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Guidelines for Registration of Intramammary Preparations for Treatment of Bovine Mastitis

Guideline No. 25
Last revised August 1996

Contents

Part 1

A GENERAL COMMENTS

The following protocols are designed to service as a guide for commercial organisations aiming to produce field trial data to support registration of Intramammary products for the treatment of bovine mastitis.

It is recommended that the company applying for product registration contract the product evaluation to an institution which can service the contract under the direction supervision of a consulting veterinary surgeon. The consulting veterinarian should have experience in mastitis research, investigation and, if possible, product testing, and will be responsible for the quality control of the treatment and mil collection procedures during the product evaluation.

An alternative arrangement would be for a private veterinarian to accept the contract and then sub-contract the microbiology and clinical pathology to a government, university or suitably accredited private pathology laboratory. Regardless of the arrangement for conducting the trial, the trial must be approved by an appropriate Animal Care and Ethics Committee.

The relevant local government adviser (Veterinary Officer, Diary Adviser, Animal Health Officer) and private veterinarian should be consulted during the design of the evaluation process and during selection of suitable farmers and herds for the study. Farmer and herd selection should take into account the following factors:

  • Farmer’s cooperation (farmers should be fully informed as to the experimental design and the procedures involved in the trial before obtaining their cooperation).
  • Previous experience of the farmer in sterile milk collection
  • Herd size
  • Type of milking shed
  • Involvement in herd teating, bulk milk cell counting, individual cow cell counting (ICCC) for monitoring sub-clinical mastitis levels, antibiotic sensitivity patterns of isolates of Staphylococcus aureus from the herd, as well as good records of cases of clinical mastitis and treatment (and response) are all important
  • Active involvement in a mastitis control program supervised by a veterinarian
  • Active implementation of a culling policy
  • Availability of a regional veterinary laboratory, or bacteriology laboratory with expertise and interest in veterinary mastitis research and/or investigation

Use of seasonal calving herds, particularly for evaluation of dry cow products, may be more convenient for coordinating treatments and sample collection. However, herds calving year round may also be used.

Residue trials, if necessary, are best undertaken in experimental herds on dairy research institute farms and under veterinary supervision.

B PRINCIPLES

B1 The trial should use cases of clinical or sub clinical mastitis which can demonstrate the effectiveness, or otherwise, of the test product. Chronic or recurrent cases should be eliminated from the trial.

B2 The number of false positive cases of clinical and sub-clinical mastitis that are included in the trial should be minimal. Effective use of herd records, farmer and laboratory expertise, and training in sample collection technique will minimise the inclusion of false positives in the trial.

B3 Specified withholding periods should be based on trial data, and be consistent with Maximum Residue Limits.

C LABEL GUIDELINES FOR INTRAMAMMARY INFUSIONS

C1 Introduction

The following are guidelines for labels of Intramammary products. These guidelines should be read in conjunction with the Code of Practice for Labeling of Veterinary Chemical Products.

C2 General

C2.1 The Label should clearly state, as part of the product name an in the label claim, that the product is for “lactating cows” or “dry cows”

C2.2 Industry accepted coloured lettering is to be used in the product name denoting “lactating cow” in blue, and “dry cow” in red

C2.3 A withholding period (WHP) statement for both meat and milk must be included on all labels (syringe, carton, and leaflet). It should be worded in accordance with the Code of Practice for Labeling of Veterinary Chemical Products.

  • The required milk withholding period statements for milk are specified under the headings C3 ‘Dry Cow Products and C4 ‘Lactating Cow Products
  • It is essential to include a slaughter WHP so that meat from treated cows does not exceed the maximum residue level. This statement will be different from the milk withholding statement, and should read as follows:

DO NOT USE less than […] days before slaughter for human consumption

C3 Dry Cow Products

C3.1 The directions should clearly specify that the product is formulated for use in cows at the end of lactation, i.e: dry cow therapy

C3.2 Explicit directions should be given on the following points:

  • Indications for treatment
  • Time for treatment (immediately after lasting milking of seasons)
  • Disinfection of teat
  • Sanitary handling of product (the wording may vary depending on the type of container)
  • Administration of product, including instructions on milking out, or not milking our, the udder prior to administration
  • Massaging of teat and udder
  • Post treatment precautions
  • Conditions for storage of products

C3.3 Directions based on residue analysis in a significant number of animals should be given as to the minimum number of days of dry period

C3.4 The statement

“DO NOT USE in lactating cows or within […] days of calving” must appear on all labels as a restraint statement following the directions for use heading

C3.5 Unless residue analysis to the contrary is provided, a carton and/or leaflet will need to have a statement to cover inadvertent use of dry cow therapy in lactating animals or prior to calving. This is to appear in the directions of use information and read as follows:

If [product] is accidentally administered to a lactating animal or within […] days of calving, contact the [product distributor/prescribing veterinarian] for advice.

C3.6 It is essential to include a meat withholding period statement for cow’s intended for slaughter as outlined in Section C2.3

C3.7 The appropriate milk withholding period for dry cows is to read as follows:

DO NOT USE in lactating cows or within […] days of calving. After calving, colostrum or milk from treated dry cows MUST NOT BE USED for human consumption or processing for […] hours ([…] milkings).

Very small labels may be able to have a variation to the above statement but this will be assessed on a case by case basis by the NRA.

C4 LACTATING COW PRODUCTS

C4.1 The label should include directions on the following points:

  • Indications for treatment
  • The optimal treatment regime, that is, the number of treatments
  • The interval between treatments, and milking out instructions
  • Disinfection of the teat
  • Sanitary handling of product
  • Administration of the product
  • Massaging of quarter if necessary
  • Teat dipping
  • Conditions for storage of product

C4.2 The milk withholding period for lactating cow products should be stated in both hours and milking, e.g:

Milk collected from cows within […] hours ([..] milkings) following treatment MUST NOT BE USED for human consumption or processing. This milk should not be fed to bobby calves.

C4.3 It is essential to include a WHP so that meat from treated slaughtered cows does not exceed the maximum residue level. This statement will be as noted in C2.3.

D REFERENCES

Blood DC and Radostits OM (1989) in Veterinary Medicine, 7th Ed, Bailliere Tindall, London

Browning JW, Mein Ga, Barton M, Nicholls TJ and Brightling P (1990). Efficacy of antibiotic therapy at drying off on mastitis in the dry period and early lactation. Aust Vet J.. 67:440-442

Browning JW, Mein Ga Brightling P, Nicholls TJ and Barton M. (1994). Strategies for mastitis control: dry cow therapy and culling. Aust Vet J.. 71:179-181

Dawson DJ and Feagon JT (1960). The use of Brilliant blue F.C.F. in Intramammary penicillin Preparations. Aust J Dairy Tech., 15:160

FAO. Specifications for the identity and purity of some antibiotics. FAO Nutrition Meeting Report Series No. 45A, WHO/Food Add./69:34

Feagon JT, Griffin AT and Bray R (1965). An improved test for detection of marker dyes in milk. Aust J Dairy Tech., 20:22

Naylor J (1960). The incidence of penicillin in Australian milk supplies. Aust J Dairy Tech., 15:153

Neave FK (1975). Diagnosis of mastitis by bacteriological methods alone. In proceedings of the International Dairy Federation Seminar on Mastitis Control, Brussels, Belgium, 1975

Novak NF, Filmore TM and Parson (1984). Dyke marked antibiotics for lactating cow mastitis therapy. J Dary Sci, 67:1841

Sears PM, Gonzalex RN, Wilson DJ, and Han HR (1993). Procedures for mastitis diagnosis and control. In “The Veterinary Clinics of North America, Volume 9, Number 3”: Food Animal Practice, Update on Bovine Mastitis, ed Anderson KL

Snedecor GW and Cochran WC. In “Statistical Methods” Iowa State University Press, Ames Iowa, USA

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PART II

GUIDELINES FOR TESTING INTRAMAMMARY ANTIBIOTICS FORMULATED FOR USE IN NON-LACTATING (DRY) COWS

D Introduction

D1.1 The purpose of testing dry cow products is to establish the efficacy of the product in eliminating specified bacterial pathogens from infected quarters during the non-lactating period. Validation should be conducted in commercial herds using sufficient numbers of sub-clinically infected quarters to yield statistically significant results.

D1.2 The principle for testing dry cow antibiotics is to compare the efficacy of the test product with that of an accepted and proven dry cow preparation. Trial incorporating an untreated control group of infected quarters will usually decrease the precision of therapy trials owing to the problems associated with spontaneous recovery in untreated quarters. Comparison of the test product with a reference product minimises the difficulties due to diagnostic errors and herd differences in response rate, and makes possible comparisons between trials. It also overcomes the reluctance of the farmer to leave an infected quarter untreated.

D1.3 The efficacy of the product in preventing acquisition of new infections during the dry period will need to be evaluated if the applicant wishes to market the product with such a claim.

D1.4 Whilst Australian efficacy data is desirable, it is not mandatory. However, local trials may be necessary if overseas efficacy data does not address the Australian criteria, particularly in the area of trial design, bacterial strains, bacterial definition of infection and sampling protocols.

D2 Selection of Cows

Cows will be selected for inclusion in the trial on the basis of a high probability of their being sub-clinically infected. Individual cows included in the trial should be reliably identified (e.g: by heel strap, ear tag, freeze brand)

D2.1 Cows can be presumptively classified as sub-clinically infected in the period two weeks before drying off using herd records (ICCC, Rapid Mastitis Test, conductivity and/or enzyme analysis). This analysis should be made by the consulting veterinary surgeon in consultation with the farmer. Cows treated with dry cow therapy in the previous dry period for clinical or sub-clinical infection should be excluded. Cows treated in the current seas for clinical mastitis, but not treated for infection in the previous dry period, may be included for consideration as candidates for bacteriological testing.

D2.2 The infection status of cows identified in D2.1 should be evaluated by bacterial culture of quarter samples. Cows should be sampled by an experienced milk sampler, ideally at three consecutive milkings or on three consecutive days, using the technique described in D3.4. Three samples collected over a five day period would be acceptable although less desirable.

D2.3 The last sample should be taken five clear working days before the prospective dry cow treatment is to be administered so as to allow final laboratory compilation of results, interpretation of findings, and allocation of cows to trial groups.

D2.4 Milk samples submitted for bacteriological examination should be processed as described in D3.5. Cows should be classified as infected on the basis of two out of three bacteriological culture of the aseptically collected milk proving positive for the same bacterial mastitis pathogen (Neave, 1975). This approach produces a 1% false positive rate and will allow for optimal assessment of product efficiency.

D2.5 Evaluation of antibiotic products for effectiveness in preventing new infections in the dry period is not indicated under most Australian conditions. However, if deemed necessary, selection of cows for this component of product performance should be undertaken by presumptive identification of a group of uninfected cows by reference to herd records and ICCC/enzyme analysis as in D2.1, and made available for bacteriological examination as in D2.2. Half these cows can be treated with product and half can serve as untreated controls. Such a study will likely require a large sample size to detect significant differences between treated and untreated animals, since the incidence of new infections in the dry period is unlikely to be high.

D3 Experimental Design

The objective of testing is to assess the effectiveness of the product administered during the dry period in removing sub-clinical infections caused by specific bacterial pathogens. The efficacy of the product will be assessed by measuring the rate of intramammary infections in infected cows following treatment. The product under test (Treatment) will be compared with a commercially available antibiotic product (Control), the efficacy of which has been demonstrated by extensive use in field trials under varied conditions. If practicable, the trial should be conducted as a “blind” trial, by having the tubes containing the Treatment and Control preparations unbranded and identified only by a code letter or number that is assigned and known only by an individual not involved in administering the product to the experimental animals. The tubes containing Treatment and Control preparations should be of similar nozzle design, and both preparations should be administered similarly with respect to site of deposition within the teat canal, to minimise differences in the rate of acquisition of new infections associated with treatment technique.

D3.1 Treatment Groups

D3.1.1 Infected cows should be randomly assigned to two groups of equal size (Treatment and Control). Details of parity and quarter infection rate should be recorded for each animal. Since individual animals may have from 1-4 quarters classified as infected, the final number of infected quarters in the treatment and control groups may differ slightly.

D3.1.2 Cows in the Treatment group will received a dose of the test compound in all four quarters, while cows in the Control group will receive a dose of the reference product in all four quarters.

D3.2 Sample Size and Statistical Analysis

The experimental unit for this trial should be an individual quarter. Treatment and Control preparations should be randomly allocated to infected cows. Data should be collected from a minimum of 3 herds. A sufficient number of quarters should be tested overall to demonstrate that the efficacy of the test product is within 10% of that of the control product where the efficacy of the control product is at least 40% (that is, the proportion of quarters that show a bacteriological cure following treatment with the test product should be no more than 10% below the proportion of quarters showing a bacteriological cure following treatment with the control product). As a guide, the z-test comparison of proportions (Snedecor and Cochran) is an appropriate statistical test to use to compare treatments, and the sample size should be established to ensure that the power of the test is at least 0.80, with a set at 0.05. Under these conditions, Figure 1 Represents the sample sizes (number of quarters per experimental group) required to compare treatment efficacy for different levels of efficacy expected for the control product.

D3.3 Treatment Method

Treatment should be supervised by the consulting veterinarian (or their delegate to the satisfaction of the consulting veterinarian). This step is critical to the success of the trial, as errors of identification and aseptic technique are most likely to occur at this point.

D3.3.1 Before treatment can be instituted, the teats must be cleaned effectively. Washing should be restricted to the teats and the base of the teats only, to minimise contaminating run off after the teats have been dried. Excess water should be dried off, using a separate paper towel for each cow.

D3.3.2 The teat furthest from the operator should be wiped with an individual tissue soaked (not dripping) in 70% alcohol. Laboratory bench wipes are most suitable as they contain little lint, are strong and do not hold excessive alcohol. A downward motion from the base of the teat to the end should be used.

D3.3.3 The end of the teat should be turned towards the operator and, using a clean section of tissue, the teat orifice should be cleaned with at least twenty circular scrubbing motions. The cleaning of each teat and teat orifice should proceed accordingly, cleaning first the teat furthest from the operator, before moving to the nearest, to minimise the change of contamination through accidental hand contact with disinfected teat orifices.

D3.3.4 The contents of the appropriate tube or syringe should be inserted into the teat. The teat orifice should be held closed while the preparation is massaged up into the streak canal and quarter to ensure adequate dispersion.

D3.3.5 The teats should then be sprayed or dipped using a registered teat disinfectant.

D3.3.6 All treated cows should be inspected 24 hours post treatment for signs of peracute Intramammary infection, and then at last weekly for the duration of the trial to monitor for the presence of adverse effects associated with treatment.

D3.4 Sample Collection and Submission

Resolution of infection due to dry cow therapy should be assessed by measuring the infection status of treated cows in the early post-partum period. The arrangements for sample collection for experimental animals in the period immediately following calving should ensure that:

  • Cows are first sampled ideally at the first milking after calving, and at least within 2 days of calving
  • Samples are collected at 3 consecutive milkings, or on 3 consecutive days
  • Samples are collected by a trained operator (veterinarian, laboratory staff, farmer)
  • Samples reach the laboratory no more than 24hrs after collection, following storage and transport at 1-6°C

Professional collection teams, (veterinary practitioners, government field staff or laboratory staff) are best involved in milk collection if large numbers of cows are to be sampled together, to minimise disruption to the milking schedule and to streamline the collection process.

D3.4.1 Laboratory submission sheets should be designed that give details of arm, cow and quarter identification, date of calving, date and time of collection of the sample, and any abnormal findings. These sheets will facilitate the collection process for the farmer, while ensuring adequate records for the evaluation of the product.

D3.4.2 Careful planning of an identification systems for collection tubes will minimise workload during collection and will limit the likelihood of confusion of samples after collection.

D3.4.3 To collect the samples, the teats should be cleaned as described in section D3.3.

D3.4.4 Samples (of approx. 5 to 7 mL) should be collected from each quarter into a sterile 10 mL tube. The closest teat must be sampled first to prevent accidental contamination of the teat orifice by the milk collector’s hand.

D3.4.5 The first two squirts of milk should be discarded in order to flush organisms from the teat canal. Then the sample should be drawn into the tube, which should be held nearly horizontal, about 2 cm from the tip of the teat. The stream should be directed against the wall of the sample tube to prevent frothing. Samples should be stored at 1-6°C as soon as possible after collection.

D3.5 Bacteriology and Data Recording

Standard aseptic technique must be observed in processing samples in the bacteriology laboratory.

D3.5.1 The contents of each sample container should be resuspended immediately before sampling. One hundred microlitres (100µl) of milk from each quarter sample should be inoculated onto a sheep blood agar plate (1 sample per plate) and spread over the surface of the plate.

D3.5.2 Plates should be incubated at 37°C for a minimum of 18 hours. After incubation, bacterial colonies should be identified, and presumptive pathogens identified to the species level. The results of primary culture (number of colony types and approximate number of colonies) should be recorded. These results should be used early in the trial to identify and correct any problems in sample collection technique that may manifest in a high rate of contamination of milk samples.

D3.5.3 The presence of one or more colonies of a mastitis pathogen (commonly Staphylococcus species or Streptococcus species; (Blood and Radostits, 1989) is considered indicative of infection. A quarter should be recorded as infected if at least two of the three milk samples collected immediately post-calving prove positive on culture with at least one colony of the same pathogen. A quarter should be recorded as uninfected if no more than one of the three samples proves positive on culture for a bacterial pathogen.

D3.5.4 The antibiotic sensitivity of all bacterial isolates selected from pre-treatment samples should be evaluated and the results recorded. Standard MIC or Disc Diffusion methodology can be used for this purpose.

D3.5.5 An individual cow and quarter record should be compiled for each cow in the trial, and should contain the following information:

  • Farm identification
  • Cow identification
  • Age
  • Lactation
  • Production records
  • Incidence of clinical mastitis
  • Previous dry cow treatments (refer to D2.1)
  • Bacteriology results for the pre-treatment samples
  • Somatic cell count/enzyme analysis results for the pre-treatment (pre-calving) samples
  • Antibiotic sensitivity profile (including with respect to the test product) for isolates of Staphylococcus aureus, or other nominated pathogen
  • Final classification of the results into cure or non cure
  • Adverse effects associated with treatment

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PART III

GUIDELINES FOR TESTING INTRAMMARY ANTIBIOTICS FORMULATED FOR USE IN LACTATING COWS

L1 Introduction

L1.1 The purpose of testing lactating cow products is to evaluate the efficacy of the product in eliminating specified bacterial pathogens from clinically infected quarters during lactation. Testing should be conducted in commercial herds using sufficient numbers of clinically infected quarters to yield statistically significant results.

L1.2 The principle of evaluating lactating cow antibiotics is to compare the efficacy of the test product with that of an accepted and proven lactating cow preparation. Trials incorporating an untreated control group of infected quarters will usually decrease the precision of therapy trials owing to the problems associated with spontaneous recovery in untreated quarters. Comparison of the test product with a reference product minimises difficulties due to diagnostic errors and herd differences in response rate, and makes possible comparisons between trials. It also overcomes the reluctance of farmers to leave an infected quarter untreated.

L1.3 Whilst Australian efficacy data is desirable, it is not mandatory. However, local trial may be necessary if overseas efficacy data does not address the Australian criteria, particularly in the area of trial design, bacterial strains, bacterial definition of infection and sampling protocols.

L2 Selection of Cows

Cows with clinical mastitis will be selected for inclusion in the trial on condition of having no history of treatment in the previous dry period for clinical or sub-clinical infection, or previously treated clinical mastitis. Individual cows included in the trial should be reliably identified (e.g: by heel strap, ear tag, freeze brand).

L.2.1 Farmer assessment of clinical condition should be used to include animals in the trial. However, retention of all animals in the trial should be based on visual and microscopic assessment of milk, and test results including bacteriological culture and somatic cell count. The presence of pathogenic Staphylococcus species or Streptococcus species, or other mastitis pathogens, associated with high somatic cell count and/or abnormalities in the milk sample, should be used to classify a quarter with clinical mastitis. In cases of severe clinical mastitis, the farmer’s veterinarian should be consulted.

L2.2 A sample of milk should be collected, before treatment, from each quarter of cows identified with clinical mastitis (see L3.4), and submitted for laboratory examination.

L3 EXPERIMENTAL DESIGN

The efficacy of the product will be assessed by measuring the rate of Intramammary infections in infected quarters treated during the lactation period. The product under test (Treatment) will be compared with a commercially available antibiotic product (Control), the efficacy of which has been demonstrated by extensive use in field trials under varied conditions. If practicable, the trial should be conducted as a “blind” trial, by having the tubes containing the Treatment and Control preparations unbranded and identified only by a code letter or number that is assigned and known only by an individual no involved in administering the product to the experimental animals.

L3.1 Treatment Groups

L3.1.1 Infected cows should be randomly assigned to two groups of equal size (Treatment and Control). Details of parity and quarter infection rate should be recorded for each animal. Since individual animals may have from 1-4 quarters classified as infected, the final number of infected quarters in the treatment and control groups may differ slightly.

Note: Depending upon the experimental design healthy quarters may be treated, either L3.1.2 or L3.1.3 may be used after discussion with the NRA to decide which treatment regimen is the most appropriate.

L3.1.2 Cows in the Treatment group will receive a dose of the test compound in all four quarters, while cows in the Control group will receive a dose of the reference product in all four quarters.

L3.1.3 Alternately, cows in the treatment group will receive a dose of the test compound in the infected quarters while cows in the control group will receive a dose of the reference product in the infected quarter.

L3.2 Sample Size

The experimental unit for this trial should be an individual quarter. Treatment and Control preparations should be randomly allocated to infected cows. Data should be collected from a minimum of 3 herds. A sufficient number of quarters should be tested overall to demonstrate that the efficacy of the test product is within 10% of that of the control product where the efficacy of the control product is at least 40% (that is, the proportion of quarters that show a bacteriological cure following treatment with the test product should be no more than 10% below the proportion of quarters showing a bacteriological cure following treatment with the control product). As a guide, the z-test comparison of proportions (Snedecor and Cochran) is an appropriate statistical test to use to compare treatment, and the sample size should be established to ensure that the power of the test is at least 0.80, with α set at 0.05. Under these conditions, Figure 1 represents the sample sizes (number of quarters per experimental group) required to compare treatment efficacy for different levels of efficacy expected for the control product.

L3.3 Treatment Method

Treatment should be undertaken by the farmer, following directions provided by the manufacturer. A sample of milk should be collected before treatment (see L3.4 below) for laboratory examination to confirm the diagnosis of clinical mastitis. Treatment will normally follow milk collection; at which time the teat orifices should be sterile. If there is any doubt, the teats should be cleaned as described below (see L3.4).

L3.3.1 The contents of the appropriate tube or syringe should be inserted into the teat. The teat orifice should be held closed while the preparation is massaged up into the teat canal and quarter to ensure good dispersion.

L3.3.2 The teats should then be sprayed or dipped using a registered teat disinfectant.

L3.4 Sample Collection and Submission

Resolution of infection should be assessed by measuring the infection status of quarters following treatment. The arrangements for sample collection from animals in the trial should ensure that:

  • Samples are collected at each of three consecutive milkings, or on 3 consecutive days, starting 14 days after the last treatment
  • Samples are collected by a trained operator (veterinarian, laboratory staff, farmer)
  • Samples reach the laboratory no more than 24hrs after collection, following storage and transport at 1-6°C.

Professional collection teams, (veterinary practitioners, government field staff or laboratory staff) are best involved in milk collection if large numbers of cows are to be sampled together, to minimise disruption to the milking schedule and to streamline the collection process.

L3.4.1 Laboratory submission sheets should be designed that give details of farm, cow and quarter identification, date of calving, date and time of collection of the sample and any abnormal findings. These sheets will facilitate the collection process for the farmer, while ensuring adequate records for the evaluation of the product.

L3.4.2 Careful planning of an identification system for collection tubes will minimise workload during collection and will limit the likelihood of confusion of samples after collection.

L3.4.3 To collect the samples, the teats must be cleaned effectively. Washing should be restricted to the teas and the base of the teats only; to minimize contaminating run off after the teats have been dried. Excess water should be dried off, using a separate paper towel for each cow.

L3.3.4 The teat furthest from the operator should be wiped with an individual tissue soaked (not dripping) in 70% alcohol. Laboratory bench wipes are most suitable as the contain little lint, are strong and do not hold excessive alcohol. A downward motion from the base of the teat at the end should be used.

L3.4.5 The end of the teat should be turned towards the operator and using a clean section of tissue, the teat orifice should be cleaned with at least twenty circular scrubbing motions. The cleaning of each teat and teat orifice should proceed accordingly, cleaning first the teats furthest from the operator, before moving to the nearest, to minimise the chance of contamination through accidental hand contact with disinfected teat orifices.

L3.4.6 Samples (of approx. 5 to 7 mL) should be collected from each quarter into a sterile 10mL tube. The closest teat should be sampled first to prevent accidental contamination of the teat orifice by the milk collector’s hand.

L3.4.7 The first two squirts of milk should be discarded in order to flush organisms from the teat canal. Then the sample should be drawn into the tube, which should be held nearly horizontal, about 2cm from the tip of the teat. The stream should be directed against the wall of the sample tube to prevent frothing. Samples should be stored at 1-6°C as soon as possible after collection.

L3.5 Bacteriology and Data Recording

Standard aseptic technique must be observed in processing samples in the bacteriology laboratory.

L3.5.1 The contents of each sample container should be resuspended immediately before sampling. One hundred microlitres (100μl) of milk from each quarter sample should be inoculated onto a sheep blood agar plate (1 sample per plate) and spread over the surface of the plate.

L3.5.2 Plates should be incubated at 37°C for a minimum of 18 hours. After incubation, bacterial colonies should be identified, and presumptive pathogens identified to the species level. The results of primary culture (number of colony types and approximate number of colonies) should be recorded. These results should be used early in the trial to identify and correct any problems in sample collection technique that may manifest in a high rate of contamination of milk samples.

L3.5.3 The presence of one or more colonies of a mastitis pathogen (commonly Straphylococcus species or Streptococcus species; Blood and Radostitis, 1989) is considered indicative of infection. A quarter should be recorded as infected (non-cure) if at least two of the three milk samples collected 21 days after treatment prove positive on culture with at least one colony of the same pathogen. A quarter should be recorded as uninfected (cure) if no more than one of the three samples proves positive on culture for a bacterial pathogen.

L3.5.4 The antibiotic sensitivity of bacterial isolates selected from pre-treatment samples should be evaluated and the results recorded. Standard MIC or Disc Diffusion methodology can be used for this purpose.

L3.5.5 An individual cow and quarter record should be compiled for each cow in the trial, and should contain the following information.

  • Farm identification
  • Cow identification
  • Age
  • Lactation
  • Production record
  • Clinical appearance for each quarter or each infected quarter
  • Bacteriological results of the pre-treatment milk samples
  • Somatic cell count/enzyme analysis results for the pre-treatment samples
  • Bacteriology results from three milk samples collected 21 days after treatment
  • Somatic cell count/enzyme analysis for post-treatment samples.
  • Antibiotic sensitivity profile (including with respect to the test product) for isolates of Staphylococcus aureus, or other nominated pathogen
  • Final classification of the results into cure or non cure
  • Adverse effects associated with treatment

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PART IV

Determination of end point for antibiotic excretion in milk following Intramammary treatment of lactating cows

R1 Introduction

Antibiotic detection in the milk from udders infused with Intramammary preparations can be accomplished by direct and indirect methods. Direct methods are conventional microbiological assays where known amount of test samples are applied to bacterial cultures with known antibiotic sensitivity patterns. Direct detection of antibiotics in milk requires trained laboratory personnel and appropriate equipped laboratory.

For indirect detection of antibiotics in milk retrieved from treated udder, such preparations are “marked” with approved dyes and then infused into the mammary gland. The dye is excreted in milk with end points equal to those of antibiotic (Novak et al. 1984). The potential advantage of dye marking is that it immediately alerts the dairy farmer and the consumer to antibiotic contamination by visually discolouring milk.

This section describes detailed guidelines for dye marker techniques that should be used to assess the excretion of antibacterial agents administered to diseased lactating udders. Some manufacturers may already by in possession of such data, which can be submitted together with other data specified in previous sections of this document. However, the data should clearly indicate the procedure used to obtain the data on dye-marker trials.

R2 Objective

The aim of providing this data to demonstrate the dye marker-antibiotic relationship during excretion following treatment of the infected lactating udder.

R3 Method

R3.1 Cow Numbers

Due to considerable variations between different quarters of the udder of the same cow as well as between cows the trial should be carried out in a minimum of 6 cows.

R3.2 The use of mastitic udder trials: For practical reasons, the use of diseased quarters is not mandatory. However, at least some cows included in the trial should be selected on the basis of one or more quarters having high somatic cell counts.

R3.3 Cows selected for use in these trials should have received no antibiotic treatment for 4 weeks prior to the trials.

R3.4 Stage of lactation

Cows with average volume of milk production for the specified breed should be selected as far as possible. The submission should clearly indicate the stage of milk production and the actual yield to the nearest 0.02kg of each treated quarter in each milking during the period of the trials.

R3.5 Sampling technique

  • Efficient quarter milkers should be utilized for this purpose. Trials are to be conducted on a quarter basis, rather than a whole udder basis.
  • Cows to be used for these trials should be accustomed to the quarter milking equipment for several days prior to trials.

R3.6 The submission should give detailed milking procedures and sampling techniques used.

R4 Analysis of samples

The laboratory performing the analyses may wish to freeze the samples before testing. Analysis should not be delayed more than a few days. Care should be taken to avoid contamination with penicillinase-producing organisms that may cause gross inaccuracy of the tests. If possible, it is recommended that the participating laboratory be involved in bacteriological examination of milk samples.

R5 The number of treatments

The manufacturer should make specific recommendations concerning the frequency and duration of treatment in respect to each product. Each quarter used for this study will be treated according to these recommendations. In cases where the manufacturer does not make such recommendations, three tubes must be administered at 24 hour intervals. The first post treatment sample is taken at the first milking following the last treatment.

Three cows are to be treated in one quarter and the following samples should be collected at all post-treatments samplings:

  1. Milk from the treated quarter
  2. Pooled milk from the 3 non-treated quarters
  3. Whole udder sample

Three cows to be treated in two quarters and the following samples taken at all post-treatment samplings:

  1. Milk from first treated quarter
  2. Milk from the second treated quarter
  3. Pooled milk from the two untreated quarters
  4. Whole udder sample

R6 Duration of the trials

In case of a product, which the manufacturer wishes to put on open sale, the objective of these trials is to determine end points for excretion for both dye and antibiotic. The trial must therefore continue until these points have been reached.

In the case of a product, which is intended for sale on a prescription basis and which is not to be dye-marked, the antibiotic end point must be determined so that an appropriate withholding time can be printed on the label.

Since the trial may be concluded before analysis begins, manufacturers must draw upon previous experience and published reports to estimate the appropriate number of samples, which will be required.

R7 Measurement of dye concentration

A numerical concentration can be obtained from the number of serial dilutions with dye-free milk required to match the sample with milk standards containing known concentrations of Brillian Blue F.C.F.. Dawson and Feagan (1960) recommended 4 standards to be set up (0.25, 0.5, 0.75 and 1.0 ppm). Care should be taken to compare milk from initial milkings with standard having the highest concentration. All measurements should be made as accurately as possible.

All dye concentration measurements should be made under standard “daylight” lighting conditions; usina white containers to hold the coloured milk samples.

For the purpose of this trial, a visual end point of 0.125 ppm should be adequate. There would, however, be no difficulty in carrying further observations with one of the simple anion exchange resin methods which have been developed for use by dairy factory personnel, and which will permit detection of 0.016 ppm by untrained operators, while 0.005 ppm can be detected with refined version of technique. Such an ion exchange technique which could be adopted as a standard is described by Feagan et al (1965).

R8 Measurement of antibiotic concentration in milk

Antibiotic measurements should be made by a 2quantitative assay technique capable of detecting the antibiotic in quarter milk samples at a concentration low enough to ensure that it will not be detectable in herd milk by the most sensitive assay technique. Ideally these trials will be made by the most sensitive technique available.

If the manufacturer is at all uncertain as to the acceptability of the proposed method to be used, advice should be sought from NRA before the trials are conducted.

The following assay procedures could be employed in trials of products containing penicillin:

  1. The technique described in the document “Specification for the Identification and purity of some Antibiotics”, FAO Nutrition Meetings Reports Series No. 45A, WO/Food Add./69:34. This is a quantitative plate technique and test organism in Sarcina lutea (ATCC 9341). The method has a sensitivity of 0.01 i.u./mL for benzylpenicillin-Na in milk.
  2. The Standard Association of Australia has described a filter paper disc method (AS 1095) and the test organism recommended in Bacillus stereothermophilus. It is a quantitative technique, which enables penicillin to be detected at or above 0.0025i.u./mL in milk.

Any other technique that will enable to detect 0.005 i.u./mL or lower concentrations of penicillin will be acceptable.

R9 The submission of trial data

Complete and impartial submission of results is required. The concentration of dye-marker and antibiotic should be presented in tabular from, showing clearly the milking at which these two constituents become undetectable by the method employed.

R10 Formulation details

Full information must be provided on the following:

  1. Identity and concentration of all antibiotics and other active constituents
  2. Identity and volume of base
  3. Type of Brilliant Blue F.C.F (micronised or crystalline)
  4. Quantity of dye in each dose