Management

Evaluating mastitis treatment trials – what have we learned?

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Mastitis occurs on virtually all dairy farms and is almost always caused by bacterial infections of individual mammary gland quarters [1-3]. Many cases of mastitis are subclinical but some cases progress to a clinical state. Clinical mastitis is defined based on occurrence of an inflammatory response that is visually detectable and includes abnormal milk with or without swelling in the mammary gland or systemic illness [4]. While most cases of clinical mastitis do not result in an obviously sick cow, the majority of cows with clinical mastitis are treated with antibiotics because abnormal milk must be discarded and farmers believe that use of antibiotics will help to rapidly achieve a clinical cure [5]. Historically, the vast majority of clinical cases of mastitis have been treated with antibiotics simply based on detection of inflammation (clinical signs) but many of these treatments are unnecessary as the infections have cleared before the milk returns to normal [6]. Several studies have shown that use of antibiotics to treat non-severe clinical mastitis cases that are culture negative or Gram-negative is often unnecessary as these cases have a high rate of spontaneous cure [7-14]. However, most cases of mastitis are treated with antibiotics and treatment protocols vary amoantng countries based on access to approved antibiotics and historical treatment practices. Evaluation of treatment success is often based the perception of how rapid the milk returns to normal, but that outcome is not a good indicator of treatment success as it has little variation and is not predictive of bacterial clearance [15]. To gain a better understanding of our knowledge about mastitis treatment, I recently reviewed 20 years of studies describing clinical trials that used antibiotics to treat clinical mastitis and this paper briefly summarizes some highlights from that paper [16].

Clinical mastitis treatment trials included in the review

Eligible studies were identified by searching publicly available literature databases for papers that included keywords or titles such as mastitis, bovine, clinical, randomized, treatment and non-inferiority. Papers were excluded if they were based on experimental challenges (rather than natural infections), were evaluating dry cow treatment, evaluated non-antibiotic alternative treatments, or were published in a language other than English. Based on reduced incidence of contagious mastitis pathogens (like Streptococcus agalactiae and Staphylococcus aureus), and increased incidence of environmental mastitis pathogens, only studies published since 2000 were considered. Despite the importance of mastitis to cow welfare and farm profitability, only 26 studies that evaluated antibiotic treatments of clinical mastitis met inclusion criteria (an average of 1.3 published studies per year). Studies were performed in the U.S. or Canada (n = 9)[7, 12, 17-23], European Union (n = 8)[14, 24-30], New Zealand (n = 6)[31-36], Brazil (n = 2)[37, 38] and Mexico (n =1)[39]. A wide variety of antibiotic treatments were evaluated (Figure 1) and none of the studies were replicated. The 26 studies included 65 comparison groups that were distributed as intramammary (IMM) treatment using a single antibiotic (n = 28), IMM treatment using a combination of antibiotics (n = 13), IMM treatment combined with systemic antibiotics (n = 5), systemic antibiotics only (n = 11), anti-inflammatories (n = 2) and negative controls (n = 6). Almost all products were β-lactam class antimicrobials with similar spectrums of activity and IMM treatments containing cephalosporins (1st – 4th generation) were the most evaluated products. Many of the treatments evaluating similar antibiotics varied the number of treatments or treatment days or evaluated different outcomes making comparisons of efficacy among studies virtually impossible.

Understanding the impact of study design

Different types of experimental designs were used to evaluate treatments and these differences make it difficult to compare results among studies. The goal of antibiotic therapy is to enhance bacteriological clearance and the value of antibiotic therapy is based on the difference between the “spontaneous cure rate” (without antibiotics) and the “treatment cure rate (with antibiotics). The spontaneous cure rate is a result of a successful immune response and expectations for spontaneous cure vary depending on characteristics of the bacteria that are causing the infection. For example, expectations of spontaneous bacteriological clearance for IMI caused by Staph. aureus are very low (<20%) while expectations of spontaneous bacteriological clearance for IMI caused by E. coli are very high (>85%). The bacteriological cure rates that we measure after antibiotic treatment are a combination of both spontaneous cures and the effect of treatment, thus studies that do not include a non-treated control group are unable to truly estimate the effect of the treatment. The “gold standard” for determining efficacy is a randomized trial that includes a non-treated control group, because that ensures that we can measure the spontaneous cure rate and determine the additional benefit due to use of the antibiotic. Based on reluctance to leave mastitis untreated, researchers have been hesitant to perform studies that include a non-treated control group. Very few (n = 6) mastitis trials performed over the last 20 years included a non-treated control group and 5 of these studies included only cases caused by Gram-negative bacteria or were culture negative at detection. Most studies compared outcomes of cows receiving different treatments and did not include non-treated cases. Some researchers stated that their objective was to prove that different antibiotics had similar efficacy and others stated that they wanted to determine that a specific treatment protocol was better (“superior”) than a comparison treatment. Unfortunately, the lack of non-treated control groups in most studies, makes it impossible to separate the effect of treatment from spontaneous cures and most studies did not demonstrate differences (“superiority”) among treatments that were compared. The inclusion of non-treated cases in future studies would help to better define the role of antimicrobial therapy for treatment of bovine mastitis.

Understanding how case inclusion criteria impact study results

Bovine mastitis is caused by bacterial infection of the udder, but we recognize the disease by detection of the inflammatory response that occurs after the infection. The clinical signs (abnormal milk, swollen udder, etc.) are not specific for a particular bacterial infection but occur due to the immune response that is attempting to eliminate the infection. In many instances, the immune response is successful, and the bacteria are eliminated before the signs of inflammation (the clinical case) are detected. Culture negative milk samples are commonly found in about 25-35% of cows with clinical signs of mastitis and these cases have a very good prognosis as they are often the result of these spontaneous cures [7, 8]. When culture negative cases are evaluated together with culture positive cases, researchers will overestimate benefits of treatment as many cases will have achieved bacteriological clearance before the case is treated [7]. Conversely, estimates of efficacy may be reduced by inclusion of cases that are caused by bacteria that are outside the spectrum of activity of the antibiotic (intrinsically resistant to the antibiotic) or when bacteria have virulence characteristics that result in low expectations of cure (like many Staph aureus cases). In the trials that were reviewed, most outcomes were evaluated regardless of etiology and most comparison groups included culture negative cases (Figure 2). As most studies did not include a non-treated control group, outcomes of most studies include both spontaneous and treatment cures, and it is likely that the impact of treatment is overestimated.

The importance of evaluating outcomes on a pathogen specific basis is especially important for studies that are designed to demonstrate non-inferiority among treatments. If a large proportion of culture-negative and non-severe Gram-negative cases (with high expectations for spontaneous cure) are included in trials, only a small proportion of cases will be expected to benefit from treatment. In these instances, it is almost mathematically impossible to arrive at any finding other than non-inferior (similar results for both products). In the trials that were reviewed, there were 6 non-interiority trials and 4 of them evaluated cases regardless of etiology (including culture-negative cases). All 6 studies concluded that the “new treatment” was non-inferior (or inconclusive) to the comparator treatment. It is important to recognize that all non-inferiority studies evaluated commercially available products which underwent efficacy trials which included non-treated controls groups, prior to approval. Results of the approval process infer an acceptable level of efficacy for pathogens that were included in the approval trials. In future studies, researchers should recognize that outcomes of non-inferiority trials must be evaluated on a pathogen specific basis, outcomes of culture negative cases should not be mixed with outcomes of culture positive cases and enrolled cases should be within the expected spectrum of activity of the antibiotics that are being compared.

Outcomes used to evaluate treatments

Clinical trials almost always report bacteriological cure but there was little consistency in other outcomes (Figure 3). Clinical outcomes included clinical cure, post-treatment SCC, culling (herd retention), post-treatment milk yield, occurrence of new IMI and recurrence of another clinical case. Few studies reported most clinical outcomes and less than half of the studies reported a significant difference among treatment groups for any outcome.

Bacteriological cure was the most reported outcome and was determined by comparing isolation of bacteria from milk samples collected at detection of the case to isolation of the same bacteria from milk samples collected at various intervals after treatment was completed. Definitions of bacteriological cure varied and were sometimes based on results of a single milk sample collected at some interval after treatment, or based on results of milk samples collected several times at varying intervals. Among treatment groups, bacteriological cure ranged from 27% to 95% (Figure 4), but comparisons among studies should not be performed due to differences in the distribution of pathogens and sampling periods. Overall, the means and ranges of bacteriological cure were 69% (27-95%) for IMM antimicrobial therapies (n = 35), 68% (33-91%) for systemic treatment or systemic and IMM therapies combined (n = 13) and 60% (38-87%) for non-treated cases (n = 6).

Statistically significant differences in bacteriological cures among treatments were reported in only 4 trials while 19 studies reported no differences (or non-inferiority). There was no evidence of a strong relationship between bacteriological cure and clinically relevant outcomes. Thus, while achieving bacteriological cure is the goal of antimicrobial therapy, the finding of differences in bacteriological cure did not appear to be associated with differences in important clinical outcomes. The failure to link bacteriological cure to clinical outcomes is likely a consequence of study design issues, or a result of including culture negative cases and multiple etiologies within comparison groups (which may mask true effects of antibiotic therapy). It is important to remember that without a non-treated control group, bacteriological cure is the combined total of spontaneous and treatment cures, and the value of antimicrobial therapy is greatest when expectations for spontaneous cure are low, and expectations of therapeutic cure are high.

All but 2 studies reported “clinical cure,” but definitions of “clinical cure” varied among studies. Most researchers defined clinical cure based on observations of when milk (and/or the udder) returned to normal appearance, but the day of observation ranged from 2 to 28 days after treatment and some researchers used single observations, while others observed milk multiple times. Clinical cure occurs when inflammation diminishes and was least when researchers performed observations before day 3 [14, 24]. When clinical cure was based on visual observations performed after day 3, it ranged from 25% to 98% with a median value of 81%. In most cases of clinical mastitis, inflammation is self-limiting and lasts about 5-6 days, regardless of bacteriological cure [7, 12, 15]. While achieving clinical cure is a primary goal of treatment, this outcome has little variation and is not useful to determine effectiveness of antimicrobial therapy. There is almost no evidence that selection of an antimicrobial has a significant impact on this outcome and clinical cure should not be used to compare treatments of make decisions about efficacy.

Post-treatment SCC was the 3rd most reported outcome (Figure 3) and only 4 of the 15 studies that evaluated it reported significant differences based on treatment group. Like other outcomes, several definitions and various sampling periods were used to define this outcome. Some studies assessed SCC at the quarter level while others used composite milk, thus low SCC milk from unaffected quarters likely artificially reduced SCC values in some studies. Like other outcomes, post-treatment sampling was performed from 7 – 90 days after treatment, and all studies that assessed SCC at multiple periods reported a gradual decline in SCC as time passed. Somatic cell count responses are a practical outcome that can be used as an indicator of treatment success, but a gradual (rather than immediate) decline should be expected, and short-term SCC values (<21 d) should not be used to make decisions about administration of additional treatments. If composite milk samples are used, a lower threshold (<150,000 cells/mL) may help prevent misclassification of on-going subclinical infections that can result after failure to achieve bacteriological cure.

Only a few studies evaluated other important clinical outcomes. Recurrence of another clinical case was reported in several studies [7, 12, 20, 32] and a few used recurrence in their definition of clinical cure (or “clinical failure”)[33, 35]. In the few studies that reported recurrence it ranged from about 5 – 30% and was strongly associated with risk factors such as parity (older cows are at greater risk of recurrence), etiology (culture positive are at greater risk as compared to culture negative), and increased milk yield. While two studies reported significant differences in recurrence based on treatment, this outcome is influenced by many other factors and should be interpreted cautiously.

Other important outcomes include post-treatment milk yield and retention within the herd, but these outcomes require prolonged follow-up and are rarely evaluated. Of the 26 studies, only 6 evaluated milk yield and 8 reported culling. Both outcomes are economically important on dairy farms and inclusion of longer-term outcomes such as these would strengthen future trials. Milk yield was reported in 4 studies that included a non-treated control group but only 1 reported a significant difference (non-treated cows had the greatest post-treatment milk production [17]). While 8 studies reported retention (culling) only 1 identified a significant difference among groups [7, 12, 14, 18, 20, 22, 30]. Both outcomes are difficult to assess and are influenced by many factors. The nature of mastitis trials results in most being non-blinded which allows farmers to cull cows that don’t have a withholding period if they have not received antimicrobial therapy. Both milk yield and herd retention are important clinical outcomes that influence economic performance of dairy cows and future researchers are encouraged to include these outcomes in studies evaluating mastitis treatments.

Conclusion

A limited number of mastitis treatment trials have been performed over the last 2 decades and differences in trial design, failure to separate outcomes of cases caused by different pathogens, combination of both culture negative and culture positive cases for analysis and lack of non-treated controls have resulted in little evidence to draw conclusions about differences among treatments. Most trials performed in the last 20 years have not demonstrated significant differences in most microbiological or clinical outcomes. The ability of non-inferiority trials to differentiate among treatments is limited when mixed etiologies and culture negative cases are combined for analysis. Bacteriological cures have not varied among IMM antibiotics approved for treatment of clinical mastitis caused by Gram-positive organisms and other characteristics of approved products (dosing interval, withholding period, price etc.) can be used to make treatment decisions. Etiology is strongly associated with the likelihood of spontaneous cure and should be determined before selection of antibiotic treatments as the spectrum of antimicrobial activity of approved products should be appropriate for the etiological agent. Cases of non-severe clinical mastitis that are culture-negative when detected or caused by E. coli rarely benefit from antimicrobial therapy and use of antimicrobials to treat these cases should be considered on a cow-by-cow basis. There is little evidence of causal associations between bacteriological cure and many clinical outcomes. Evaluation of continued decline in quarter-level SCC appears to be the most reliable farm-level indicator of success.

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Text: Pamela L. Ruegg , Michigan State University, US

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