Guidelines for testing of intramammary preparations for treatment of bovine mastitis

This is a guideline about the sorts of information an applicant can submit to address the safety and efficacy criteria for intramammary preparations for the treatment of bovine mastitis. It also provides guidance on how the information might be presented and analysed. For further information on the safety and efficacy criteria, refer to the guidelines on making an application.

The following protocols are designed to serve as a guide for commercial organisations aiming to produce field trial data to support the registration of intramammary products for the treatment of bovine mastitis. The guidelines for labels of intramammary products can be found under the Veterinary Labelling Code.

1. General considerations

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

The principle for testing dry or lactating cow antibiotics is to compare the efficacy of the test product with that of an accepted and proven dry or lactating cow preparation, respectively. Trials incorporating an untreated control group of infected quarters are usually less precise because of 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 farmers to leave an infected quarter untreated.

The efficacy of the product in preventing the acquisition of new infections during the dry period should be demonstrated if the applicant wishes to market the product with such a claim.

While Australian efficacy data are desirable, these are not mandatory. However, local trials may be relevant if overseas efficacy data do not address the Australian criteria, particularly in the area of trial design, bacterial strains, bacterial definition of infection and sampling protocols.

Trials should be approved by an appropriate animal care and ethics committee and conducted under the direct supervision of a veterinarian with experience in mastitis research, investigation and, if possible, product testing.

The use of seasonal calving herds, particularly for the 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.

Selection of cows

Cows should be selected for inclusion in the trial on the basis of a high probability of their being sub-clinically infected or the presence of clinical mastitis. Chronic or recurrent cases should be eliminated from the trial. Cows treated with dry cow therapy in the previous dry period for clinical or subclinical infection should be excluded.

 Individual cows included in the trial should be reliably identified (for example, by heel strap, ear tag or freeze brand).

Experimental design

The objective of testing dry cow products is to assess the effectiveness of a product administered during the dry period in removing subclinical infections caused by specific bacterial pathogens. The purpose of testing lactating cow products is to evaluate the efficacy of the products in eliminating specified bacterial pathogens from clinically infected quarters during lactation. The efficacy of the product should be assessed by measuring the rate of intramammary infections in infected cows following treatment or in infected quarters treated during the lactation period.

The product under test (the treatment) should be compared with a commercially available antibiotic product (the positive 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 the treatment and control preparations should be of similar nozzle design, and both preparations should be administered similarly with respect to the site of deposition within the teat canal, to minimise differences in the rate of acquisition of new infections associated with the treatment technique.

Treatment groups

Infected cows should be randomly assigned to 2 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 one to 4 quarters classified as infected, the final number of infected quarters in the treatment and control groups may differ slightly.

Cows in the treatment group should receive a dose of the test compound in all 4 quarters, while cows in the control group should receive a dose of the reference product in all 4 quarters.

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

Note: Depending upon the experimental design, healthy quarters may be treated. Applicants may apply to the APVMA for technical assistance on which treatment regimen is the most appropriate

Sample size and statistical analysis

The experimental unit for the 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 & 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 α set at 0.05. There should be a table in the report representing the sample sizes (number of quarters per experimental group) recommended to compare treatment efficacy for different levels of efficacy expected for the control product.

Treatment method

Before treatment can be instituted, the teats should 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.

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 because 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.

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 20 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 chance of contamination through accidental hand contact with disinfected teat orifices.

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.

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

All treated cows should be inspected 24 hours after treatment for signs of peracute intramammary infection, and then at least weekly for the duration of the trial to monitor for the presence of adverse effects associated with treatment.

Sample collection and submission

For dry cow products, the resolution of infection should be assessed by measuring the infection status of treated cows in the early postpartum period. For lactating cow products, the resolution of infection should be assessed by measuring the infection status of quarters following treatment.

To collect the samples, the teats should be cleaned as described in the Treatment method section.

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

The first 2 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 horizontally, 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.

The arrangements for sample collection from animals in the trial should ensure that:

• for dry cow products, cows are first sampled ideally at the first milking after calving, at least within 2 days of calving
• for lactating cow products, cows are sampled ideally starting 14 days after the last treatment
• samples are collected at 3 consecutive milkings, or on 3 consecutive days
• samples are collected by a trained operator (veterinarian, laboratory technician or farmer)
• samples reach the laboratory no more than 24 hours after collection, following storage and transport at 1 to 6 °C.

It is best to involve professional collection teams (veterinary practitioners, government field staff or laboratory staff) 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.

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 facilitate the collection process for the farmer while ensuring adequate records for the evaluation of the product.

Careful planning of an identification system for collection tubes minimises the workload during collection and limits the likelihood of confusion of samples after collection.

Bacteriology and data recording

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

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

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 the sample collection technique that may manifest in a high rate of contamination of milk samples.

The presence of one or more colonies of a mastitis pathogen (commonly Staphylococcus species or Streptococcus species—Blood & Radostits 1989) is considered indicative of infection. For dry cow products, a quarter should be recorded as infected (non-cure) if at least 2 of the 3 milk samples collected immediately after calving prove positive on culture, with at least one colony of the same pathogen. For lactating cow products, a quarter should be recorded as infected (non-cure) if at least 2 of the 3 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 3 samples proves positive on culture for a bacterial pathogen.

The antibiotic sensitivity of all bacterial isolates selected from pre-treatment samples should be evaluated and the results recorded. Standard minimum inhibitory concentration or disc diffusion methodology can be used for this purpose.

An individual cow and quarter record should be compiled for each cow in the trial, and should contain the following information, where applicable:

• Farm identification
• Cow identification
• Age
• Lactation
• Production records
• Incidence of clinical mastitis
• Clinical appearance of each quarter or each infected quarter
• Previous dry cow treatments (refer to the Selection of cows section)
• Bacteriological results for the pre-treatment samples
• Somatic cell count or enzyme analysis results for the pre-treatment samples
• Bacteriological results for 3 milk samples collected 21 days after treatment
• Somatic cell count or enzyme analysis for the 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

2. Special considerations for testing intramammary antibiotics formulated for use in non-lactating (dry) cows

• Cows can be presumptively classified as sub-clinically infected in the 2-week period before drying off using herd records (ICCC, rapid mastitis test, conductivity and/or enzyme analysis). The analysis should be made by the consulting veterinary surgeon in consultation with the farmer.
• Cows treated in the current season for clinical mastitis, but not treated for infection in the previous dry period, may be included for consideration as candidates for bacteriological testing.
• The infection status of cows should be evaluated by bacterial culture of quarter samples.
• Cows should be sampled by an experienced milk sampler, ideally at 3 consecutive milkings or on 3 consecutive days, using the technique described in the Sample collection and submission section. Three samples collected over a 5-day period would be acceptable although less desirable.
• The last sample should be taken 5 clear working days before the prospective dry cow treatment is to be administered in order to allow final laboratory compilation of results, interpretation of findings, and allocation of cows to trial groups.
• Milk samples submitted for bacteriological examination should be processed as described in the Bacteriology and data recording section. Cows should be classified as infected if 2 out of 3 bacteriological cultures of the aseptically collected milk prove positive for the same bacterial mastitis pathogen (Neave, 1975). This approach produces a 1% false positive rate and should allow for optimal assessment of product efficiency.
• 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, the 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 or enzyme analysis and made available for bacteriological examination. Half of the cows can be treated with the product and half can serve as untreated controls. Such a study should use 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.

3. Special considerations for testing intramammary antibiotics formulated for use in lactating cows

• A sample of milk should be collected before treatment, from each quarter of cows identified with clinical mastitis (see the Sample collection and submission section) for laboratory examination to confirm the diagnosis of clinical mastitis.
• The farmer’s 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.

4. Part IV: Determination of endpoint for antibiotic excretion in milk following intramammary treatment of lactating cows

Antibiotics in the milk from udders infused with intramammary preparations can be detected by direct and indirect methods. Direct methods are conventional microbiological assays where known amounts of test samples are applied to bacterial cultures with known antibiotic sensitivity patterns. Direct detection of antibiotics in milk should be carried out by trained laboratory personnel using appropriate equipped laboratory.

Indirect detection involves ‘marking’ such preparations with approved dyes and then infusing them into the mammary gland. The dye is excreted in milk with endpoints equal to those of the antibiotic (Novak et al. 1984). The potential advantage of dye marking is that it immediately alerts the farmer and the consumer to antibiotic contamination by visually discolouring the milk.

This section provides 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 have 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 from dye-marker trials.

Objective

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

Method

Cow numbers: Due to the considerable variations between different quarters of the udder of the same cow as well as between cows, the trial should be on a minimum of 6 cows.

Use of mastitic udder trials: At least some cows included in the trial should be selected on the basis of one or more quarters having high somatic cell counts.

Cows selected for use in the trial should have received no antibiotic treatment for 4 weeks prior to the trial.

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 0.02 kg of each treated quarter in each milking during the period of the trial.

Sampling technique:

• Efficient quarter milkers should be used since the trial is to be conducted on a quarter basis, rather than a whole udder basis.
• The quarter milking equipment should be used with the cows for several days prior to the trial so they become accustomed to it.

The submission should give details of the milking procedures and sampling techniques used.

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, the participating laboratory should be involved in the bacteriological examination of milk samples.

Number of treatments

The manufacturer should make specific recommendations concerning the frequency and duration of treatment for each product. Each quarter used for the study should be treated according to those recommendations. In cases where the manufacturer does not make such recommendations, 3 tubes should be administered at 24-hour intervals. The first post-treatment sample should be taken at the first milking following the last treatment.

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

• milk from the treated quarter
• pooled milk from the 3 non-treated quarters
• a whole udder sample.

Three cows should be treated in 2 quarters and the following samples taken at all post-treatment samplings:

• milk from the first treated quarter
• milk from the second treated quarter
• pooled milk from the 2 untreated quarters
• a whole udder sample.

Duration of trial

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

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

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

Measurement of dye concentration

A numerical concentration can be obtained from the number of serial dilutions with dye-free milk, which should match the sample with milk standards containing known concentrations of Brilliant Blue F.C.F. Dawson and Feagan (1960) recommended that 4 standards be set up (0.25, 0.5, 0.75 and 1.0 ppm). Care should be taken to compare milk from initial milkings with the 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, and white containers should be used to hold the coloured milk samples.

For the purpose of the trial, a visual endpoint 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 that have been developed for use by dairy factory personnel, which will permit the detection of 0.016 ppm by untrained operators (0.005 ppm can be detected with refined versions of technique). An anion exchange technique that could be adopted as a standard is described by Feagan et al. (1965).

Measurement of antibiotic concentration in milk

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

Manufacturers that are at all uncertain as to the acceptability of the proposed method to be used may wish to seek technical assistance from the APVMA before conducting the trials.

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

• The technique described in the document Specification for the Identification and purity of some Antibiotics, FAO Nutrition Meeting 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.
• 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 0.0025 i.u./mL in milk.

Any other technique that enables the detection of 0.005 i.u./mL or lower concentrations of penicillin is likely to be acceptable.

Submission of trial data

Complete and impartial submission of results should be provided. The concentration of dye marker and antibiotic should be presented in tabular form, showing clearly the milking at which these 2 constituents become undetectable by the method employed.

Formulation details

Full information should be provided on the following:

• identity and concentration of all antibiotics and other active constituents
• identity and volume of base
• type of Brilliant Blue F.C.F. (micronised or crystalline)
• quantity of dye in each dose.

5. References

Blood, DC & Radostits, OM 1989, in Veterinary medicine, 7th edn, Bailliere Tindall, London.

Browning, JW, Mein, GA, Barton, M, Nicholls, TJ & Brightling, P 1990, Efficacy of antibiotic therapy at drying off on mastitis in the dry period and early lactation, Aust Vet J, vol. 67, pp. 440–42

Browning, JW, Mein, GA, Brightling, P, Nicholls, TJ & Barton, M 1994, Strategies for mastitis control: dry cow therapy and culling, Aust Vet J, vol. 71, pp. 179–81.

Dawson, DJ & Feagon, JT 1960, The use of brilliant blue F.C.F. in intramammary penicillin preparations, Aust J Dairy Tech, vol. 15, p. 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 & Bray, R 1965, An improved test for detection of marker dyes in milk, Aust J Dairy Tech, vol. 20, p. 22.

Naylor, J 1960, The incidence of penicillin in Australian milk supplies, Aust J Dairy Tech, vol. 15, p. 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 & Parson 1984, Dye marked antibiotics for lactating cow mastitis therapy, J Dairy Sci, vol. 67, p. 1841.

Sears, PM, Gonzalex, RN, Wilson, DJ & Han, HR 1993, Procedures for mastitis diagnosis and control, in Anderson, KL (ed.),Veterinary Clinics of North America, vol, 9, no. 3: Food animal practice: Update on bovine mastitis.

Snedecor, GW & Cochran, WC, in Statistical methods, Iowa State University Press, Ames, Iowa.