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Comparison of the efficacy of cloxacillin alone and cloxacillin combined with an internal teat sealant for dry-cow therapy
  1. H. T. Newton, BVSc, PhD, MRCVS1,
  2. M. J. Green, BVSc, DCHP, MRCVS2,
  3. H. Benchaoui, DVM, PhD3,
  4. V. Cracknell, BVetMed, MRCVS3,
  5. T. Rowan, BVSc, CBiol, MIBiol, PhD, MRCVS3 and
  6. A. J. Bradley, MA, VetMB, PhD, DCHP, MRCVS1
  1. 1 School of Veterinary Science, University of Bristol, Langford, Bristol BS40 5DU
  2. 2 School of Veterinary Medicine and Science and Department of Mathematical Sciences, University of Nottingham, Sutton Bonington Campus, Sutton Bonington LE12 5RD
  3. 3 Veterinary Medicine Research and Development, Pfizer Ltd, Sandwich, Kent CT13 9NJ
  1. Correspondence to Dr Bradley

Abstract

All the quarters in the cows with high somatic cell counts in 10 herds were treated at drying off with either 600 mg cloxacillin or 600 mg cloxacillin and 4 g of an internal teat sealant containing 65 per cent bismuth subnitrate. The quarters were sampled daily for bacteriological tests for the three days before drying off and twice after calving to establish whether they were infected. Clinical mastitis cases were monitored from drying off until 100 days after calving. The odds of a quarter being bacteriologically negative after calving or developing clinical mastitis in the first 100 days after calving were investigated by multilevel logistic regression. The quarters treated with the internal sealant and cloxacillin were significantly more likely to be bacteriologically negative in the immediate period after calving and were significantly less likely to suffer clinical mastitis during the first 100 days after calving than the quarters treated with cloxacillin alone. There was more variation between cows than between herds in the underlying risk of an infection after calving, but there was more variation between herds than between cows in the underlying risk of clinical mastitis during the 100 days after calving.

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    BLANKET dry-cow therapy is used at the end of lactation and has two aims. The first is to cure existing intramam-mary infections and the second is to prevent the establishment of new infections during the dry period (Huxley and others 2002). Traditionally, antibiotic dry-cow therapy has aimed to cure existing infections caused by contagious pathogens, and antibiotic dry-cow intramammary products therefore have a predominantly Gram-positive spectrum of activity.

    Over the past 30 years, the incidence and aetiology of mastitis in the uk has changed. The environmental pathogens, such as Streptococcus uberis and Escherichia coli, have increased in importance, whereas the incidence of the contagious pathogens, such as Staphylococcus aureus and Streptococcus agalactiae, has decreased (Bradley 2002). As early as 1950 it was recognised that the rate of new infections was high around the time of involution, when the rate of new infections during the first 21 days of the dry period was found to be 6·25 times higher than the rate of new infections during the previous lactation (Neave and others 1950). More recently, the importance of infections with environmental pathogens during the dry period has been identified, cows being particularly susceptible at both the beginning (Oliver 1988) and the end of the dry period (Oliver and Sordillo 1988). The importance of these infections and their impact on clinical mastitis has been established under field conditions in the uk (Bradley and Green 2000). It is thought that the infections gain access to the gland via the teat canal, which is likely to be open or incompletely closed particularly during involution and around the time of colostrogenesis (Oliver 1988, Oliver and Sordillo 1988).

    It has been established that the failure of the keratin plug to form during the dry period is an important risk factor, quarters with ‘open’ teat ends or incomplete keratin plugs being 1·7 times more likely to develop new infections during the dry period (Dingwell and others 2003). In a New Zealand study, 45 to 55 per cent of quarters were identified as ‘open’ seven days after drying off, and 97 per cent of the clinical infections that were identified in the 21 days after drying off occurred in these open quarters (Williamson and others 1995). The recognition of the protective effect of the keratin plug and the increasing importance of the environmental pathogens have revived interest in the potential value of internal teat sealants. One such sealant (OrbeSeal; Pfizer) when used alone, has been shown to be more effective at preventing new infections with E coli and all major pathogens combined than an infusion of 250 mg cephalonium (Cepravin Dry Cow; Schering-Plough) in cows selected on the basis of a low somatic cell count (Huxley and others 2002). The same teat sealant was similarly effective under field conditions in New Zealand (Woolford and others 1998).

    However, although internal teat sealants have been shown to be effective in preventing new infections in the dry period in quarters that are uninfected at drying off, their use in infected quarters has been less well studied. There is a strong rationale for the use of an internal teat sealant in combination with intramammary antibiotics in cows with high cell counts, because although the cure rate of infections is about 80 per cent when antibiotics are used at drying off (Gruet and others 2001), there is a significant risk of new infections during the late dry period, either because the spectrum of activity of the dry cow antibiotic is inadequate, or because its efficacy wanes as its concentration decreases (Bradley and others 2003). Woolford and others (1998) found that there was no significant benefit in using an internal teat sealant in combination with antibiotic under New Zealand's pasture-based management systems, but in the usa Godden and others (2003) reported significant reductions in the prevalence of intramammary infections after calving, in the incidence of mastitis in the first 60 days of lactation, and in somatic cell counts, when an antibiotic and an internal teat sealant were used in combination. However, neither of these studies investigated the use of the antibiotic and sealant products in combination in cows that were infected at drying off.

    The purpose of this study was to investigate whether, under uk field conditions and in cows deemed to be infected at drying off, an internal teat sealant in combination with antibiotic dry-cow therapy would reduce intramammary infections after calving or the incidence of clinical mastitis in the first 100 days of lactation.

    MATERIALS AND METHODS

    Herd selection

    Ten herds were selected in the south west of England on the basis that they recorded individual cow somatic cell counts (scc) monthly, maintained adequate records and were likely to comply with the protocol of the study.

    Cow selection

    Cows were eligible for the study if they were in good health as determined by a clinical examination, had four functional quarters free of significant teat lesions, and had had an scc exceeding 200,000 cells/ml in the last month and in at least two of the three monthly recordings before drying off. They could not have received systemic or intramammary antibiotics or anti-inflammatories in the 30 days before the last milking and had to have an expected dry period lasting at least 42 days.

    Sampling protocol

    Duplicate samples for microbiology and a single sample for scc were collected aseptically by the method described by Huxley and others (2002) from all the quarters on five occasions: two days before drying off (D−2), one day before drying off (D−1), on the day of drying off (D0), four days after calving (C+4), and between eight and 11 days after calving (C+8−11).

    The samples were collected within an hour of routine milking, and transported in a cool box to the laboratory, where the samples for bacteriology were stored at below −20°C and the samples for scc analysis were stored at between 2 and 8°C. The samples were batched and sent weekly to an accredited laboratory for microbiological and scc analysis.

    Product administration and randomisation

    After sampling on the day of drying off, the teats were rescrubbed with cotton wool soaked in 70 per cent ethanol and allowed to dry; 600 mg of benzathine cloxacillin (Orbenin Extra Dry Cow [oedc]; Pfizer Animal Health) was infused into the teat cistern of each quarter and massaged into the udder, and 4 g of the teat sealant containing 65 per cent bismuth subnitrate in a mineral oil base (OrbeSeal; Pfizer Animal Health) was then instilled into the teat sinus of two of the quarters. The treatments were allocated randomly so that one front and one hind quarter of each cow received the internal teat sealant. All the quarters were then dipped in a solution containing 2800 ppm available chlorine (Chlorisept; Davidson Group) and the cow was confined to a loafing yard for 30 minutes. The cows were managed according to the normal husbandry practices of each farm. Quarters that received oedc only were coded as ‘at’ and quarters that received both oedc and OrbeSeal were coded as ‘ct’.

    Clinical mastitis

    Clinical cases of mastitis were recorded and sampled throughout the dry period and for the first 100 days of the next lactation. Herdspersons were trained to identify cases of mastitis and in the aseptic sampling technique. Any case of clinical mastitis diagnosed by the herdsperson was sampled either by the herdsperson or by the investigator before it was treated. Clinical samples were stored frozen on the farm at below −20°C before they were collected and sent to the laboratory for analysis.

    Analysis of milk samples

    The samples for somatic cell counts were sent weekly to onmerit Laboratories for analysis by the Fossomatic method.

    The samples from cases of clinical mastitis and the second of the two samples collected for microbiology on each occasion were sent to Compton Paddock Laboratories for bacteriological analysis; 10 μl of the secretion was inoculated on to blood agar and Edward's agar; 100 μl of secretion was inoculated on to MacConkey's agar to enhance the detection of Enterobacteriaceae. The plates were incubated at 37°C and read after 24, 48 and 72 hours. The organisms were identified and quantified by standard laboratory techniques (Quinn and others 1994, NMC 1999). The isolation of a pathogen was recorded as an infection regardless of the number of colony forming units present.

    Additional data collected

    Each cow's parity, last recorded milk yield, breed, previous history of clinical mastitis and scc records for the previous three months were recorded when it was enrolled. Any concomitant disease and mastitis were recorded during the dry period and the first 100 days of lactation.

    Definition of terms used for analysis

    Intramammary infection at drying off or after calving

    A quarter was defined as infected at drying off if a specific pathogen had been cultured at least once before drying off, that is, at D−2, D−1 or D0. An infection after calving was defined as the isolation of a pathogen from either of the two samples taken after calving. A sample was defined as contaminated if more than three pathogens were cultured from it, and in this event, the duplicate sample was cultured (Bradley and Green 2000).

    New infection

    A new infection was defined as the culture of a pathogen in either of the samples taken after calving that was not present in any of the three samples taken before drying off.

    Cure of infection during the dry period

    A cure during the dry period was defined as the absence of a pathogen in both the samples taken after calving that had been present in one or more of the three samples taken from that quarter before drying off.

    Successful outcome of dry-cow therapy

    A successful outcome of the dry-cow therapy was defined as a quarter that had no pathogens cultured from it in either of the samples taken after calving. A successful outcome therefore included quarters that had had no pathogens isolated before drying off and did not develop a new infection between drying off and up to 11 days after calving, and quarters that were infected before drying off but were cured and did not acquire a new infection.

    Clinical mastitis

    Clinical mastitis was defined as the presence of an abnormal secretion and/or udder changes (heat, pain and/or swelling) as diagnosed by the herdspersons.

    Minor pathogen

    A minor pathogen was defined as either a coagulase-negative Staphylococcus species or a Corynebacterium species.

    Statistical analyses

    The data were collated and analysed by using Excel and Access 2000 (Microsoft) and Statistix 8 (Analytical Software). A uni-variable analysis was carried out by using the chi-square test (Anon 2003).

    Multilevel logistic regression models were developed with either the absence of an intramammary infection after calving (‘successful outcome’) or clinical mastitis on one or more occasions within the first 100 days after calving as the response variables. Random effects were included for ‘cow’ (level two) and ‘farm’ (level three) to account for correlations within the data, that is, quarters within cows and cows within farms. The potential confounding factors of quarter position, days in milk, milk yield at drying off and parity were tested and left out of the final models if they did not have an important effect on the values of the coefficients or the biological interpretation. The models took the general form: Embedded Image where the subscripts i, j and k denote the ith quarter, the jth cow and the kth farm respectively; μijk is the fitted probability of the response in quarter i of cow j on farm k; α is the regression intercept; TXijk covariate ‘treatment’ (either ct or at); β1 coefficient for TXijk; QTijk covariate ‘quarter position’ (right hind, left hind, right fore or left fore); β2 coefficient for QTijk; vk random effect due to residual variation between farms; ujk random effect due to residual variation between cows; σ2v farm level variance; σ2u cow level variance.

    The parameters were estimated and the fit of the models was assessed by the methods described by Browne (1998) and Green and others (2004). In order to obtain robust estimates of the variance components, Markov chain Monte Carlo procedures were used in a Bayesian context (Browne 1998, Snijders and Bosker 1999), using Winbugs (Spiegelhalter and others 2000). ‘Vague’, that is, flat priors were specified for fixed-effect coefficients (∼Normal distribution [mean=0, variance=106]). After an assessment of the effect of different ‘vague’ priors for random effect parameters (Spiegelhalter and others 2004), priors with a uniform distribution (0, 10) were used in the final models for the random effect standard deviations.

    RESULTS

    The 10 herds ranged in size from 90 to 350 cows in milk. The latest monthly bulk milk scc (bmscc) when the cows were enrolled ranged from 102,000 cells/ml to 273,000 cells/ml.

    The herd size, the number of cows recruited from each herd and the geometric means of the last five monthly bmscc are shown in Table 1.

    Of the 313 cows originally enrolled, 30 were withdrawn for reasons other than mastitis and were not included in the analysis; eight had received antibiotic or anti-inflammatory treatments for reasons other than mastitis, 11 had died or been sold for reasons other than mastitis, eight aborted or were not in calf, and three missed some samples. The remaining 283 cows each contributed two quarters to each of the treatments. They had a mean lactation number of 4·2 (range one to 14) and their mean last recorded milk yield was 12 litres (range 3 to 30 litres). Their three previous monthly sccs before drying off ranged from 11,000 cells/ml to 7,915,000 cells/ml. Further information on the lactation number, drying off yield and scc of the cows is summarised in Table 2.

    The numbers of intramammary infections at drying off, after calving, the dry period cures and new dry period infections are shown on a pathogen-specific basis in Tables 3, 4 and 5. Seventeen samples were defined as contaminated, and in these cases the results are based on the duplicate sample.

    Univariable analysis

    There were no significant differences in the prevalence of different pathogens at drying off between the two treatment groups, apart from the quarters that were defined as infected with coliforms other than E coli; significantly more of the quarters given the combined treatment were infected with coliforms other than E coli. After calving there was a trend for fewer of the quarters given the combined treatment to be infected with minor pathogens (111/566 v 87/566; P=0·06).

    The numbers of quarters that were classified as uninfected at drying off, uninfected after calving, that developed a new infection during the dry period, or were cured during the dry period are shown in Table 6. More of the quarters given the combined treatment were cured during the dry period and fewer new infections occurred in them than in the quarters treated with antibiotic alone, but the differences were not statistically significant. When the quarters were classified as ‘uninfected after calving’ there was a strong trend towards a difference between the groups (P=0·059).

    In the quarters given the combined treatment there were 23 cases of clinical mastitis in 23 quarters between drying off and 100 days in milk, compared with 50 cases of clinical mastitis in 45 quarters in the group treated with antibiotic alone; the differences in the numbers of mastitis episodes and quarter cases were both significant (P<0·01). A summary of the pathogens isolated from the cases of mastitis is given in Table 7. In the quarters treated with antibiotic alone there were significantly more cases of clinical mastitis caused by S uberis (14/552 v 4/562) (P=0·02) and coagulase-positive staphylococci (7/559 v 1/565) (P=0·03) than in the quarters given the combined treatment.

    Statistical models

    A summary of the model for a successful dry period outcome (absence of bacterial growth after calving) is shown in Table 8. The quarters given the combined treatment were significantly more likely to have a successful outcome, with an odds ratio (or) of 1·42 (95 per cent credibility interval 1·05 to 1·91), than the quarters treated with antibiotic alone. There was more residual variation in the probability of a successful dry period outcome between cows within farms, than between farms.

    A summary of the model for clinical mastitis in the first 100 days of lactation is shown in Table 9. The quarters given the combined treatment were significantly less likely to develop clinical mastitis in the first 100 days of lactation, with an or of 0·47 (95 per cent credibility interval 0·26 to 0·82) than the quarters treated with antibiotic alone. There was more residual variation between farms, than between cows within farms in the probability of clinical mastitis in the first 100 days of lactation.

    Both logistic regression models provided a good fit with the data.

    DISCUSSION

    This is the first study of the efficacy of an antibiotic dry cow product combined with a separate internal teat sealant under uk field conditions. There were significantly fewer infected quarters after calving and there was significantly less clinical mastitis in early lactation in the group treated with the combination when the data were analysed with a multivariable model. These outcomes can be appreciated by the farmer and most accurately indicate the ‘overall efficacy’ of the product.

    The reduction in the incidence of clinical mastitis was probably the most important finding because it suggests that a real clinical improvement can be made by using combination therapy in this way. The results agree with the results of Godden and others (2003) who reported that the odds of a combination-treated quarter experiencing mastitis in the first 60 days of lactation was 0·67 (95 per cent confidence interval 0·48 to 0·93) compared with a quarter treated with cloxacillin alone. A reduction in the risk of clinical mastitis in early lactation of 33 to 50 per cent therefore appears possible.

    The relative importance of between farm (as opposed to between cow) variation was greater in the model for clinical mastitis than it was in the model for the success of dry period therapy. This suggests that cow factors were more influential in determining whether an infection was present after calving, but that farm factors were more influential in determining whether a clinical episode occurred. Such farm factors could include nutrition (Weiss and others 1990, Hogan and others 1993), management (Barkema and others 1999), or intercurrent disease (Suriyasathaporn and others 2000), although no specific farm factors were investigated in this study. The difference in the relative importance of the variation between farm and cow with respect to the two outcomes could also be associated with different pathogens causing intramammary infections and clinical episodes of mastitis (Barkema and others 1997).

    In contrast, the analysis indicated that the susceptibility to acquiring an infection during the dry period was governed more by cow factors than farm factors. Several such factors have been shown to affect the likelihood of new infections in the dry period, including the rate of formation of the keratin plug (Williamson and others 1995, Dingwell and others 2004), parity (Zadoks and others 2001, Huxley and others 2002, Dingwell and others 2002, 2004, Green and others 2005), the length of the dry period (Dingwell and others 2002) and the milk yield at drying off (Dingwell and others 2002, Huxley and others 2002). The contrast between the importance of cow (for infection) and farm (for clinical disease) is indicative of the balance that probably exists between infection and immunity as determinants of clinical disease.

    The finding that the rate of new infections was not significantly different between the treatment groups contrasts with the findings of Huxley and others (2002), who found that quarters treated with OrbeSeal were significantly less likely to acquire a new infection with E coli, all Enterobacteriaceae, and all major pathogens combined than quarters treated with an antibiotic; the difference could be due to the different criteria used to select the cows in the two studies, or to different pathogens and epidemiology on the farms studied. In this study the cows had higher sccs than the cows in the study by Huxley and others (2002), which looked exclusively at cows with a low scc. In a study in New Zealand, Woolford and others (1998) reported no differences between the numbers of new infections acquired during the dry period by sealed quarters, antibiotic-treated quarters, or quarters treated with both an antibiotic and an internal sealant. This lack of effect may be associated with the different environmental conditions in New Zealand in comparison with the uk and illustrates the importance of local factors such as environment, farm and cow in studies of the efficacy of dry-cow therapy. Godden and others (2003) found that a significantly smaller proportion of quarters treated with both an antibiotic and a sealant developed a new infection between drying off and one to three days after calving, but that this significant benefit was lost by six to eight days into lactation; they considered that this was probably associated with the high levels of ‘self cure’ observed in early lactation, particularly for coagulase-negative staphylococci. In this study there was no significant difference between the treatment groups when their infection status was examined at four days or eight to 11 days after calving. However, when the success of dry cow therapy was assessed in terms of having an uninfected quarter on these two occasions the combination-treated quarters were significantly more likely to have had a successful outcome than the quarters treated with antibiotic alone (or 1·42, 95 per cent credibility interval 1·05 to 1·91). It may be argued that freedom from an intramammary infection at calving is a sensible and biologically valid method for assessing the efficacy of dry-cow therapy, because it takes into account both of its aims, namely to cure existing infections and prevent new infections (Oliver and Sordillo 1988).

    More of the quarters treated with the combination were cured and fewer of them developed new infections during the dry period than among the quarters treated with antibiotic alone. Although the differences were not statistically significant they suggest that reinfection with the same pathogen during the dry period may be important, and could lead to a reduced ‘apparent’ cure rate. In a similar study in the usa there were significantly fewer new infections in combination-treated quarters than in quarters treated only with antibiotic at up to three days in milk, and although the difference was no longer significant by six to eight days in milk the trend remained. In the same study the cure rate from drying off to one to three days in milk was not significantly different, but there were more cures in the combination-treated group (91·3 v 88·2 per cent) (Godden and others 2003). Both these results support the hypothesis that reinfection with a pathogen indistinguishable from that isolated before drying off may mask, or reduce, the apparent cure rate. This may divert emphasis away from the prevention of new infections, even in cows deemed to be infected at drying off, as indicated by the reduced incidence of clinical mastitis and the increase in the number of quarters that were not infected after calving, that is, had a successful dry period outcome. Molecular typing may make it possible to differentiate strains of bacteria that are indistinguishable at the species level, and quantify the extent of reinfection with the same species (Zadoks and others 2003).

    The timing of the collection of milk samples and the definition of intramammary infections are subjects of debate. However, in this study, the same sampling techniques and measures of infection status were used in both groups, and the likelihood of any of bias was therefore low. In addition, the collection of samples after milking would have decreased the sensitivity of detecting an infection, but increased its specificity; thus the positive predictive value of the results would have been higher than for premilking samples and there would have been fewer multiple isolates (Sears and others 1991). The implication is that it is more likely that the infections detected both before drying off and after calving were true intramammary infections, even though the infection was diagnosed on the basis of a single isolate.

    ACKNOWLEDGEMENTS

    The authors would like to acknowledge the farmers and herdspersons for their cooperation and the staff at Compton Paddock Laboratories for bacteriological analysis.

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