of raw milk in pasteurised milk 1(PasLite) 2Measures phosphatase activity of raw meat on surfaces and in cooked samples (CHEF Test)