Use of dry period treatment is one of the important components of an effective mastitis control programm. The present study was planned to evaluate the comparative efficacy of oxytetracycline and enrofloxacin as systemic dry period therapy in the control of bubaline mastitis. For this purpose, twenty seven pregnant dry buffaloes were selected and divided into three equal groups. Animals in group G1 were treated with Enrofloxacin (Inj. Encure10TM; Nawan Laboratories, Pakistan) @ 2.5 mg/kg body weight intramuscularly at 14 days and 7 days prior to expected date of parturition. Group G2 were treated with Oxytetracycline-HCl (Inj. Oxy-KakTM LA; Kaksian, Pakistan) @ 11 mg/kg body weight intramuscularly at 14 days and 7 days prior to expected date of parturition and group G3 served as non medicated control. Samples of mammary secretions were collected aseptically 14 days prior to expected calving and milk samples from each quarter aseptically collected at day 7 and 14 post calving. The efficacy of treatments was evaluated through prevalence of clinical and subclinical mastitis before, at and after parturition and bacteriological cure rate. Prevalence of clinical mastitis and quarter wise prevalence of subclinical mastitis post calving after systemic dry period therapy with enrofloxacin and oxytetracycline were lower than control group. Postpartum cure rate of infected quarters at day 14 were 91.67% with enrofloxacin treatment groups, 70% with oxytetracycline treatment groups and 21% with control groups. The cure rate among the groups was significantly different (p< 0.05). The highest cure rate belonged to enrofloxacin group which was significantly higher than that of control (p< 0.05), followed by oxytetracycline group which was also significantly higher than that of control (p< 0.05); however there were no significant difference between enrofloxacin groups and oxytetracycline groups ( p> 0.05). Results suggested that dry period antibiotics therapy offers the best opportunities to remove existing, persistent intramammary infections and preventing new intramammary infections.
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Key Words: buffalo, Mastitis, dry period therapy,
Mastitis is one of the most economically important diseases of milch animals and also causes the changes in glandular tissues affecting quality and quantity of milk (Ullah et al., 2005).
Mastitis can be prevented when an effective control program is adopted. Dry period therapy is an essential part of this control program. The goal of mastitis control during the dry period is to have as few infected quarters as possible at the next calving (Eberhart, 1986). At present, the most effective means of achieving these objectives is to administer dry period antibiotic therapy during dry period. (Cousins et al., 1980; Radostitis et al., 2007). The use of antibiotic dry cow therapy and the treatment of intramammary infection during dry period has been a basis for mastitis management and control (Bradley, 2002). Dry cow therapy eliminates existing intra-mammary infections approximately 70 to 98% during the dry period and preventing new intra-mammary infections by 50 to 75%; a fundamental part of a successful mastitis control programme (Eberhart, 1986; Janosi and Huszenicaza, 2001; Petzer et al., 2009).
Cure rate is higher in dry period than during lactation, higher concentration of drugs can be used, reduce new intramammary infections during dry period, damaged tissue may regenerate prior to calving, incidence of clinical mastitis at caving is also reduced (Nickerson and Owens, 1994). During the dry period, elimination of infection with an antibiotic is easier than during lactation as the drug is not milked out and a higher and more uniform concentration of antibiotics is maintained in the udder. In addition there are no economic losses due to discarding of antibiotic containing milk (Sandholm et al., 1995). Dry cow antibiotic treatment is more efficacious than lactation therapy and has less risk of milk residue (Berry and Hillerton, 2002; Bradley and Huxley, 2003).
The intramammary route is the route of choice for delivery of dry period therapy and has the advantages of being well reached. Its disadvantages are the risks of both physiological and anatomical damage to the streak canal and inoculation of organisms at the time of infusion (Bradley and Huxley, 2003). Systemic dry period therapy may have advantages, better distribution of drug in the udder tissue which may lead to better cure of intra-mammary infections (Ziv, 1980) and avoidance of new infections which is possible risk at administration of intramammary infusion (Boddie and Nickerson, 1986). Systemic administration could simplify dry cow therapy routine. It would also eliminate the risk of introducing infections through non-sterile intramammary injection (Soback, 1988). Systemic administration of antibiotics at drying off or some weeks before parturition seems to be effective treatment for intra-mammary therapy, which may advisable for practice (Nickerson et al., 1999; Bolourchi et al., 1995; Zecconi et al., 1999). The most efficacious time for treatment seems to be 7 to 14 days prior to the expected calving. Treatments earlier than 14 days prior to calving may allow time after the treatment for new intramammary infections to occur prior to calving (Oliver et al., 2003)
Materials and Methods
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A total of twenty seven dry pregnant buffaloes were selected from the different livestock farms and randomly allocated to three equal groupââ‚¬â„¢s viz. G1, G2 and G3. Samples of mammary secretions were collected aseptically 14 days prior to expected calving. Collection of mammary secretions from each quarter was done aseptically according to National Mastitis Council guidelines (Hogan et al., 1999). Each teat end was scrubbed vigorously with cotton gauze soaked with 70 percent ethyl alcohol. Immediately following mammary secretions collection, antibiotic treatments were given to the animals. Animals in group G1 were treated with Enrofloxacin (Inj. Encure10TM; Nawan Laboratories, Pakistan) @ 2.5 mg/kg body weight intramuscularly at 14 days and 7 days prior to expected date of parturition. Group G2 were treated with Oxytetracycline-HCl (Inj. Oxy-KakTM LA; Kaksian, Pakistan) @ 11 mg/kg body weight intramuscularly at 14 days and 7 days prior to expected date of parturition and group G3 served as non medicated control.
Ten ml of milk samples from each quarter aseptically collected at day 7 and 14 post calving. The collected samples will be shifted to microbiological laboratory, college of Veterinary and Animal Sciences, Jhang for isolation and biochracterisation of prevalent mastitis pathogens. Procedure described by National Mastitis council Inc., USA (1990) was followed for the collection of milk samplers. Sterile vial of 15 ml capacity were used. Each teat end was scrubbed vigorously with cotton gauze soaked with 70 percent ethyl alcohol. A separate swab was used for each teat. While holding the vials as horizontal as possible, the cap was removed without touching inner surface and held with the inner surface facing downwards.
Precalving secretion samples and postcalving milk samples were processed for bacteriology within 24 hour of collection following storage at 4ÂÂ°C. Microbiological procedure described by National Mastitis Council Inc., USA (1990) was followed for culturing the samples and identification of mastitis pathogens. The mastitic milk samples were gently shaken 8 times to get a uniform dispersion of the pathogens. Using a platinum-rhodium loop, 0.01 ml of milk sample was streaked onto blood agar plates. Four quarter milk samples were cultured on a 100 mm plate by plating individual quarter sample on one quadrant of plate and were incubated at 370 C for 48 hours. A quarter was considered to be infected if 5 or more similar colonies were present on plate. (Roberson et al., 1988) The quarter should be considered infected if milk secretions samples contain one of mastitis pathogens before treatment. The absence of the same bacteria at sampling 7 and 14 days following parturition were interpreted as a bacteriological cure otherwise in the presence of the bacteria the mammary quarter remained uncured. An uninfected quarter at drying off that was infected at calving was considered to indicate new intramammary infections. A quarter that was infected at drying off but infected with another organism at calving also indicated new intramammary infections.
The cultural and morphological characteristics of primary growth were studied by examination of colony characteristics and preparation of smears from different colonies. These smears were stained with Gramââ‚¬â„¢s staining method and examined under the microscope. The primary growths were purified by frequent sub culturing on selective and differential media. The selective and differential media used were MacConkeyââ‚¬â„¢s agar (for streptococcal species) and blood agar (hemolytic species).
Each of the isolate was identified on the basis of cultural and morphological characteristics, motility, hemolytic and biochemical properties as described (Cruickshank et al. 1975). Catalase positive. Gram positive coccal isolates presumptively identified as staphylococcus or micrococci. The genus of bacteria determined on the basis of colony morphology, Gram stain, hemolysis pattern, catalase test, and esculin reaction. Gram-positive, catalase-positive isolates were further tested using a tube-coagulase test. Coagulase-positive isolates were defined as Staphylococcus aureus and coagulase-negative isolates as CNS. Gram-positive, catalase-negative isolates were CAMP-tested: esculin-positive and CAMP-positive or negative isolates were defined as Strep. uberis; esculin-negative and CAMP-negative isolates were defined as Streptococcus dysgalactiae; and esculin negative and CAMP-positive isolates were defined as Streptococcus agalactiae. Gram-negative rods were subcultured on MacConkey. Corynebacterium spp. was defined as small gram-positive rods organized in distinctive "pallisades". Organism other than staphylococci identified by routine biochemical tests (National Mastitis Council Inc., 1990).
Percent prevalence of mastitis was calculated in all groups. Cure rate of infected quarter among groups were calculated using chi square tests comparing treated groups and the control. Each treatment group was also compared with other group and with the control group using two proportional Z test. P< 0.05 considered as significant and P> 0.05 considered as non significant (Steel et al., 1997).
Prevalence of clinical mastitis at parturition
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Out of total 27 animals 3(11%) were clinically mastitic at parturition. Among these no animal were positive in enrofloxacin group (0 %), one in oxytetracyclin group (11.11%) and two in control group 2(22.22%) were positive. Visual examination of udder and teats followed by palpation revealed that udder quarter were asymmetric due to swelling clinical symptoms swelling of udder presence of visible changes (e.g., clots or discoloration) of mastitic milk, pain, hotness, and firmness were observed. Out of 144 quarters 7(9.72%) were infected. No quarter was infected at parturition in enrofloxacin group (0 %), two quarters were infected in oxytetracyclin group (5.56%), and in control group 5(13.89%) were positive for mastitis. Percent Prevalence of clinical mastitis at parturition is higher in control group as compared to dry period antibiotics treatment groups.
4.2 Prevalence of subclinical mastitis after parturition
Out of 27 animals 7(25.93%) were subclinical mastitic after parturition at day 7 post calving. Among these with enrofloxacin treatment were 1(11.11%), with oxytetracycline 2(22.22%) and in control group 4(44.44%) as depicted in Table 4.3. Quarter wise prevalence of subclinical mastitis was 14(38.89%) after parturition at day 7 post calving. Among these with enrofloxacin treatment group were 1(2.78%), with oxytetracycline were 3(8.33%) and in control group 10(27.78%) quarters were infected. Results showed that prevalence of subclinical mastitis is higher in control group as compared to treatment groups. Similarly quarter wise prevalence of subclinical mastitis at day 14 post calving was 11(30.56%). Among these with enrofloxacin treatment group were 1(2.78%), with oxytetracycline were 3(8.33%) and in control group 7(19.44%) quarters were infected. Animals that had no observable mastitis symptoms in milk or mammary lobes while one or more querters detected as infected with mastitis pathogens were classified as having subclinical mastitis
4.3 Prevalence of clinical mastitis after parturition
Out of 27 animals only 2(7.41%) were having clinical mastitis after parturition at day 7. Among these no animal was infected with clinical mastitis in enroflaxacin and oxytetracycline treatment group. No quarter was infected clinically in these groups. In control group 2(8.33%) animals clinically positive. Neither any animal nor any quarter was found to be infected at 14 days post calving.
4.4 Postpartum cure rate of infected quarters
Postpartum cure rate of infected quarters at day 14 were 91.67% (11/12) with enrofloxacin treatment groups, 70% (7/10) with oxytetracycline treatment groups and 21% (3/14) with control groups. The cure rate among the group were significantly different (p=0.001, p< 0.05). The highest cure rate belongs to enrofloxacin group which was significantly higher than that of control (p=0.0002 p< 0.05), followed by oxytetracycline group which was also significantly higher than that of control (p=0.008 p< 0.05); however there were no significant difference between enrofloxacin groups and oxytetracycline groups (p=0.90 p> 0.05) as depicted in Table 4.8
The present study was the first, to the best of our knowledge designed to compare the efficacy of oxytetracycline and enrofloxacin as systemic dry period therapy in the control of bubaline mastitis. This study was performed on twenty seven dry pregnant buffaloes. This was based on objectives that dry period antibiotics therapy before parturition eliminates the existing intramammry infections and preventing new intramammary infections. Prevalence of clinical mastitis and quarter wise prevalence of subclinical mastitis post calving after systemic dry period therapy with enrofloxacin were 0% and 2.78% which was lower than control group. Prevalence of clinical mastitis and quarter wise prevalence of subclinical mastitis post calving after systemic dry period therapy with oxytetracycline were 11.11% and 8.33% which is lower than control group. In control group, prevalence of clinical mastitis and quarter wise prevalence of subclinical mastitis post calving were 22.22% and 27.78% which is higher than systemic dry period treatment groups. Cure rates of infected quartes after parental enrofloxacin were 91.67 %, with oxytetracycline were 70% and in control group spontaneous were 21%. These findings are consistent with Soback et al. (1990) who observed similar findings that systemic dry cow therapy using norfloxacin nicotinate, which possesses large distribution volume, long half life, and is highly active against the pathogen involved, was more effective than the other treatments. Both enrofloxacin and oxyteracycline are well distributed into the body fluids and should be able to reach the site of infection. But enrofloxacin has better results than oxytetracycline. In other study, Hovareshti et al. (2007) get similar results with injection of tylosin intramuscularly 83%, with intramuscularly enrofloxacin injection 70% while comparing with dry cow intramammary preparations 93%. However dry cow intramammary preparations were the most effective methods to cure the intramammary infections. However in large dairy herds, it is almost impractical, tendios, dangerous and not expectable by the farmers. Additionally, this may introduce environmental bacteria and fungi into the quarters by unsanitary manipulation of udder. The risk of antibody residue was lower in systemic treatment as compared to intramammary infusions. Similarly better efficacy obtained with systemic dry cow therapy by (Smith and Hogan 1995; Marco, 1996). There was no significant difference on cure rate % of intramammary infections between intramammary and systemic antibiotics combinations, systemic antibiotic only or between systemic antibiotic and intramammary antibiotic only.
Systemic antibiotics dry period therapy can be cost effective especially in herds where subclinical mastitis is a chronic problem. Our results, however, agree with previous reports using a macrolide antibiotic, tylosin, 2 weeks before the expected day of calving by (Zecconi et al. 1999). Systemic administration of antibiotics could simplify dry period therapy routine. It would also eliminate the risk of introducing infections through non-sterile intramammary injection. Systemic dry period therapy had so far reported inconsistent results with (Soback et al. 1990a; Erskine et al. 1994; Smith and Hogan, 1998; Nickerson et al. 1999; Zecconi et al. 1999). In contrary to this study Shpigel et al. (2006) observed very low cure rate after systemic cefquinome treatment was comparable to the spontaneous cure rate observed in untreated controls in previous studies. The unfavorable results of the cefquinome systemic dry period therapy might reflect inadequate pharmacokinetic properties of the drug regarding poor udder penetration in subclinical mastitis and short antimicrobial effect. In another study Erskine et al. (1994) compared efficacy of intramuscular oxytetracycline as a dry period treatment for Staphylococcus aureus mastitis. Cure rate of infected quarter with oxytetracycline treatment group were (34 quarters, 10 cures) 29.4%) 30 day post calving and 21.2% (7 of 33) of the quarters were considered to be cured, 60 days post calving and got inferior result from the present study. One reason might be, they used dry period antibiotics therapy at drying of, dry period length may be long and animal regain infections during dry period. Similarly Nickerson et al. (1994) compared parenteral administration of dry cow therapy by subcutaneous injection of tilmicosin at 5 mg/kg of body weight at dry-off and again 4 days later was ineffective against intramammary infections caused by Staphlococcus aureus (9.1% cure rate). Parker et al. (2008) used of an internal teat canal sealant and teat dipping as dry period therapy in heifers to reduced the post calving intramammary infections prevalence and pathogen associated with clinical mastitis by decreasing the incidence of new infections over high risk peripartum period. It may be a useful method for preventing new intramammary infections but it can not cure existing intramammary infections.
In the present study prevalence of staphylococcus aureus, Streptococcus agalactiae, E. coli, coagulase negative staphylococci and corynebacterium were at day 7 and 14 postpartum were 0% 0%, 0%, 2.78% and 0% respectively in enrofloxacin groups. Cure rate of staphylococcus aureus, Streptococcus agalactiae, E. coli, and corynebacterium were 100% while in coagulase negative staphylococci was 66.66%. In oxytetracycline groups prevalence of staphylococcus aureus, Streptococcus agalactiae, E. coli, coagulase negative staphylococci and corynebacterium post calving were 5.56%, 2.78%, 0%, 2.78%, and 0% respectively. Cure rate staphylococcus aureus was 66.66%, Streptococcus agalactiae 0%, E. coli 100%, and coagulase negative staphylococci 50%. In the control group prevalence of staphylococcus aureus, Streptococcus agalactiae, E. coli, coagulase negative staphylococci and corynebacterium post calving were 19.44%, 8.33%, 5.56%, 5.56% and 2.78 % respectively. Spontaneous cure rate of staphylococcus aureus was 12%, Streptococcus agalactiae 0%, E. coli 0%, coagulase negative staphylococci 33.33%, and corynebacterium 0%. Almost similar cure rate obtained Petzer et al (2009) using intramammary product containing (Cephalexin 250 mg and neomycin sulphate 250 mg). The cure rate of varied from 94.4 % for Staphylococcus aureus, 100% for Streptococcus agalactiae and Streptococcus dysgalactiae, 78.1% for coagulase negative staphylococci and 100% for the other minor pathogens. The quarter new infection rate during the dry period was 17.44 % in control groups. These results are almost similar with the present study.
In the present study, four new infections (11.11%) were also occurred in the control group but no new infections appeared in the treated group. These findings consistent with Natzke (1971) that the development of new quarter infections during the dry period without dry period therapy, are 10 to 15%. These infections can reduce milk production by 36% during the first month of the subsequent lactation. Approximately 33 to 50% of the subclinical infections rates, postpartum result in clinical symptoms of mastitis during the lactation; 50% of these new infections occur in cows that were previously infected in at least one quarter (Healed et al., 1977). Osteras et al. (1991) recorded new intramammary infections rates during the dry period of between 13.1 % and 24.0 %. However, most other studies which were in agreement with the findings of present study described lower rates of between 4 % and 14 % (Natzke, et al., 1975; Oliver, 1988; Schukken, et al., 1993; Smith et al., 1985; Williamson et al., 1995). The expected rate of new intramammary infections during the dry period in bacteria negative quarters that were untreated was reported by Eberhart (1986) to vary between 8 % and 12 % while Berry and Hillerton (2002) reported new infection rates of 34.4 % in untreated cows compared with 10.3 % in treated cows. The reduction of new intramammary infections during the dry period with dry period therapy has been estimated at between 50 % to 80 % (Eberhart, 1986).
The optimum production and maximum daily yield of milk can only be achieved if mastitis is prevented at herd level by adopting guidelines of mastitis control program. The dry period offers the best opportunity during the lactation cycle to remove existing, persistent intramammary infections, though unfortunately this opportunity is tempered by an increased risk of new intramammary infections. Dry period antibiotics therapy reduces this risk. Dry period antibiotics therapy has the dual purpose of treatment and prevention. Systematic dry period antibiotics therapy is recommended for herds with a high infection rate. It is better, easy to adopt and friendly to farmers. Dry period antibiotic therapy is also suggested in herds with low somatic cell counts and low prevalence of contagious mastitis pathogens, to minimize the new dry period infections by environmental pathogens which can result in a high incidence of clinical mastitis in the early lactation. Dry period antibiotic therapy administered within 1 to 3 wk before expected calving might have best results in our conditions. In order to achieve the best cure rates it is important to select an appropriate antibiotic that have high absorption in udder.
Table 4.8: Day 14 post calving quarter based cure rate of infections treated with enrofloxacin and oxytateracycline
Total Number of Quarter
Number of Quarter Infected Before Treatment
Number of Quarter Cured
Cured Rate %age