Mycoplasmal mastitis in dairy cows in the Moghan region of Ardabil State , Iran

Mastitis in dairy cows caused by mycoplasmas was first reported in England in 1960 and in the United States of America in 1961. Subsequently the organisms have been reported in other countries such as Canada, Japan, Israel, Australia, New Zealand and many European countries. Mycoplasmas are now regarded as being an important and economically significant cause of mastitis in dairy cows in various parts of the world. Different species of mycoplasmas have been shown to cause udder infections in dairy cows, with Mycoplasma bovis being responsible for most of the mastitis outbreaks. Characteristics of this disease are its highly infectious and contagious nature, complex clinical presentation, and extreme resistance to treatment. Mastitis caused by Mycoplasma species has highly variable signs, ranging from mild clinical cases to complete loss of milk production. The 2 characteristics of most Mycoplasma mastitis infections of individually affected cows are failure to respond to therapy, and spread of the infection from one quarter of the udder to another. Milk transfer during milking is the most common way Mycoplasma mastitis is spread between cows. Mycoplasma species have been isolated from the respiratory and urogenital tracts of asymptomatic, healthy cattle. Under certain circumstances Mycoplasma has been associated with pneumonia, ear and eye infections, vaginal infections and joint disease in cows and calves. It has been suggested that the spread of Mycoplasma spp. from an infected organ system to the mammary gland may occur. Infected cows may remain infected for life, regardless of therapy. Culture of bulk-tank and cows’ milk samples is a useful procedure to determine the existence of Mycoplasma-infected cows in a herd. The milk of cows with clinical mastitis is often watery, tan to brown in colour, and often has a flaky or sandy sediment. The affected quarter of the udder is firm and appears swollen, as if the cow had recently freshened. Results of milk culture for Mycoplasma are reported after 3 and 7 days of being plated in the laboratory. A portion of the milk sample is placed in a pre-incubation broth for 48 hours, then plated onto a special medium and incubated in a low-oxygen (microaerophilic) environment. Until 1998, Mycoplasma mastitis had not been recognized in the State of Ardabil or the Moghan region in spite of repeated attempts to culture organisms from milk samples of mastitis cases refractory to treatment. In 1998, however, 44 cases with classical signs of mycoplasmal mastitis, based on culture-positive milk samples, were reported (Veterinary data 2002, Iran Veterinary Organization, Ardabil City). M. bovis was recovered from milk samples in these cases. A survey of bulk-tank samples and cows’ milk samples was therefore initiated to determine the prevalence of mycoplasma infections in a random sample of Moghan dairy herds, and to establish whether the organism was significantly distributed in dairy cows in Ardabil State. Four dairy farms, A, B, C, D, with >550 animals/herd were chosen for this study. Strict aseptic procedures were used when collecting milk samples to avoid contamination with bacteria present on the skin of the cow, on the hands of the sampler and in the barns. Sampling was monitored as follows: A, teats and udders were cleaned and dried before attempting to collect sterile samples; B, teats were grasped with one hand, and then vigorously scrubbed with cotton moistened with alcohol; C, the cap of each sterile, labelled vial was loosened and held at an angle under the udder; D, 2–3 streams were milked onto the floor; E, each vial was carefully opened and 2–3 streams of milk directed into it; F, neither the ends of the teats nor fingers were allowed to come into contact with the open end of the vial or the inside of the cap; G, caps were replaced and the samples refrigerated; H, quarter samples (1 quarter per vial) were collected from quarters affected with clinical mastitis; I, the farm name, date of collection and source of the sample were written on each vial. Samples from bulk tanks on each farm were collected as follows: The tank was agitated for at least 5 minutes. A sterile milk straw or sterile dipper was used to obtain each sample. A sterile vial was used to contain the sample and filled to about three-fourths. Samples were not collected from the valve at the bottom of tank. The samples were placed on ice or refrigerated immediately for transportation to the laboratory. Milk samples were streaked over onehalf of a plate and bulk-tank samples over an entire plate. Plates were examined for colonies under low power on a standard microscope and colonies were identified by their shape. Growth could be seen after

Mastitis in dairy cows caused by mycoplasmas was first reported in England in 1960 and in the United States of America in 1961.Subsequently the organisms have been reported in other countries such as Canada, Japan, Israel, Australia, New Zealand and many European countries.Mycoplasmas are now regarded as being an important and economically significant cause of mastitis in dairy cows in various parts of the world.
Different species of mycoplasmas have been shown to cause udder infections in dairy cows, with Mycoplasma bovis being responsible for most of the mastitis outbreaks.Characteristics of this disease are its highly infectious and contagious nature, complex clinical presentation, and extreme resistance to treatment 5,8,14 .Mastitis caused by Mycoplasma species has highly variable signs, ranging from mild clinical cases to complete loss of milk production.The 2 characteristics of most Mycoplasma mastitis infections of individually affected cows are failure to respond to therapy, and spread of the infection from one quarter of the udder to another.Milk transfer during milking is the most common way Mycoplasma mastitis is spread between cows 2,7,9,10 .
Mycoplasma species have been isolated from the respiratory and urogenital tracts of asymptomatic, healthy cattle.Under certain circumstances Mycoplasma has been associated with pneumonia, ear and eye infections, vaginal infections and joint disease in cows and calves.It has been suggested that the spread of Mycoplasma spp.from an infected organ system to the mammary gland may occur.Infected cows may remain infected for life, regardless of therapy 1,3,6,7 .Culture of bulk-tank and cows' milk samples is a useful procedure to determine the existence of Mycoplasma-infected cows in a herd 1,8 .The milk of cows with clinical mastitis is often watery, tan to brown in colour, and often has a flaky or sandy sediment.The affected quarter of the udder is firm and appears swollen, as if the cow had recently freshened 1,3,6,7 .Results of milk culture for Mycoplasma are reported after 3 and 7 days of being plated in the laboratory.A portion of the milk sample is placed in a pre-incubation broth for 48 hours, then plated onto a special medium and incubated in a low-oxygen (microaerophilic) environment 1,5,7,12 .Until 1998, Mycoplasma mastitis had not been recognized in the State of Ardabil or the Moghan region in spite of repeated attempts to culture organisms from milk samples of mastitis cases refractory to treatment.In 1998, however, 44 cases with classical signs of mycoplasmal mastitis, based on culture-positive milk samples, were reported (Veterinary data 2002, Iran Veterinary Organization, Ardabil City).M. bovis was recovered from milk samples in these cases.A survey of bulk-tank samples and cows' milk samples was therefore initiated to determine the prevalence of mycoplasma infections in a random sample of Moghan dairy herds, and to establish whether the organism was significantly distributed in dairy cows in Ardabil State.
Four dairy farms, A, B, C, D, with >550 animals/herd were chosen for this study.Strict aseptic procedures were used when collecting milk samples to avoid contamination with bacteria present on the skin of the cow, on the hands of the sampler and in the barns.Sampling was monitored as follows: A, teats and udders were cleaned and dried before attempting to collect sterile samples; B, teats were grasped with one hand, and then vigorously scrubbed with cotton moistened with alcohol; C, the cap of each sterile, labelled vial was loosened and held at an angle under the udder; D, 2-3 streams were milked onto the floor; E, each vial was carefully opened and 2-3 streams of milk directed into it; F, neither the ends of the teats nor fingers were allowed to come into contact with the open end of the vial or the inside of the cap; G, caps were replaced and the samples refrigerated; H, quarter samples (1 quarter per vial) were collected from quarters affected with clinical mastitis; I, the farm name, date of collection and source of the sample were written on each vial.
Samples from bulk tanks on each farm were collected as follows: The tank was agitated for at least 5 minutes.A sterile milk straw or sterile dipper was used to obtain each sample.A sterile vial was used to contain the sample and filled to about three-fourths.Samples were not collected from the valve at the bottom of tank.The samples were placed on ice or refrigerated immediately for transportation to the laboratory.
Milk samples were streaked over onehalf of a plate and bulk-tank samples over an entire plate.Plates were examined for colonies under low power on a standard microscope and colonies were identified by their shape.Growth could be seen after 3 days of incubation at 37 °C in a moist 10 % CO2 incubator, but 7 days of incubation were needed for full development of colonies.Incubation was continued for 7 days before plates were considered negative 1,7,12,13 .For pre-inrichment, 0.1 ml of each milk sample was diluted in 3 serial, 10-fold dilutions of mycoplasma enrichment broth (modified Hayflick broth, Hi-media Laboratories, Biomed, Mumbai, India) containing 15 % horse serum, DNA extract and fresh yeast.)The serial dilutions were incubated at 37 °C for 48 hours to allow for the enrichment of samples 1,7 .
Selective inhibitors of bacterial growth were added to the mycoplasma broth and agar.Initial samples were processed using ampicillin, methicillin, bacitracin and thallium acetate 1,7 .For improved control of bacterial contamination during enrichment, cefoperazone was added in addition to the above inhibitors throughout the rest of the study.For culture detection, enhanced growth in broth followed by culture on a mycoplasma agar medium 7 .Identification of the colonies was accomplished by an indirect immunoperoxidase test 1 .Recommended antisera were used for each of the Mycoplasma spp. 1,5.
Mycoplasma bovis was isolated from 39 (48.75 %) of the clinical mastitis samples.The percentage of positive samples per farm are given in Table 1.Mycoplasma bovis was also isolated from 48 of the bulk-tank samples.No other Mycoplasma species were isolated from any of the samples.
This investigation was specifically aimed at determining whether Mycoplasma udder infections were of any significance in dairy herds in the Moghan region of Ardabil State.The possible role of other causative organisms was not included in the study.The findings of this study indicate that almost 50.0 % of the clinical mastitis cases were caused by Mycoplasma bovis.
It is likely that, using bulk-tank milk, the true occurrence of Mycoplasma is underestimated because cows that recover from an initial infection may stop shedding Mycoplasma organisms or shed the organisms in such low numbers that they go undetected in these samples.In addition, bulk-tank samples generally represent only healthy, untreated cattle within a herd, since milk from unhealthy or treated cattle is kept separately.Also, to ensure accurate screening of a dairy herd, routine sampling of bulk-tank milk is required 11,12,13 .
There is no laboratory or epidemiological data available for this disease in Iran.Veterinary data in Iran from 2002 ( Iran Veter-inary Organization,Tehran), showed that 96.84 % of dairies tested positive for Mycoplasma when a single bulk-tank milk sample was cultured using standard culturing methods.The species isolated most frequently was M. bovis (92 % of farms) followed by very small but virtually equal numbers of M. californicum, M. alkalescens and M. bovigenitalium.States in the Western region had a greater percentage of farms with positive Mycoplasma cultures than farms in the Midwest, Northeast, and Southeast regions.In addition, large herds (500 animals or more) were more likely to have positive Mycoplasma cultures than medium-sized (100-499) or small herds (<100).All of the 23 States had at least 1 farm with a positive Mycoplasma bulk-tank milk culture.These data showed that Mycoplasma infections can be economically devastating to dairies because of the contagious nature of the organism and its resistance to therapy.Early detection and segregation or culling of Mycoplasma-infected cattle is essential in this area, and also throughout Iran.In this survey we described the need for an epidemiological approach to the investigation of disease problems in Iran.
The most effective method of preventing an outbreak of Mycoplasma mastitis is to screen all introduced cattle by collecting milk samples at freshening (heifers) or prior to mingling purchased cattle with the home herd. 4,6n conjunction with the screening of newly introduced heifers and cows, bulktank milk should be cultured at least once a month.In herds with 500 or more cows, a bulk-tank culture may not be accurate enough to detect a single infected cow.In this case, culturing a sample from a smaller group of cattle (e.g. an individual pen sample) is recommended on a monthly basis. 11,13In herds that are expanding or when routine purchasing of heifers is carried out, screening of bulktank and/or pen samples twice a month should be considered.In addition to the screening of purchased cattle, and bulktank or pen sampling, the implementation of proper milking techniques is critical for minimising the spread of Mycoplasma in a parlour.Proper milking techniques that minimise the spread of Mycoplasma include the use of gloves, minimising the amount of milk on hands and towels while moving from cow to cow, and using a 1 % iodine post-milking teat dip 4,11 .
This investigation confirmed the importance of Mycoplasma bovis as a significant pathogen in dairy herds as reported in other parts of the world.The study also highlighted the importance of conducting surveys in dairy herds so as to determine the prevalence and significance of mycoplasmal infections.