Evaluation of a Cow-Side Test for Detection of Gram-Negative Bacteria in Milk from Cows with Mastitis

Waage, S., P. Jonsson and A. Franklin: Evaluation of a cow-side test for detection of Gram-negative bacteria in milk from cows with mastitis. Acta vet. scand. 1994, 207-211.– A modified Limulus amebocyte lysate (LAL) cow-side test was evaluated under field conditions. The principle of the test is to visualize reactions between test components and endotoxin from the cell wall of Gram-negative bacteria. The practical purpose is to detect such bacteria in mastitic milk. Secretions from 789 udder quarters with clinical mastitis were examined by the LAL-test. Parallel quarter milk samples were sent to a mastitis laboratory for microbiological examination. Eleven veterinary surgeons in three veterinary districts in Norway performed the field investigations. Results of the LAL-test and culture agreed in 93% of all milk samples tested, agreement measured by kappa being 0.63. The sensitivity of the test in detecting Gram-negative bacteria was 63%, while the specificity was 97%. The predictive value of a positive test result was 70%, the figure being somewhat higher (75%) when the material was limited to milk samples without antibiotic residues. The predictive value of a negative test result was 95%. The LAL-test is considered to constitute a valuable cow-side test for the veterinary practitioner, aiding the selection of antibacterial drug of choice for the initial treatment of clinical mastitis.     

Evaluation of a portable test system for assessing endotoxin activity in raw milk

The aim of the present study was to compare endotoxin activities detected in raw milk samples obtained from cattle by a commercially available portable test system (PTS) and traditional microplate limulus amebocyte lysate (LAL)-based assay, which determined activities using a kinetic turbidimetric (KT) assay. Raw milk samples were obtained from 53 and 12 dairy cattle without and with clinical mastitis, respectively. Comparison between the KT and PTS was performed by the Friedman test. The Pearson product moment correlation coefficients were calculated to evaluate associations between any two continuous variables. Linear regression model analysis was also performed to obtain the equation describing the relationship between PTS and KT assay. The endotoxin activities detected in 200- or 400-fold diluted milk samples were similar between PTS and KT assay, whereas a significant difference was observed in 100-fold diluted milk (P<0.001). The results obtained from 200- (r²=0.778, P<0.001) and 400-fold diluted milk samples (r²=0.945, P<0.001) using PTS correlated with those using KT assay. The median milk endotoxin activities in Gram-positive and Gram-negative clinical mastitis cows were 0.655 and 11,523.5 EU/ml, respectively. The results of the present study suggest that PTS as a simple and easy test to assess endotoxin activity in raw milk is efficient, simple and reproducible.     

Endotoxin levels in milk and plasma of mastitis-affected cows measured with a chromogenic limulus test

A chromogenic limulus test ("Toxicolor") was applied to cow's milk and plasma after treatment with perchloric acid to remove interfering factors. The endotoxin levels in normal cow's milk and plasma were all less than 10 pg ml-1. In acute mastitis, the milk endotoxin level averaged (1.1 +/- 0.7) X 10(3) pg ml-1 in the cases where Gram-negative bacteria were isolated, while the plasma endotoxin concentration was normal. The endotoxin levels in the quarters infected with Gram-positive bacteria were all normal, both in milk and plasma. In gangrenous mastitis due to Gram-negative bacteria, the endotoxin concentration was very high in both milk [(9.3 +/- 5.3) X 10(6) pg ml-1] and plasma (85.2 +/- 68.2 pg ml-1). In similar cases due to Gram-positive bacteria, endotoxin levels were all normal, both in milk and plasma, resembling the acute mastitis due to Gram-positive bacteria. The test was considered suitable for the diagnosis of mastitis due to Gram-negative organisms and the levels of endotoxin detected would aid in assessing the prognosis.     

奶牛静脉血内毒素浓度及其与产奶量的相关性

为调查我国当前主流饲喂方式下奶牛静脉血内毒素浓度的范围及内毒素浓度与产奶量的相关性,本试验首先验证了应用鲎试剂法定量奶牛静脉血中内毒素浓度的可行性,在此基础上,分别检测了安徽牧场 １（６０头,饲粮精粗比 ４５∶５５）、江苏牧场（１５头,按产奶量高低精粗比分别为 ２０∶８０、４５∶５５、５０∶５０）和安徽牧场 ２（１４头,饲粮精粗比 ７０∶３０）共 ８９头奶牛的尾静脉血中内毒素浓度,并进行了内毒素浓度与产奶量的相关性分析。结果表明：鲎试剂法可有效检测奶牛静脉血中内毒素浓度,３个牧场奶牛静脉血中内毒素浓度高低不等,在 ０～１．５０ＥＵ／ｍＬ范围内。奶牛静脉血中内毒素浓度与产奶量之间的相关性皆不显著（Ｐ＞０．０５）,但安徽牧场 ２的相关系数（０．１９５２）大于安徽牧场 １（０．０２０５）和江苏牧场（０．０３７６）。结果提示,鲎试剂法可用于定量检测奶牛血中内毒素浓度,在我国当前的饲喂方式下,奶牛血液中普遍含有内毒素,静脉血内毒素浓度与产奶量无显著相关性。     

Development and evaluation of an enzyme-linked immunosorbent assay for endotoxin in milk

A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection of endotoxin in milk samples. Bovine and rabbit antisera raised in response to vaccination with the J5 mutant of Escherichia coli 0111:B4 were used. Antiserum to this mutant has been shown to be cross-reactive with endotoxin from other gram-negative organisms. Known quantities of endotoxin were added to milk samples to generate a standard curve. Acid treatment of whole milk enhanced the detection of endotoxin as compared to untreated whole milk, skim milk and chloroform-treated milk. Milk samples from experimentally induced mastitic cows were then assayed for endotoxin content. Recovery of endotoxin, as measured by ELISA, positively correlated with the amount of endotoxin infused and the time post-infusion of sampling. However, when endotoxin from these samples was quantitated using the Limulus Amebocyte Lysate (LAL) assay, readings tended to increase, suggesting false-positive reactions with the LAL assay. Milk samples from cases of clinical mastitis were assayed by ELISA with 64% of these showing measurable levels of endotoxin. While further studies of this assay are needed, refinements may produce an assay important for clinical applications.     

Biological effects of sonicated suspension and phenol-water extract of Mycoplasma mycoides subspecies mycoides in goats.

Strain Y3343 isolated from a goat with septicemia and polyarthritis was studied. The strain was virulent and induced septicemia, polyarthritis and coagulopathy in two goats. Limulus amebocyte lysate active material was present in plasma, but not in higher titre in inoculated goats. Sonicated mycoplasma material induced a dramatic somatic cell response in the mammary gland of cows and goats and marked clotting of the cows' milk, but it did not clot limulus amebocyte lysate or kill chick embryos. Phenol-water extract clotted limulus amebocyte lysate and induced somatic cell response in cows but not in goats. The phenol-water extract did not kill chick embryos, was not pyrogenic in rabbits or goats, and did not induce generalized Shwartzman reaction or change the leukocyte kinetics in rabbits. It therfore appears that the virulence mechanisms of strain Y3343 can not be explained on the basis of factors with strong endotoxin activity.     

Intracecal endotoxin and lactate during the onset of equine laminitis: a preliminary report.

Cecal fluid from two adult horses was assayed by the limulus amebocyte lysate system for endotoxin before and after carbohydrate overload of the gastrointestinal tract. There were increases in cecal fluid endotoxin concentrations at the 3-, 6-, and 12-hour samplings when compared with base-line values. Concomitant cecal fluid lactate concentrations and pH values increased and decreased, respectively. Both horses subsequently developed clinical signs of acute laminitis.     

Preliminary study of appearance of endotoxin in circulatiory system of sheep and cattle after induced grain engorgement.

Edotoxin was detected, using the limulus amebocyte lysate (LAL) test, in the blood of 3 sheep and 1 steer which had been experimentally "overfed" (induced grain engorgement) with a mixture of corn and oats (2:1). The 1st postfeeding blood samples were collected 24 hours after overfeeding. In 2 sheep and 1 steer, the 24-hours blood samples were test positive. In 1 sheep which died, the 48-hour blood sample was the 1st test-positive sample. In all cases, pre-overfeeding blood samples were taken just before overfeeding.     

Measures taken by Veterinarians in Sweden in Cases of Bovine Mastitis

A questionnaire concerning the diagnosis and treatment of bovine mastitis was sent to all 350 Swedish food animal practitioners. 287 (82%) of the questionnaires were returned. One of the main aims was to establish if Swedish food animal practitioners used a common therapeutic regime that could be used as a control treatment in future clinical trials.It was found that many factors of importance for the clinical diagnosis of mastitis such as body temperature, duration of the symptoms etc. often were not considered. On the other hand 60–70% of the practitioners regularly took milk samples to obtain a bacteriological diagnosis. Approximately 40% of the veterinarians cultured the milk samples in their home laboratory.Basic measures like frequent emptying of the udder were recommended by only 40–50% of the veterinarians.All responding field veterinarians used the systemic route for administering antibiotics when treating cases of acute, clinical bovine mastitis. The drug of choice, initially, in these cases was benzylpenicillinprocain, which was used by 65–75% of the veterinarians. Twentyfive percent used a broadspectrum antibiotic, most commonly a combination of penicillin and streptomycin. A minority (5%) directed their initial therapy towards gramnegative bacteria.About 30–40% supported the systemic therapy with intra-mammaries. Other drugs such as NSAID, corticosteroids and oxytocin was used, on a regular basis, by only about 10% of the practitioners.     

Antimicrobial activity of bovine bactericidal permeability-increasing protein-derived peptides against gram-negative bacteria isolated from the milk of cows with clinical mastitis

OBJECTIVE: To evaluate antimicrobial activity of bovine bactericidal permeability-increasing protein (bBPI)-derived synthetic peptides against mastitis-causing gram-negative bacteria. SAMPLE POPULATION: Bacterial isolates from the milk of cows with clinical mastitis. PROCEDURES: 3 peptides were synthesized with sequences corresponding to amino acids 65 to 99 (bBPI(6,599)) or 142 to 169 (bBPI(142,169)) or the combination of amino acids 90 to 99 and 148 to 161 (bBPI(9,099, 148,161)) of bBPI. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of these peptides against bacterial isolates from cows with mastitis were determined by use of a standardized broth microdilution assay. The ability of these peptides to retain their antimicrobial activity in serum and milk was also evaluated. Finally, bacterial lipopolysaccharide (LPS)-neutralizing activity of these peptides was assayed with the Limulus amebocyte lysate test. RESULTS: Of the 3 peptides tested, bBPI(9,099, 148,161) had the widest spectrum of antimicrobial activity, with MIC and MBC values ranging from 16 to 64 Mg/mL against Escherichia coli, Klebsiella pneumoniae, and Enterobacter spp and from 64 to 128 Mg/mL against Pseudomonas aeruginosa. None of the peptides had any growth-inhibitory effect on Serratia marcescens. The antimicrobial activity of bBPI(9,099, 148,161) was inhibited in milk, but preserved in serum. Finally, bBPI(142,169) and bBPI(9,099, 148,161) completely neutralized LPS. CONCLUSIONS AND CLINICAL RELEVANCE: bBPI(9,099, 148,161) is a potent neutralizer of the highly proinflammatory molecule bacterial LPS and has antimicrobial activity against a variety of gram-negative bacteria. The ability of bBPI(9099,148161) to retain antimicrobial activity in serum suggests a potential therapeutic application for this peptide in the management of gram-negative septicemia.     

Ability of hematologic and serum biochemical variables to differentiate gram-negative and gram-positive mastitis in dairy cows

Medical records of 142 dairy cows with clinical mastitis were examined to determine whether hematologic or serum biochemical results could be used to distinguish between mastitis episodes caused by gram-negative bacteria (n = 78) from those caused by gram-positive bacteria (n = 64). Signalment, historic information, hematologic and serum biochemical results, milk culture results, and outcome (discharged from hospital or died) were obtained from the medical records. Cows with gram-negative mastitis had significantly (P < .01) lower blood leukocyte, segmented neutrophil, monocyte, and lymphocyte counts and had higher blood hemoglobin concentrations and hematocrits than did cows with gram-positive mastitis. Serum urea nitrogen was the only serum biochemical result associated with pathogen type, and it was higher in cows with gram-negative mastitis than in those with gram-positive mastitis. Mortality rate (25% overall) did not differ between groups. Logistic regression indicated that routine hematologic analysis (segmented neutrophil count, monocyte count, and hemoglobin concentration) was an accurate predictor of gram-negative mastitis, with a sensitivity of .93, a specificity of .89, and an overall accuracy of 91%. The values for sensitivity and specificity were higher than those previously reported for clinical tests differentiating mastitis episodes caused by gram-negative bacteria from those caused by gram-positive bacteria. Our results indicate that routine hematologic analysis is useful for predicting pathogen type in dairy cows with clinical mastitis, thereby facilitating treatment decisions.     

Efficacy of targeted 5-day combined parenteral and intramammary treatment of clinical mastitis caused by penicillin-susceptible or penicillin-resistant Staphylococcus aureus

Combined parenteral and intramammary treatment of mastitis caused by Staphylococcus aureus was compared to parenteral treatment only. Cows with clinical mastitis (166 mastitic quarters) caused by S. aureus treated by veterinarians of the Ambulatory Clinic of the Faculty of Veterinary Medicine during routine farm calls were included. Treatment was based on in vitro susceptibility testing of the bacterial isolate. Procaine penicillin G (86 cases due to beta-lactamase negative strains) or amoxycillin-clavulanic acid (24 cases due to beta-lactamase positive strains) was administered parenterally and intramammarily for 5 days. Efficacy of treatments was assessed 2 and 4 weeks later by physical examination, bacteriological culture, determination of CMT, somatic cell count and NAGase activity in milk. Quarters with growth of S. aureus in at least one post-treatment sample were classified as non-cured. As controls we used 41 clinical mastitis cases caused by penicillin-susceptible S. aureus isolates treated with procaine penicillin G parenterally for 5 days and 15 cases due to penicillin-resistant isolates treated with spiramycin parenterally for 5 days from the same practice area. Bacteriological cure rate after the combination treatment was 75.6% for quarters infected with penicillin-susceptible S. aureus isolates, and 29.2% for quarters infected with penicillin-resistant isolates. Cure rate for quarters treated only parenterally with procaine penicillin G was 56.1% and that for quarters treated with spiramycin 33.3%. The difference in cure rates between mastitis due to penicillin-susceptible and penicillin-resistant S. aureus was highly significant. Combined treatment was superior over systemic treatment only in the beta-lactamase negative group.     

Prevalence of coagulase-negative staphylococci in bovine mastitis in Zimbabwe

This study was carried out to determine the prevalence of coagulase-negative staphylococci in clinical and subclinical mastitis in commercial and small-scale farms in Zimbabwe. Thirty five quarter milk samples from clinical mastitis cases and 371 quarter milk samples from cows with subclinical mastitis were cultured for bacterial pathogens. The most frequent pathogens isolated in clinical mastitis were the enteric bacteria (31.4%), followed by coagulase negative staphylococci (22.9%) and then Staphylococcus aureus (17.1%), whereas in subclinical mastitis S. aureus (34.2%) and coagulase-negative staphylococci were (33.2%) the most common. Bacillus species were only isolated in milk samples from subclinical mastitis. Coagulase-negative staphylococci were observed in mixed infections with other bacteria in only 2.2 of the 406 milk samples from clinical and subclinical mastitis where they were isolated together with Bacillus species in 6 of the 9 mixed infection cases. About 95% of the milk samples from which 131 coagulase-negative staphylococci were isolated had correspondingly high somatic cell counts. The coagulase-negative staphylococci isolated most frequently were S. chromogenes (7.9%), S. epidermidis (7.4%) and S. hominis (5.9%). They were all associated with high somatic cell counts. All the coagulase-negative staphylococci isolates were susceptible to cloxacillin and erythromycin, and more than 90% of the isolates were susceptible to neomycin, penicillin and streptomycin. The highest resistance was to tetracycline (17.6%), followed by lincomycin (13.7%). About 8% of the isolates were resistant to both penicillin and streptomycin.     

Evaluation of a rapid coliform detection kit from clinical mastitis milk using colloidal gold nanoparticle–based immunochromatographic strips

The accurate identification of mastitis‐causing bacteria assists in effective management by both dairy farmers and veterinarians and can be used to implement the selective use of antimicrobials for treatment. The purpose of this study was to evaluate the ability of our developed anti–ribosomal protein-L7/L12 antibody–coated immunochromatographic strip (ICS) test to detect coliforms in milk by comparing the results with the bacteriological culture method. We investigated the performance of the ICS test as compared with the bacteriological culture method using 308 milk samples from clinical bovine mastitis. First, to determine the optimal ICS test cutoff point for detecting coliform mastitis, we developed a receiver-operating characteristic curve. The result showed that the cutoff point was at 0.5 of our index. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value of the ICS test were 81.3%, 84.8%, 69.2%, and 91.54%, respectively. As the clinical signs increased in severity, the F-measure, a weighted harmonic mean of the sensitivity and overall PPV performance, increased. Because it is especially important to treat clinical mastitis appropriately in the early stages of detection, the ICS test, which can be used by both dairy farmers and veterinarians on dairy farms, is considered to be a useful tool for detecting coliform mastitis, which often presents with severe signs.     

Mastitis in lactating camels (Camelus dromedarius) in Afar Region, north-eastern Ethiopia.

Quarter milk samples (n = 543) from 152 traditionally managed lactating camels (Camelus dromedarius) in Afar Region, north-eastern Ethiopia were examined to determine the prevalence of camel mastitis and identify its bacterial causes. Out of 152 camels examined, 19 (12.5%) were diagnosed as clinical mastitis cases based on clinical signs and bacteriological examinations. Of the 257 California Mastitis Test (CMT) positive quarter milk samples 162 (63.0%) yielded pathogenic bacteria. A positive correlation was observed between CMT positive results and presence of major pathogens in camel milk samples. The main mastitis pathogens isolated were Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus agalactiae, S. dysgalactiae, and other species of streptococci, Pasteurella haemolytica and E. coli. Results of the present study suggest that mastitis in Afar camels is prevalent, Gram-positive cocci are the major isolates from camel milk samples and the CMT can be used as a screening test for the detection of mastitis in camels.     

Evaluation of the California mastitis test to detect an intramammary infection with a major pathogen in early lactation dairy cows

The purpose of this study was to evaluate the usefulness of the California mastitis test (CMT) to detect an intramammary infection caused by a major mastitis pathogen in early lactation cows. The gold standard used for comparison was bacteriological culture of single milk samples. The sensitivity (82.4%) and specificity (80.6%) of a positive CMT were highest on the 4th day of lactation.     

Clinical and bacteriological response to treatment of clinical mastitis with one of three intramammary antibiotics

AIM: To compare the proportions of clinical and bacteriological cure of glands of dairy cows diagnosed with clinical mastitis, following treatment with one of three different intramammary antibiotic preparations. METHODS: Cows from dairy cow herds (n=28) across New Zealand which were diagnosed with clinical mastitis in one or more glands at any stage of lactation were randomly assigned at the cow level within sequentially presented groups of three animals to be treated with one of three intramammary antibiotics. The treatments were 1 g procaine penicillin, 0.25 g cefuroxime, and a combination of 1 g procaine penicillin and 0.5 g dihydrostreptomycin (DHS). All treatments were infused on three occasions at 12-hourly intervals. Duplicate milk samples were collected for bacteriology before initial treatment, and 21-42 days later. Logistic regression or generalised linear mixed models were used to analyse the proportion of cows or quarters retreated for mastitis within 30 days of initial treatment ('clinical treatment failure'), and the proportion of glands from which bacteria were isolated initially but from which the same bacterial species was not re-isolated ('bacteriological cure'). RESULTS: The annual herd average cumulative incidence rate of clinical mastitis was 12.7 cases/100 cows. The incidence rate was higher in young (2-year-old) and old (> or = 9-year-old) cows relative to 3- and 4-year-old cows, and was higher in Friesian than Jersey or crossbred cows. Streptococcus uberis was the pathogen most commonly isolated, and its relative prevalence declined with time postpartum. Cows treated with cefuroxime were more likely (p<0.01) to be re-treated for clinical mastitis in the 30 days after initial treatment than cows treated with procaine penicillin or procaine penicillin and DHS. Bacteriological cure occurred in 74% of treated glands and there was no difference in the proportion of cures among the treatments (p=0.4). The proportion of cures was lower when treatment occurred 28-72 days after calving (p<0.01) and if a major pathogen was isolated (p<0.001). CONCLUSIONS AND CLINICAL RELEVANCE: There was no benefit in terms of clinical or bacteriological cure rate in treating clinical mastitis cases with the combination of procaine penicillin and DHS compared to treatment with procaine penicillin alone. The proportion of clinical mastitis cases re-treated differed among herds, and more cows treated with cefuroxime were retreated within 30 days of initial treatment. However, the bacteriological cure proportion was the same among the treatments. Cure proportions were lower in cows from which major mastitis pathogens were isolated and when treatment commenced 28-72 days after calving.     

A comparative efficacy trial between cefuroxime and cloxacillin as intramammary treatments for clinical mastitis in lactating cows on commercial dairy farms

AIMS: To assess the efficacy of a commercial intramammary preparation containing cefuroxime as a treatment for clinical mastitis in lactating dairy cows. METHODS: Clinical mastitis cases (n=440) were identified during early to mid lactation on 36 seasonally-calving commercial dairy herds in south-western Victoria, Australia, that ranged in size from 140 to 550 cows. A milk sample for bacterial culture was collected from each affected quarter prior to treatment. Cases were treated using a commercial intramammary antibiotic product containing either 250 mg cefuroxime or 200 mg cloxacillin, according to the manufacturer's recommendations, and the recommended milk with-holding period was observed. The assessment of clinical cure of each case was made by the farm owner/manager at the end of the milk with-holding period. Post-treatment milk samples were taken from clinically cured quarters at each of three consecutive milkings, commencing 7 days after the end of the milk with-holding period and submitted for bacterial culture. RESULTS: Pathogenic bacteria were isolated from 252/416 (60.6%) pre-treatment milk samples submitted from eligible cases. Streptococcus uberis was the most frequent isolate, being cultured from 31.7% of cases. Staphylococcus aureus was isolated from 18.3% of pre-treatment samples, and Esherichia coli from 7.0%. The clinical cure rate for all eligible cases was 81.7%. There was no significant difference in clinical cure rates between cases treated with cefuroxime (186/225=82.7%) and cases treated with cloxacillin (154/191=80.6%). The trial had 80% power to detect a significant difference if the actual cure rates differed by at least 12.6%. There was a superior clinical response rate (p=0.04) for mastitis cases from which E. coli was isolated that were treated with cefuroxime (18/19=95%) compared with cloxacillin 6/10=60%), but case numbers were low (n=29). The overall bacteriological cure rate for the trial was 70% (69/98 cases assessed). There was no significant difference (p=0.27) in bacteriological cure rate between cases treated with cefuroxime (42/56=75%) and cases treated with cloxacillin (27/42=64%). CONCLUSIONS: This trial demonstrated that cefuroxime was an effective intramammary treatment for clinical mastitis and had similar clinical and bacteriological cure rates to cloxacillin, an accepted industry-standard product. Activity against Gram-negative and Gram-positive bacteria was demonstrated for cefuroxime which, along with the economic benefits of less discarded milk compared with other antibiotic preparations, indicated that cefuroxime is likely to be an appropriate intramammary antibiotic for the treatment of clinical mastitis in commercial dairy herds during early to mid lactation in Victoria, Australia.     

奶牛急性乳房炎病原菌的分离与鉴定

[目的]为了掌握奶牛急性乳房炎致病菌的种类.[方法]通过采集病牛乳汁样品3份,分别对其病原茵进行分离与鉴定.检测[结果]表明:检出4种共13株细菌,其中致病性金黄色葡萄球菌6株;停乳链球菌5株;大肠埃希茵1株,乳房链球茵1株.[结论]该场奶牛的急性乳房炎是由致病性金黄色葡萄球菌、停乳链球菌、乳房链球菌和大肠杆菌共同引起...     

Acute coliform mastitis in buffaloes (Bubalus bubalis): Clinical findings and treatment outcomes

This report was delineated to study the clinical, bacteriological and therapeutic aspects concerned with acute coliform mastitis in buffaloes. Bacteriological examination of 80 quarter milk samples obtained aseptically from 56 buffaloes with acute mastitis revealed that coliform bacteria was the most common pathogen (45 cases) followed by Staphylococcus aureus (seven cases) then Streptococcus uberis (three cases), and Streptococcus agalactiae (one case). Clinically, hotness, swelling and painful reaction with serous excretion containing clots was recorded in buffaloes with coliform mastitis. The efficacy of ceftiofur was evaluated in the treatment of buffaloes with acute coliform mastitis. Parenteral ceftiofur neither improved clinical signs nor returned milk to pre-infection production level, whereas intramammary ceftiofur and combination of intramammary with parenteral ceftiofur improved the clinical signs in 10/15 and 12/15 buffaloes, respectively. On quarter level, 3/17, 12/17 and 15/21 quarters recovered in groups received parenteral, intramammary and combination therapy, respectively. This study demonstrates that systemic ceftofur is not effective in the treatment of clinical coliform mastitis in buffaloes.