Impact of intramammary tilmicosin infusion as a dry cow therapy
Abstract
Three hundred subclinically infected quarters of 259 Holstein cows infected with gram-positive bacteria were selected via quota sampling based on the California Mastitis Test (CMT) result and were divided randomly and equally into treatment and test groups. Quarters of test group (n = 150 in 128 cows) were treated with an intramammary infusion of tilmicosin, and quarters of the control group (n = 150 in 131 cows) were treated with cloxacillin as a traditional intramammary infusion of dry cow (DC) ointment. Cows with more than one infected quarter were randomly assigned to the same group, and adjacent quarters were treated the same. The milk samples of all quarters were obtained, and bacterial cultures and somatic cell count (SCC) were tested before dry cow therapy (DCT) (50 ± 15 days before parturition), and finally on day 2 of the next lactation. Results have shown that total bacteriological cure rates on day 2 of the next lactation were 45% and 78%, (p = .01), new infection rates were 43.3% and 56.6%, and SCC was (6.732 × 105 ± 3.124 × 105) and (5.025 × 105 ± 2.935 × 105), (p > .05) in test and control groups, respectively. Tilmicosin had less effect on reducing IMI due to Corynebacterium bovis, and had no effect on Streptococcus agalactiae, but had a potent effect against Staphylococcus aureus. It was concluded that tilmicosin alone should not be infused as an alternative to conventional dry cow therapy. However, it had a significant effect against S. aureus, and the potential of tilmicosin to treat S. aureus IMI should be confirmed in further studies.
1 INTRODUCTION
Treatment of dry cows with an intramammary antibiotic is an important therapeutic process in mastitis prevention protocols (Constable, Hinchcliff, Done, & Grünberg, 2017). The increase of new infection rate in quarters that receive no DCT may be as high as 10% to 12% (Eberhart, 1986). The reduction in this rate achieved by DC therapy has been estimated to be 50% to 75% (Boddie & Nickerson, 1986; Schukken, Vanvliet, Vandegeer, & Groomers, 1993; Smith, Todhunter, & Schoenberger, 1985). The purpose of a mastitis control program during the dry period is to have as few infected quarters as possible at the next lactation (Eberhart, 1986). So the elimination of the existing intramammary infection (IMI) at the end of lactation, as well as the prevention of a new IMI during the dry period must be performed. Systemic, intramammary and combination antibiotic therapy have been used to control gram-positive bacterial infections due to streptococci and especially Staphylococcus aureus during the dry period) Boddie & Nickerson, 1986; Erskine & Gombas, 1994; Constable et al., 2017). DCT is generally considered as being less successful in eliminating IMI caused by S. aureus, compared with the elimination of other pathogens. An actual cure rate of S. aureus may vary between 20% and 78% (Dingwell et al., 2003). Variations of cure rate may be explained by pathogen, cow, herd, and finally, treatment factors. Aging, increasing SCC, the duration of infection, bacterial colony counts in milk before treatment, and the number of infected quarters decrease cure rate (Dingwell et al., 2003).
Reported cases of reduced IMI at the beginning of the dry period are more than the end of it. (Smith et al., 1985). Staphylococcus aureus infections in hind quarters have a lower cure rate compared with front quarters (Dingwell et al., 2003). Intramammary infections of penicillin-resistant S. aureus strains have a lower cure rate when treated with either β-lactam or non-β-lactam antibiotics (Barkema, Schukken, & Zadoks, 2006; Constable et al., 2017; Dingwell et al., 2003).
Many antibiotics, such as tylosin, enrofloxacin, oxytetracycline, cloxacillin, nafcillin, and cephapirin benzathine, were used successfully via parenteral (IM or IV) or intramammary infusion (Adams, 2001; Erskine, Cullor, Schaellibaum, Yancey, & Zecconi, 2004). Antibiotic resistance is a perpetual challenge in the drug selection, and so the evaluation and the recommendation of new antibiotics are very important to prevent emergence of drug resistance. Given the importance of avoiding or at least reducing treatment with antibiotics, many studies have been conducted (Scherpenzeel et al., 2014). Compared with mass DC therapy, selective therapy in low SCC cows (SCC < 150,000 cells/ml for primiparous and <250,000 cells/ml for multiparous cows) has some advantages such as reduced use of antibiotics (Scherpenzeel et al., 2014). Macrolides (e.g., erythromycin, spiramycin, tilmicosin) and lincosamides (e.g., pirlimycin) are commonly used for the treatment of intramammary infections due to S. aureus (Adams, 2001; Erskine et al., 2004). The reported resistance levels of S. aureus to macrolide antibiotics are much lower than for penicillin and range from 14% to 17% based on phenotypic testing (Erskine et al., 2004). Tilmicosin is a long-acting macrolide antibiotic (Modric, Webb, & Derendorf, 1998) effective in ewes' clinical mastitis (Naccari, Martino, Giofrè, Passantino, & De Montis, 2003). It is demonstrated that a single dose of tilmicosin (10 mg/kg body weight, subcutaneously) can be used in therapy of bacterial mastitis caused by S. aureus, Staphylococcus epidermidis and other coagulase-negative staphylococci (CNS) with a good efficacy and safety (Naccari et al., 2003). Moreover, the greater susceptibility of these staphylococci to tilmicosin in comparison with various other antibiotics tested in vitro (cephalothin, cloxacillin, norfloxacin, gentamicin, tetracycline, thiamphenicol) makes it an excellent choice for the treatment of this infection (Naccari et al., 2003).
The ability of S. aureus to evade phagocytosis by neutrophils develops resistance to the host immune response, except in the case of antibiotics such as tilmicosin that penetrates to the intracellular space of somatic (epithelial and phagocytes) cells and affects the intracellular bacteria (Jánosi et al., 2001). The in vitro interactions of tilmicosin with bovine phagocytes and epithelial cells suggest an integral role in the clinical efficacy (Scorneaux & Shryock, 1999). Because the concentrations of tilmicosin are generally low in bovine serum, many authors have evaluated the interactions of tilmicosin with three types of bovine phagocytes and mammary gland epithelial cells to provide an understanding of potential clinical efficacy. The uptake of tilmicosin in mammary gland cells depends on cell viability, temperature, and pH, but does not depend on metabolic inhibitors or anaerobiosis. Moreover, lipopolysaccharide exposure increases tilmicosin uptake by the bovine mammary macrophages and epithelial cells (Scorneaux & Shryock, 1999). Some researchers examined the Voltammetry (electrochemical) behavior of the interaction between tilmicosin and bovine serum albumin and figured out that the intramammary infusions might be more efficient than the systemic therapy with tilmicosin to remedy IMI (Biçer & Özdemir, 2011).
The aim of this research was to study the influence of an intramammary infusion of tilmicosin in dry cows to reduce the rate of IMI in the dry period, and new infections in the next lactation.
2 MATERIALS AND METHODS
Quarters of 500 late pregnant Holstein cows on a Fashapoye dairy farm located around Tehran province were checked out with a CMT solution (DeLaval; subclinical mastitis test solution; Poland), and the involved quarters with subclinical mastitis at the beginning of drying period (CMT > 1) were selected from September to October 2014. To compare the impact of tilmicosin and cloxacillin on all degrees of subclinical mastitis, different degrees of subclinical mastitis were defined and enrolled in the study. So, DeLaval solution was used as a cell counter solution to determine the degree of subclinical mastitis, at first. DeLaval CMT solution estimates the SCC of milk samples indirectly and divides the milk samples from 0 degree (normal, SCC < 2 × 105 cells/ml), slight (trace) degree of subclinical mastitis (SCC; 150–500 × 103 cells/ml), 1 + (4–15 × 105 cells/ml), 2 + (8 × 105–5 × 106 cells/ml), to 3 + (SCC > 5 × 106 cells/ml).
2.1 Sampling procedures
Based on the results of CMT and primary bacteriological culture, 300 quarters of 259 Holstein cows, subclinically infected with gram-positive bacteria were selected via quota sampling to provide all degrees of subclinical mastitis. Milk samples of all involved quarters were collected, and the second bacteriological examination and SCC were tested on the first day of drying (50 ± 15 days before parturition), and finally on day 2 of the next lactation. Selected quarters were randomly and equally divided into the test (n = 150 in 128 cows) and the control (n = 150 in 131 cows) group on the first day of drying. Finally, quarters of the test group were treated with tilmicosin (5 ml, intramammary tilmicosin 30%, 1,500 mg, Razak, Tehran, IRAN), and quarters of the control group were treated with cloxacillin (10 ml, 500 mg, intramammary cloxalmo, DC ointment; zistkimia CO., Tehran, IRAN) as a traditional intramammary infusion of DC ointment. Cows with more than one infected quarter were randomly enrolled in one group, and adjacent quarters were treated identically within the group. Teat ends were washed and swabbed with a methyl alcohol until they were clean. Then the tip of the syringe nozzle of the dry cow (DC) ointment was inserted into the teat canal, and the antibiotic was massaged up into the teat and udder cistern. After the administration, all teats were dipped with a commercial iodine teat dip (Teat Guard, Natavest Co. Tehran, IRAN).
The sample size of this study was estimated about 44 cows based on Cochran's (1977) formula discussed by Schoenfeld and Richter (1982). To estimate the sample size, the power of the test was considered 80%, β = 0.2, α = 0.05, confidence level = 0.95, standard deviation of SCC in parturient cows was 500 × 103, and the difference between SCC of the normal and the subclinically involved quarters (μ1−μ2) was about 300 × 103.
2.2 Laboratory procedures
The number of milk somatic cells was counted based on Schalm, Carrol, and Jain (1971). Bacteriological culturing of milk samples was performed according to standards of Hogan et al. (1999). Ten microliters of each involved milk sample was spread on blood agar plates (5% defibrinated sheep blood). Plates were incubated aerobically at 37°C and examined after 24 and 48 hr. Colonies were provisionally identified based on Gram stain, morphology, and hemolysis patterns. Finally, the amount of each colony type was recorded. To collect pure cultures, representative colonies were then incubated (subcultured) on blood agar plates aerobically at 37°C for 24 hr. For further identification, catalase and coagulase production were tested in the case of gram-positive cocci.
2.3 Interpretive criteria
Specific identifications of staphylococci and streptococci were performed using commercial kits (API Staph and API 20 Strep, BioMérieux, Firenze, Italy). Gram-negative isolates were identified by using colony morphology, gram-staining characteristics, oxidase, and biochemical reactions on MacConkey's agar and API 20E (BioMérieux). Contagious pathogens such as S. aureus and S. agalactiae were considered to cause IMI if only one kind of colony was isolated with ≥100 cfu/ml. For other micro-organisms especially in the case of environmental bacteria, IMI was defined by the isolation of more than 500 cfu/ml with only lower than three colony types. Milk samples with more than three colony types or less than 500 cfu/ml colonies of micro-organisms other than S. aureus and S. agalactiae were considered as contaminated or uninfected quarters, respectively, and were excluded from the study.
2.4 Statistical analysis
The rates of intramammary and new infections were compared with the chi-square and log-linear tests between the test and the control group. One-sample Kolmogrov–Smirnov test was put into practice to evaluate the normality of the distribution of SCC, and Mann–Whitney U test was applied to compare SCC between the control and the test groups if the distribution of SCC was not normal. PASW statistics (spss Version 18, IBM Co., New York 10504-1722, USA) was used to analyze the variables.
3 RESULTS
The findings showed that the absolute and the relative frequency of infected quarters were different between the test (70/150, 46.6%) and the control (35/150, 23.3%) group on the second day of the next lactation (p = .01) (Table 1). Mean and standard deviation of somatic cell counts (SCC) were calculated in the test and control group on the first day of drying and second day of the following lactation. SCC of the test group were reduced from the first day of drying-off (8.225 × 105 ± 2.724 × 105) to the second day of the next lactation (6.732 × 105 ± 3.124 × 105, p < .05). The mean of SCC of the control group was also reduced from the first day of drying-off (8.746 × 105 ± 2.362 × 105) to the second day of the next lactation (5.025 × 105 ± 2. 935 × 105, p < .05); however, the difference between the mean of SCC in the test and control group was not statistically significant on the second day of the next lactation (p > .05). Tilmicosin had less effect on reducing intramammary infection due to Corynebacterium bovis (40% cure rate, compared with 71.4%, p = .00) and had no effect on S. agalactiae (0% cure rate, compared with 100%), but had the same effect as cloxacillin on coagulase-negative staphylococci (87.5% cure rate compared with 100%, p = .1) and had the most dominant effect on S. aureus (80% cure rate, compared with 0%) (p = .07) (Table 1).
| Isolated bacteria | Number (%) | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Tilmicosin group | Cloxacillin group | |||||||||
| At Drying-off | At New lactation | At Drying-off | At New lactation | |||||||
| n | % | n | %a | CR (%) | n | % | n | % | CR (%) | |
| S. aureus | 25 | 16.6 | 5 | 3.3 | 80 | 5 | 3.3 | 5 | 3.3 | 0 |
| CNS | 40 | 26.6 | 5 | 3.3 | 87.5 | 35 | 23.3 | 0 | 0 | 100 |
| C. bovis | 75 | 50 | 45 | 30 | 40 | 105 | 70 | 30 | 20 | 71.4 |
| S. agalactiae | 15 | 10 | 15 | 10 | 0 | 15 | 10 | 0 | 0 | 100 |
| Total bacterial isolation b | 155 | 103 | 70 | 46.6 | 54.8 | 160 | 106.6 | 35 | 23.3 | 78.1 |
| Total quarters | 150 | 100 | 150 | 100 | 53.3 | 150 | 100 | 150 | 100 | 76.6 |
- a the percent was calculated as followed: the number of specific bacterium isolation/total quarters (150) × 100.
- b there were some quarters with more than one organism.
- CR, cure rate; CNS, coagulase-negative staphylococci.
The lower new infection rates due to S. aureus (0% compared with 23.3%), Escherichia coli (0% compared with 6.6%), and Trueperella pyogenes (0% compared with 10%), (p < .05) were observed (in tilmicosin compared with cloxacillin-treated quarters, respectively). However, the levels of new infection rate due to CNS (10% compared with 3.3%), C. bovis (20% compared with 10%), and S. agalactiae (10% compared with 0%), (p < .05) were higher in tilmicosin-treated quarters. The rate of new IMI of Klebsiella pneumoniae (3.3%) was equal between the groups (Table 2). The total new infection rate was 43.3% in tilmicosin-treated quarters and 56.6% in cloxacillin-treated quarters (p < .05).
| New infections | Number (%) | |||
|---|---|---|---|---|
| Tilmicosin group | Cloxacillin group | |||
| N | %a | N | %a | |
| CNS | 15 | 10 | 5 | 3.3 |
| S. aureus | 0 | 0 | 35 | 23.3 |
| C. bovis | 30 | 20 | 15 | 10 |
| S. agalactiae | 15 | 10 | 0 | 0 |
| E. coli | 0 | 0 | 10 | 6.6 |
| K. pneumonia | 5 | 3.3 | 5 | 3.3 |
| T. pyogenes | 0 | 0 | 15 | 10 |
| Total bacterial isolation | 65 | 43.3 | 85 | 56.6 |
| Noninfected quarters | 85 | 56.6 | 65 | 43.3 |
| Total quarters | 150 | 100 | 150 | 100 |
- a The percent was calculated as followed: the number of infected quarters: total infected quarters (150) × 100.
4 DISCUSSION
Tilmicosin was more effective than cloxacillin against the consequential new infections. However, failure of tilmicosin to reduce intramammary infections of S. agalactiae must be considered crucial. Tilmicosin phosphate (20-deoxo-20-desmycosin) is a semi-synthetic macrolide antibiotic that is closely related to erythromycin and has been shown to have important interactions with bovine phagocytes and epithelial cells, demonstrating a potential role in its clinical efficacy against intracellular organisms (Adams, 2001). Spectrum activities of tilmicosin are similar to that of erythromycin, but in vitro data concern its activity against Pasteurella spp. and Haemophilus somnus. Other gram-negative bacteria are resistant to tilmicosin (Adams, 2001). However, in the present study, new E. coli infections occurred less frequently in tilmicosin-treated compared with cloxacillin-treated quarters (p < .05). Tilmicosin was also more effective than cloxacillin against intramammary infections and the new IMI of S. aureus, but had contradictory effects against CNS and no effects against S. agalactiae in the presented study.
In our study, there was a considerable lack of balance in the isolation number of S. aureus infections at the beginning of the study between the tilmicosin (n = 25, 16.6%) and cloxacillin (n = 5, 3.3%) groups. This may be a weakness and disadvantage of the study. Furthermore, tilmicosin exhibited 80% cure rate against S. aureus, but cloxacillin had no effect on it. We did not expect such a great success of tilmicosin in bacteriological cure rate against S. aureus. In some other studies, an experimentally intramammary formulation of tilmicosin was equally as effective as cephapirin benzathine for eliminating S. aureus mastitis in both, cows (64.3% cure rate) and quarter levels (78.1% cure rate) (Nickerson & Owens, 1999). Moreover, in a clinical trial, the cure rate of S. aureus infection in the dry period following the administration of tilmicosin was 67.3% and 72.5%, and following the administration of cloxacillin was 56.9% and 62.9% for cows and quarters, respectively (Dingwell et al., 2003). Cephapirin benzathine, penicillin–streptomycin, penicillin–novobiocin, and tilmicosin infused once intramammarily between day 180 and 270 of pregnancy have had similar effects against S. aureus in pregnant heifers (Moroni et al., 2005). Strain-specific characteristics can be expected to affect the probability of cure rate of S. aureus IMI (Owens & Nickerson, 2001). A strain-specific response of S. aureus infection to dry cow treatment (DCT) with tilmicosin or cloxacillin has been reported by some researchers (Dingwell et al., 2003). Ziv, Shem-Tov, Glickman, Winkler, and Saran (1995) showed that concentrations of tilmicosin >0.78 μg/ml were found in the udder secretion for 8–9 days after subcutaneous drug administration. So the half-life of tilmicosin following the intramammary administration might be somewhat as effective as a traditional dry cow ointment (Ziv et al., 1995).
The basic aim of dry cow therapy is the elimination of IMI and prevention of new IMI, not only due to S. aureus, but also due to hemolytic streptococci such as S. agalactiae (Constable et al., 2017). Such effects on S. agalactiae were not observed with intramammary tilmicosin infusion in the present study.
DC therapy with tilmicosin was also unsuccessful in some other studies. Tilmicosin (500 μg/ml) was the least effective treatment for S. epidermidis and Staphylococcus caprae, isolated from subclinical mastitis cases of two commercial Italian goat herds with 156 goats (Moroni et al., 2005). Of course, the failure of tilmicosin in goats may not be considered an important finding in cattle, because the resistant caprine bacteria are not considered major and important pathogens in cattle (Constable et al., 2017).
A high level of resistance to macrolide–lincosamide–streptogramin (MLS) antibiotics has been reported for staphylococci from the milk of cows with mastitis, especially with a high rate of the MLS phenotype in S. aureus isolates. It was concluded that MLS antibiotics should be judiciously used to treat or prevent bovine mastitis caused by staphylococci (Li, Feng, Zhang, Xue, & Zhao, 2015).
The prevalence and mechanisms of macrolide–lincosamide resistance in S. aureus isolates from cows with clinical mastitis have been studied by Wang et al. (2008). Bacteria isolated from mastitis milk samples showed a high level of resistance to erythromycin (93.1%), azithromycin (93.1%), spiramycin (41.7%), tylosin (40.3%), tilmicosin (27.8%), and clindamycin (36.1%). Macrolide–lincosamide MIC90 values were more than 128 mg/L. Inducible macrolide–lincosamide resistance phenotype was detected in 52.8% (38/72) of isolates studied by Wang et al. (2008). The occurrence of high levels of resistance to macrolide–lincosamide antibiotics among the S. aureus isolates and the high rate of inducible macrolide–lincosamide phenotype indicate that appropriate alternative antibiotics should be prescribed for treating bovine mastitis caused by S. aureus. Furthermore, significant differences in the conformations of lactone rings of 16- and 14-membered macrolides could explain why some isolates with a constitutive macrolide–lincosamide (CML) resistance phenotype were sensitive to 16-membered macrolides alone. The different interaction of the 16-membered macrolides with the 50S ribosomal subunit is also presumably the reason why the susceptibility results of tilmicosin differed from those of tyrosine and spiramycin (Wang et al., 2012). To enhance the antibacterial activity of tilmicosin, some researchers studied tilmicosin-loaded hydrogenated castor oil (HCO)-SLN (solid lipid nanoparticles) and found a sustained-release effect and enhanced antibacterial activity in vitro (Wang et al., 2012). In another study, (HCO)-SLN significantly reduced the toxicity of tilmicosin (Xie et al., 2011). The failure of tilmicosin to reduce SCC of milk samples was unexpected, because the anti-inflammatory effect of macrolides (tilmicosin and tylosin) through modulating the synthesis of several mediators and cytokines involved in the inflammatory process was proven in some studies (Cao et al., 2006). The new form of tilmicosin (solid lipid nanoparticles) requires further studies in the field of dry cow therapy.
5 CONCLUSION
It was concluded that tilmicosin alone could not be infused as an alternative to conventional dry cow therapy, because of its insufficient effectivity against S. agalactiae. However, it had a significant effect against S. aureus. As the control group for S. aureus was very small, the potential of tilmicosin to treat S. aureus IMI should be confirmed in further studies.
ACKNOWLEDGMENT
The author thanks Dr. Askary M., Dr. Faghanzadeh H., and Mr. Yazdani M. for their appreciated efforts. This study was not financially supported by any grant.
CONFLICTS OF INTEREST
The authors have no conflict of interests.
