Mycoplasmal mastitis in dairy herds

Vet Clin Food Anim 19 (2003) 199–221

Mycoplasmal mastitis in dairy herds Rube´n N. Gonza´lez, DVM, MPVM, PhD*, David J. Wilson, DVM, MS Quality Milk Production Services, Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, 22 Thornwood Drive, Ithaca, NY 14850-1263, USA

Mycoplasmas are the smallest self-replicating organisms known. They have a worldwide distribution as free-living saprophytes or as parasites of humans, mammals, reptiles, fish, arthropods, and plants [1]. Mycoplasmas lack cell walls, have simple-surface exposed plasma membranes, possess minute genomes, and have limited metabolic capabilities compared with other bacterial groups [2]. The absence of cell walls and cell wall–associated proteins renders mycoplasmas resistant to the action of antibiotics that interact with these proteins [3]. Because of their limited genetic potential, mycoplasmas usually require intimate association with mammalian cell surfaces and an enriched medium containing a peptone, yeast extract, and animal serum for in vitro growth [3]. Despite their genetic simplicity and an image as impotent microorganisms [4], mycoplasmas are considered major animal pathogens in animal production units worldwide [5]. The introduction of molecular techniques to taxonomy, including the comparison of 16S rRNA and other conserved gene sequences, as well as genomic restriction patterns have been effectively used in species and strain identification [2,6]. Mycoplasmal infections follow a chronic course because the organisms usually live in harmony with their host. Mycoplasmas exhibit a rather strict host, tissue, and organ specificity, probably reflecting their nutritionally precise nature and obligate parasitic mode of life [2]. They have been found, however, in hosts and tissues different from their normal habitats [1]. Several species of Mycoplasma have been found as the cofactors or actual cause of diseases of the respiratory and urogenital tracts, joints, and mammary gland of the bovine [7,8].

* Corresponding author. E-mail address: [email protected] (R.N. Gonza´lez). 0749-0720/03/$ - see front matter
2003, Elsevier Science (USA). All rights reserved. PII: S 0 7 4 9 - 0 7 2 0 ( 0 2 ) 0 0 0 7 6 - 2

200

R.N. Gonza´lez, D.J. Wilson / Vet Clin Food Anim 19 (2003) 199–221

Mechanisms of pathogenesis Mycoplasmas can cause chronic diseases in man and animals because of their unique and intriguing ways of interaction with host cells [5]. New techniques that were developed and adapted to mycoplasmas during the last decade have been useful in the study of these organisms at the molecular level [5]. Multiple pathways of interaction with host cells and sophisticated genetic machinery are thought to play essential roles in the pathogenesis of mycoplasmal infections [9,10]. Mycoplasmas have distinct variable surface lipoproteins (Vsps) on the outer face of the plasma membrane that are potent stimulants of the host immune response [11]. They may play a critical role as mediators for adherence to epithelial cells [2,6], as one of the mechanisms involved in mycoplasmal infection, and in maintaining parasitism and evading immune destruction [12]. The Vsps are important factors in the inflammatory process, inducing the secretion of proinflammatory cytokines including tumor necrosis factor a, interleukin 1, and interleukin 6, and are probably also involved in leukocyte recruitment to the infected tissue [2,11]. Initial studies with Mycoplasma bovis determined the existence of 4 classes of Vsps [10,12] in this organism, but later research detected the presence of at least 13 structurally related, yet clearly different, Vsps [4,13]. These Vsps experience highly dynamic and spontaneous changes in size and reversible phase variation in their expression level (ON to OFF expression states) by high-frequency rearrangement of genes [4,13]. It has also been shown that antibody to a specific Vsp (or exposure to macrophages) results in selection of phase-variant mycoplasmas with OFF expression of that Vsp [14]. Furthermore, the finding of a Vsp-unrelated membrane protein also associated with M bovis antigenic variation [15] suggested that M bovis employs two types of specialized membrane proteins for surface diversification [15]. Maintenance of diversity in propagating populations is an important microbial strategy for host adaptation and evasion of immune responses [16]. It is important to realize that the majority of the studies on pathogenesis of mycoplasmas have been undertaken using cell cultures and animal models [11]. A better understanding of the mechanisms of mycoplasmal disease in vivo, including antigenic variation and cell invasion, may lead to a change in the way that mycoplasmal disease is currently thought about [5]. Pathologic changes in the bovine udder induced by intramammary infections (IMI) because of Mycoplasma spp usually decrease milk production. Mycoplasmas are able to survive with little evidence of phagocytosis or inhibition growth in the presence of high concentrations of neutrophils in milk that are produced in response to IMI [17]. Macroscopic and histologic changes observed in slaughtered cows that were infused intramammarily with M bovis differed depending on the duration of infection.

R.N. Gonza´lez, D.J. Wilson / Vet Clin Food Anim 19 (2003) 199–221

201

During the first week of infection, a pronounced degeneration of the alveolar epithelium and a massive exudation of leukocytes including neutrophils, eosinophils, and macrophages [18,19] into the mammary tissue was observed. In chronic stages of the infection, a progressive fibroplasia around milk ducts [19], extensive alveolar atrophy, and interalveolar infiltration of plasma cells, lymphocytes, and histiocytes [18,19] were found in several areas of the affected glands. Researchers in California [20,21] reported similar observations and the formation of multiple abscesses containing viable mycoplasmas. The presence of these abscesses may play a role in the intermittent shedding observed from chronically infected cows [20,108]. M bovis is highly invasive [5]. Multiple clinical manifestations and the isolation of this organism from different body sites in the bovine reflect its ability to disseminate systemically after displacement through mucosal barriers [22]. It has been suggested that M bovis adapts to different host environments and different selective pressures by possibly altering its gene expression, including adhesin genes [5]. It is speculated that specific adhesins could be expressed only when needed, preventing an early host immune response that could block tissue colonization [5]. Mycoplasmas present in the mucous membranes of the respiratory and urogenital tracts of healthy cattle or associated with arthritis in cattle of all ages may possibly serve as a source of mammary infection to them or other cattle. Stresses such as calving, extreme temperature variations, transportation, disease, or external trauma may allow the organisms to enter other body tissues or enter directly into the mammary gland, resulting in clinical mastitis [23,24].

Mycoplasmas and mastitis M bovis is the most important agent of outbreaks of mycoplasmal mastitis in dairy cows. Therefore, most of the observations and research on mycoplasmal mastitis, as well as studies related to pathogenicity, virulence factors, and immune response have dealt with this species. Nevertheless, 11 other Mycoplasma and Acholeplasma species have been also isolated from milk: Mycoplasma alkalescens, Mycoplasma arginini, Mycoplasma bovigenitalium, Mycoplasma bovirhinis, Mycoplasma californicum, Mycoplasma canadense, Mycoplasma dispar, Mycoplasma species group 7, Mycoplasma F-38, Acholeplasma laidlawii, and Acholeplasma axanthum [7,20,25–27]. The disease produced by each Mycoplasma species is similar but may vary in severity [20]. During the last 40 years while performing diagnostic work in the authors’ laboratory, mycoplasmas different than M bovis have been found (although very infrequently) as ethiologic agents of mycoplasmal IMI and mastitis in dairy herds in several northeastern states. This is not the case in California, however, where principally M californicum and M bovigenitalium [28,29] and M canadense have also been consistently isolated [28]. M bovis, regarded as

202

R.N. Gonza´lez, D.J. Wilson / Vet Clin Food Anim 19 (2003) 199–221

the most pathogenic of the mycoplasmas in the bovine [7,8], has been associated with several production diseases in cattle, including abortion, infertility, arthritis, keratoconjunctivitis, mastitis, otitis media, pneumonia, and subcutaneous abscesses [23,28,30–35]. M bovigenitalium was the first species of mycoplasmas recognized as an agent of bovine mastitis [36]. Clinical presentation in herds and cows is similar to M bovis, although it has been reported that affected cows tend to recover over a period of a few weeks or months [28]. Several cows in two western New York dairies with M bovigenitalium IMI, however, carried the infection from the current lactation into the next lactation, at which time they were removed from the herds. Mastitis caused by M canadense has been said to be similar but not usually as severe as that caused by M bovis [28]. Apparently, spontaneous recovery occurs sooner, and spread from infected to uninfected quarters and cows is less than with M bovis [28]. Since its original isolation in 1972, M californicum has become the second most common Mycoplasma isolated from cows with mastitis in California [28]. Clinical mastitis because of M californicum has been reported as usually acute, with affected cows returning to near-normal milk production more quickly than those with M bovis [28]. A severe outbreak of mastitis in dry cows because of M californicum was reported in Ireland [37]. The authors know of only three outbreaks of M californicum diagnosed in New York, the first occurring in 1990 [38]. In the authors’ laboratory, this mycoplasmal species has also been isolated twice from cows in Vermont in 1987 and 1988. M alkalescens was first isolated from cows with mastitis in New Zealand in 1969 [39]. M alkalescens caused 27 mastitis outbreaks in California between 1978 and 1982 [28]. In the authors’ laboratory, a few herds have been diagnosed with this type of mastitis in New York in the last 5 years. The remaining Mycoplasma and Acholeplasma species have been isolated sporadically from cows with mastitis. Independent of the species involved, diseases caused by mycoplasmas are resistant to antimicrobial therapy, although they are susceptible in vitro to several antibiotics [23,40]. Acholeplasma laidlawii, frequently regarded as a nonpathogenic saprophytic contaminant resembling mycoplasmas, may be found in milk, but the source is the environment during wet weather or from the teat skin rather than from true IMI [23,24]. Researchers in Japan isolated only Acholeplasma laidlawii from 100 milk samples obtained from cows with mastitis on several farms in the Kanto area [41]. Based on biologic requirements for growth, these researchers distinguished two forms of Acholeplasma laidlawii (‘‘parasitic’’ and ‘‘saprophytic’’) but concluded that it was necessary to determine the pathogenicity, if any, of the parasitic Acholeplasma laidlawii [41]. During an outbreak of mycoplasmal mastitis in Great Britain, milk samples from 15 of 53 cows with clinical mastitis yielded mixed cultures of M bovigenitalium and Acholeplasma laidlawii [42]. Several studies to determine the pathogenicity of Acholeplasma laidlawii for the mammary gland of the bovine have showed inconsistent results, suggesting the possibility of the existence

R.N. Gonza´lez, D.J. Wilson / Vet Clin Food Anim 19 (2003) 199–221

203

of both pathogenic and nonpathogenic strains [20], a possibility that Japanese researchers [41] have already mentioned. Mycoplasmas are considered contagious mastitis pathogens that can cause clinical, subclinical, and chronic IMI. These infections usually persist through the current lactation and into subsequent lactations. Mycoplasma is most commonly spread from infected cows to uninfected cows at milking by way of milking machines or milkers’ hands. Contaminated intramammary treatments, improper teat sanitation during or at the end of lactation, and airborne transmission in poorly ventilated barns are also means of new herd infections [23,24,31,38,43]. Cattle of all ages and at any stage of lactation, as well as nonlactating cattle are susceptible. Herds with and without mycoplasmal mastitis may contain both young and mature asymptomatic carriers [23,24,28]. The young are exposed to the various mycoplasmas during calving by direct contact with the urogenital tract, from nasal discharges of the dam, and in the milk they receive from shedding animals [23,24]. Mycoplasma may be shed in nasal discharges of calves and in vaginal discharges of heifers at the time of calving [20,44,45]. Therefore, it is extremely important to realize that although a dairy is not currently experiencing mycoplasmal mastitis, the organisms may very well be present within the herd and the possibility of a mastitis outbreak always exists [20,46].

Herd diagnosis of mycoplasmal mastitis The veterinary practitioner should have an open mind and avoid preconceptions when undertaking a mastitis control program in a dairy herd. Regardless of what is described in textbooks and research publications, IMI may have a particular presentation on each farm, independent of the microorganisms that are causing them. The pathogenicity of the strain or strains of Mycoplasma involved, the susceptibility of the affected animals and their management, breakdowns in sanitation and milking equipment maintenance, and treatment procedures for mastitis play key roles for the presentation of mycoplasmal mastitis on a dairy farm. Cows of all ages and at any stage of lactation are susceptible to mycoplasmal mastitis. It has been reported that cows in early lactation seem to suffer more severely because of an increased mammary gland edema [20,23]. In the authors’ experience, a consistent gross pathology has not been seen at the moment of intervention in Mycoplasma-affected herds; this lack of consistent clinical signs has also been reported by other researchers [47]. In lactating cows, the classically seen signs are severe clinical mastitis that resists treatment, more than one mammary quarter affected (sometimes all four), a marked drop in milk production, and abnormal udder secretions that may vary from watery milk with a few clots to a colostrum-like material [23,24]. The affected cow usually lacks systemic signs of disease and

204

R.N. Gonza´lez, D.J. Wilson / Vet Clin Food Anim 19 (2003) 199–221

continues to eat and drink normally. Chronically infected cows may show a tan-colored secretion with sandy or flaky sediments that resembles cooked cereal in a wheylike fluid. Udder secretions may become purulent and last for several weeks [20,23,24,48]. In the authors’ experience, however, the great majority of milk samples positive for Mycoplasma in the laboratory do not show abnormal colors or pus. Furthermore, in several New York dairy herds, the authors isolated M bovis from cows without any clinical signs of disease that were producing between 60 and 90 pounds of milk per day [49]. Cows that continue their lactation produce less milk than expected for the current lactation, usually with normal appearance of milk but with high somatic cell counts. They may shed mycoplasmas intermittently for variable periods [20,23,24,108]. In two New York dairies, the authors followed up cows with chronic M bovis IMI; the animals were sampled once daily for periods of 4 to 6 months [50,108]. In one of the dairies, a cow in its third lactation had periods that ranged from 2 to 24 days without shedding M bovis in the first 120 days of its lactation. A few days later, this cow was sent to slaughter after developing an acute polyarthritis and being unable to stand up. In the remaining dairy, a second-lactation cow had a period of 56 days without shedding M bovis between the fourth and six month of its lactation but was again positive until the end of the lactation, shedding M bovis intermittently [50]. Infected cows may return to their expected milk production in the same lactation, remain infected in the dry period, and increase their milk production in the following lactation while shedding M bovis [51,52]. Some cows may eliminate M bovis mammary gland infections by themselves after the clinical episode, usually during the dry period [50–52]. The variable duration of clinical signs and shedder status contributes to the difficulty in predicting the outcome of infected quarters and the determination of complete bacteriologic recovery. For this reason, cows diagnosed positive for Mycoplasma should probably be considered positive for life, although this may not always be the case [20,24]. The severity and the recovery from the infection may vary within herds and between herds, depending on the species of Mycoplasma and on the relative susceptibility of the cows. Dry cows are equally susceptible to infection by Mycoplasma [20,37] but show little swelling or other signs until calving, when clinical mastitis frequently occurs. Lameness due to arthritis caused by the presence of Mycoplasma in the hocks and fetlocks of mastitic and nonmastitic cows is frequently seen in infected herds [20,23,31,38]. Somatic cell counts of cows are frequently used to monitor and evaluate a herd’s infection status [53]. Cows with over 200,000 cells/mL are considered to have subclinical mastitis and the most likely to be cultured to identify the agents of IMI in a herd [53]; however, cows have been seen that cultured positive for M bovis for almost an entire lactation and whose somatic cell counts ranged between 60,000 cells/mL and 115, 000 cells/mL (R.N. Gonza´lez and D.J. Wilson, unpublished observations). During the first mycoplasmal outbreak studied in California, M bovis was isolated from the

R.N. Gonza´lez, D.J. Wilson / Vet Clin Food Anim 19 (2003) 199–221

205

mammary gland of cows without a history of mastitis [48]. Although mycoplasmal lipoproteins are potent stimulants of the host immune response [11], the aforementioned observations show that mycoplasmal infections may be present in the mammary gland without a strong inflammatory reaction or clinical signs of the disease. There are speculations that some mycoplasmas have developed the capability of downregulating the synthesis of proinflammatory cytokines [11]. Laboratory diagnosis of mycoplasmal mastitis Microbiologic procedures are still the most common in use for routine diagnosis of IMI. They are suitable for the simultaneous detection of several Mycoplasma species that may be present in a sample. Diagnosis of infection at the herd level is usually made by isolation of mycoplasmas from either bulk-tank milk (BTM) or samples from cows with clinical and subclinical IMI [23,24,38,49,54,55]. Culture of BTM samples is a valuable procedure for screening and surveillance on a herd basis [23,24,38,54,55]; however, a negative result does not necessarily indicate the absence of Mycoplasma infections in a herd [50,56]. False-negative diagnoses may occur because dilution could mask several low-level shedders of Mycoplasma [54], the intermittent shedding of Mycoplasma by chronically infected cows [20,50,52,108], or because dairy producers withhold abnormal cows’ milk from the tank [24,38,57]. Across all herd sizes, when herds that were studied had at least one positive cow for Mycoplasma, sensitivity of a single culture of BTM samples to screen for the organisms ranged from 33% [56] to 59% [54,55]. The number of mycoplasmas in BTM (colonly-forming units per milliliter) is not predictive of the percentage of infected cows shedding the organisms [54]. Sometimes, the presence of Mycoplasma and Arcanobacterium pyogenes in BTM may also have a source in blind quarters milked by mistake by untrained milkers [45,56]. The Quality Milk Production Services recommends serial cultures to detect the presence of contagious pathogens in BTM [56]. If, however, at least three BTM samples are collected 3 to 4 days apart, are cultured for Mycoplasma, and are all negative, the probability is 70% that cows contributing milk to the tank are negative for Mycoplasma. Seven consecutive daily cultures are even better to increase the predictive value of negative BTM cultures for Mycoplasma [56]. Large numbers of mycoplasmas are usually present in milk samples from cows with clinical mastitis. A low level of excretion of mycoplasmas in the milk from latent infections or carrier cattle may impair the diagnosis of IMI [20,23,24,56]. Enhanced growth in broth incubated aerobically followed by culture on an agar medium for mycoplasmas can be used [20,49,58]. Thurmond and co-workers [58] found that the combined use of direct inoculation and pre-enrichment yielded 70% more isolates of Mycoplasma from bovine milk than direct inoculation on Hayflick medium plates alone. At the authors’ laboratory, the examination of 4116 milk samples by both

206

R.N. Gonza´lez, D.J. Wilson / Vet Clin Food Anim 19 (2003) 199–221

direct inoculation of milk on Hayflick agar plates and pre-enrichment in Hayflick broth produced only a 6% increase [49]. Therefore, pre-enrichment might not increase the isolation of Mycoplasma significantly but would increase the cost of diagnosis [20,49]. Pre-enrichment should not be used for BTM because its use may result in the isolation of more colonies of Acholeplasma laidlawii. Mycoplasmas are fragile and susceptible to drying and changes in the pH of milk [40,59,60]. Milk samples for culture of Mycoplasma must be obtained aseptically to avoid contamination, should be kept cool during transit or storage before culture, and be plated promptly to maximize isolation. Bacterial or other microorganism overgrowth is a serious problem that may affect Mycoplasma survival on samples. Even if mycoplasmas are still viable when the sample is inoculated onto the agar plate, detection of colonies can become impossible because contaminants grow more quickly than mycoplasmas, despite the presence of inhibitors (usually penicillin and thallium acetate) in the medium. When delay of more than 48 hours is anticipated, milk samples should be frozen (preferably at ÿ30C or below to assure viability) or stored in liquid nitrogen [60]. Most species of Mycoplasma that cause IMI in cows can be isolated on petri plates containing modified Hayflick medium, of which there are several variations [61]. Plates should be kept in plastic bags to prevent desiccation and stored at 4C until use. The basic medium and supplements can be purchased commercially [60]. The water to prepare the medium should be of excellent quality. Each new batch of prepared medium should be tested with field strains of all species of Mycoplasma to be cultured to ensure that the medium sustains growth [62] and does not contain any substances inhibitory to the mycoplasmas. It has been seen very frequently in the authors’ laboratory and reported elsewhere [62] that reference Mycoplasma strains (American Type Culture Collection [ATCC]) grow readily on media that will not provide for isolation of all mycoplasmas from milk samples. Procedures for culturing milk samples have been described elsewhere [60]. The plates should be incubated inverted at 35C to 37C in a moist 10% carbon dioxide incubator. A candle jar with a moist sponge may be used instead of a carbon dioxide incubator; however, some toxic products may be generated and Mycoplasma growth may be inhibited [63]. Agar plates are examined at 24 to 48 hours of incubation to detect early growth or contamination as soon as possible. Daily examination of plates under a stereomicroscope or the low power of an ordinary light microscope for the next 2 or 3 days is necessary to differentiate growth of mycoplasmas from slow-growing contaminants. The use of a stereomicroscope provides excellent resolution for observing Mycoplasma colonies on the agar surface through the bottom of the plate without opening the plate. This method of observation is much faster than the use of an ordinary microscope and avoids contamination of the agar surface with bacteria and other organisms during the repeated observations

R.N. Gonza´lez, D.J. Wilson / Vet Clin Food Anim 19 (2003) 199–221

207

necessary for the detection of growth [62]. Incubation should proceed 7 to 10 days before plates are considered negative for Mycoplasma [8,20,49]. Direct examination of stained smears is of little value because mycoplasmas do not stain with Gram stain and stain weakly with Giemsa stain. Therefore, identification of Mycoplasma relies on the demonstration of typical colonies. Mycoplasma colonies have a dense, central core that grows down into the medium. A lighter peripheral zone of surface growth surrounds this area and gives Mycoplasma colonies a ‘‘fried-egg’’ appearance on solid medium. Smaller colonies may have a smooth or coarse appearance. Confluent growth, where colonies cannot be distinguished except at the edge, usually appears in samples from cows with acute mastitis. Lack of cell wall is the reason for the fried-egg colonial morphology that is characteristic of mycoplasmal growth on solid media [3]. Bacteria that have temporarily failed to form cell walls (L-forms) can also produce colonies that also have a fried-egg appearance. b-Lactam antibiotics used in dairy cows for the treatment of mastitis could induce the transformation of mastitis pathogens such as Streptococcus agalactiae, Staphylococcus aureus, and Pseudomonas aeruginosa to the L-form status [64–66]. Differentiation between mycoplasmal and bacterial L-form colonies may be necessary and can be performed as described previously [60]. Identification of Mycoplasma species Species identification of bovine isolates is accomplished by serologic methods. A pure Mycoplasma culture is needed before any procedure can be performed. Immunofluorescence procedures are the most commonly used methods for species identification. Colonies appearing on the plates can be identified as a Mycoplasma species by staining directly on agar with homologous fluorescein-conjugated antibody [67]. Glass-slide impression of Mycoplasma colonies developing on plates can react with polyclonal antisera specific for Mycoplasma species that cause bovine mastitis. This process is followed by addition of fluorescein isothiocyanate conjugated (FITC)labeled protein G and examination for specific fluorescence on colonies by microscopy. An indirect immunoperoxidase test performed on Mycoplasma colonies [68] was modified to speed up and facilitate the identification process by including the use of blank disks (similar to those used for antibiotic susceptibility tests) [69]. The test has given excellent results, is easy to perform, and does not require fluorescence microscopy [69]. Both immunofluorescence and immunoperoxidase are species-specific tests. An immunobinding test for use directly on suspect milk or culture for detection and/or identification has also been shown to recognize 5 · 103 or more organisms per milliliter [70]. Species-specific DNA probes have been developed for the identification of some Mycoplasma species [5]. Most mycoplasmas isolated from BTM and cow milk samples are pathogenic but some may be Acholeplasma laidlawii. Therefore, speciation

208

R.N. Gonza´lez, D.J. Wilson / Vet Clin Food Anim 19 (2003) 199–221

of Mycoplasma-like colonies is recommended, especially when there are not signs of mycoplasmal mastitis in cows in the herd. Digitonin inhibition of sterol metabolism by mycoplasmas can also be used to differentiate isolates of Mycoplasma and Acholeplasma laidlawii from milk [71]. Among several sophisticated tests, the polymerase chain reaction has emerged as the procedure for routine laboratory diagnosis in the near future. It can provide high sensitivity, specificity, and speed (24 hours or less) for laboratory diagnosis; however, several practical problems including interference in the normal course of polymerase chain reaction by protein and other milk components must be solved before full-scale adoption of this diagnostic procedure [72]. A nested polymerase chain reaction assay used for testing of preservativetreated milk has been shown to be as sensitive as traditional culture of fresh or frozen milk for detection of M bovis. Viability of M bovis in preservativetreated milk, however, was found to decline steadily compared with milk samples without preservative [73]. In some situations, DNA fingerprinting of M bovis strains has found use for epidemiologic tracing or strain differentiation [31].

Epidemiology Most transfer of mycoplasmal infection within herds occurs at milking by means of fomites such as milking machines, teat cups, and milkers’ hands [20,23,24,28]. Many new herd infections occur from the introduction of replacements with infected udders [23,24,38]. Treatment of mastitis provides a good opportunity for spread from cow to cow, and even from herd to herd when rigid sanitary precautions are not followed [23,24]. An outbreak of mycoplasmal mastitis, however, may occur in previously clean herds without introduction of animals or history of previous intramammary treatment [23,24,38]. Mycoplasmas may disseminate from several body sites to the mammary gland by hematogenous or other route [20,28]. Hematogenous spread of M bovis was demonstrated when the organism was recovered from viable fetuses and calves of cows with mastitis [74,109]. Intramammary infection also followed 10 days after an intradermal injection of viable M bovis as a test for hypersensitivity [75]. Later, researchers isolated mycoplasmas from the blood of calves within a week after intratracheal inoculation [76]. When an udder infection is established, rapid spread within a herd can occur by more routine methods for spreading mastitis. This is easy to understand because large numbers of mycoplasmas may be shed in the milk before the onset of clinical mastitis, and delay in diagnosis is common [20]. The prepuce and distal urethra of apparently normal bulls were found colonized by various mycoplasmas and ureaplasma [30,43,77]. This could result in infected semen and may be a way of dissemination of these

R.N. Gonza´lez, D.J. Wilson / Vet Clin Food Anim 19 (2003) 199–221

209

organisms. Use of infected semen was shown to lower conception rates, increase services per conception, and prolong calving interval [78]. In England, M bovis was implicated as the primary etiologic agent in a severe outbreak of calf pneumonia [79]. Later in California, calves and young stock from six herds were observed for a period of 5 consecutive months, with a final sample being taken after 8 months [44]. Three herds had known current or recent mycoplasmal mastitis, and young calves were fed M bovis–infected milk. The three control herds had no known history of mycoplasmal mastitis, confirmed by extensive culturing. Calves in these herds were also fed waste milk. The mean prevalence of M bovis–in the nares of calves fed M bovis–infected milk was 34% but was only 6% in the control herds without mycoplasmal mastitis. Nasal colonization of M bovis persisted at elevated levels in animals fed M bovis–infected milk until approximately 1 year of age. Then, the nasal prevalence was comparable to calves in the control herds. Researchers suggested that heavy nasal colonization with M bovis could possibly induce pneumonia in association with other organisms and stress factors [44]. Moreover, these results demonstrate the presence of M bovis in dairy herds without M bovis mastitis [20]. Lateral transmission of respiratory infection between calves occurs, especially in poorly ventilated barns [31,38,80]. It may be airborne [31,43] and persist until the first calving [74]. On several New York dairy farms where calves, heifers, and cows shared the same barn, clinical mastitis in lactating cows was associated with exposure to calves, heifers, and cows with signs of respiratory disease [31]. Lung fluids collected during the necropsy of three calves from two farms were cultured, and M bovis was isolated in each case. On other farms, cases of clinical mastitis started within a month after calves were diagnosed as having pneumonia and arthritis were definitively diagnosed as mycoplasmal in origin [31,38,81]. Therefore, calves and young cattle also play an important role in the spread of M bovis because they can be the origin of the infection chain in a herd [82]. In California, the risk of large herds (>350 cows) having a Mycoplasmapositive bulk-tank sample was found to be 15 times greater than for small herds (<350 cows) [57]. The reason for this was thought to be a combination of several poorly understood management factors commonly found in larger California herds [57]. In contrast, size was not a risk factor in affected New York herds having 30 to 400 milking cows [38]. Data from an Ohio study suggested that the risk of Mycoplasma infection was also present in small herds [83]. In New York, the highest frequency of clinical mastitis due to mycoplasmas was found to occur during the winter, starting late in the fall, peaking in January, and decreasing by midspring [38]. A similar seasonal variation of Mycoplasma mastitis was also observed in California and was attributed to improper ventilation in barns [20]. Quality Milk Production Services investigated 140 herds with Mycoplasma mastitis problems between January 1989 and December 1995. In

210

R.N. Gonza´lez, D.J. Wilson / Vet Clin Food Anim 19 (2003) 199–221

almost all the herds, mycoplasmas were introduced when replacements (virgin heifers, pregnant heifers, or cows) were purchased and commingled with the existing herd without quarantine and bacteriologic testing [81]. In the states of New York and Pennsylvania, purchased heifers were the origin of severe mycoplasmal mastitis in previously Mycoplasma-free herds, the heifers showing clinical mastitis immediately after calving [31,38,50]. At least two outbreaks of M bovis mastitis involved cows that apparently were exposed to the organism at livestock shows. In some areas in New York where mycoplasmal mastitis epidemics occurred between 1976 and 1980, it was speculated that the disease could have been transmitted between adjacent farms by people who were in contact with infected milk, such as milkers, herd owners, milk-plant truck drivers, and veterinarians [38]. Contaminated equipment, treatment devices, clothing, sampling meters, or any type of improperly cleaned material could have served as a fomite vehicle [23]. Another aspect involved in the epidemiology of mycoplasmal mastitis that has not been adequately studied is the survival of mycoplasmas in different environments and different dairy herd management systems. It is known that exposure of the mycoplasmal membrane to the environment makes mycoplasmas sensitive to drying, osmotic changes, and the accumulation of harmful metabolites [59]. Conversely, the lack of a rigid cell wall makes mycoplasmas less sensitive than true bacteria to the effects of freezing and thawing [59]. Early studies in Italy evaluated the persistence of M bovis on several sterile and nonsterile materials contaminated with milk obtained from naturally infected cows and experimentally infected goats, respectively [84,85]. Materials used in the studies included milk dried on glass, stainless steel, wood, natural rubber, corn straw (corn plant leaves that remain on the ground when corn is harvested), cotton stuff (material that was available in some regions of Italy after the industrial processing of cotton), synthetic sponge, manure, and drinking water. At 23C to 28C, M bovis survived in manure for 236 days in the dark and 145 days in the light. At 37C, M bovis survived for 108 days. In drinking water, M bovis persisted for 23 days at 23C to 28C in the dark and only a day in the light, whereas survival in water at 37C was less than a day [85]. The survival of M bovis in water implies that possibly it can be transported from places such as the milking parlor to other locations on the farm [47]. For the remaining materials, the maximum range of persistence was from less than a day (wood) to 9 days (cotton stuff) in the dark at 23C to 28C. With the exception of sterilized manure and cotton stuff where M bovis survived for 37 days and 18 days, respectively, at 23C to 28C in the dark, there were little differences in the survival periods for the other sterile materials compared with the nonsterile materials [84]. In Germany, using sterilized materials contaminated with M bovis, researchers demonstrated that at 4C, the organism survived 57 days on sponges, 54 days in milk, 20 days on straw, and 17 days on wood and in water. At 20C and 37C, the survival

R.N. Gonza´lez, D.J. Wilson / Vet Clin Food Anim 19 (2003) 199–221

211

period for M bovis on those materials was 1 to 2 weeks and 1 week, respectively [82,86]. In more recent studies in Japan [87], researchers inoculated several strains of Mycoplasma into liquid media supplemented with or without horse serum and yeast extract. They measured mycoplasmal persistence post inoculation at 4C, 30C, 37C, and room temperature (2C–33C during May– December). For M bovis, M arginini, Acholeplasma laidlawii, and Acholeplasma axanthum, the survival period ranged between 59 and 185 days and it was not significantly influenced by temperature. Medium components and temperature influenced the survival periods for M bovigenitalium (7–86 days) and M bovirhinis (7–36 days). In another experiment, strains of M bovigenitalium, M bovis, M bovirhinis, M arginini, and Acholeplasma laidlawii persisted on dry paper disks for at most 28, 126, 154, 56, and over 168 days at 4C, respectively. At 30C, strains of M bovis, M bovirhinis, M arginini, and Acholeplasma laidlawii survived for 28, 84, 56, and over 168 days, respectively. In an outdoor environment (April–December, 0–36C), strains of M bovirhinis and Acholeplasma laidlawii survived for at most 28 and 14 days, respectively. Survival of mycoplasmas was also influenced by the pH of the suspension used prior to application to the disks, with mycoplasmal strains persisting longer at pH 7.5 than at pH 6.5 [87]. In Florida, M bovis has been isolated from dirt in lots with recently calved cows, as well as from cooling ponds [88,89]. In a herd with a history of many outbreaks of mycoplasmal mastitis, M bovis has been regularly isolated from its cooling pond for several years [89]. In the year 2000, mycoplasmal mastitis outbreaks were attributed to a high concentration of mycoplasmas in the cooling ponds due to several factors including lack of rain, evaporation of water, and a failure of dairy producers to add fresh water to the ponds as recommended [89]. Mycoplasmas can survive for variable periods on teat skin [90] and in a variety of environmental conditions as the aforementioned studies suggest. The practice of amputating the teats of unproductive mammary quarters and the subsequent drainage of material containing live mycoplasmas can contribute to the spread of mycoplasmas in the farm environment. Bedding material, the area under the shades in dry lots, and cooling ponds can be the source of new mycoplasmal infections, providing that conditions of adequate temperature and moisture and avoidance of sunlight are provided. More precise information regarding the biology of mycoplasmas in dairy farm environments could therefore help to understand its importance in the epidemiology, prevention, and control of this type of mastitis and other related pathologies. Prevention and control There is no treatment for mycoplasmal mastitis. Control of the disease when it is present in a herd relies on identification of infected cows by

212

R.N. Gonza´lez, D.J. Wilson / Vet Clin Food Anim 19 (2003) 199–221

culture of composite or quarter milk samples from all milking and dry cows in the herd [23,24]. All cases of clinical mastitis should also be cultured, as well as all animals at calving, including heifers. If they are kept in the herd, Mycoplasma-infected cows must be segregated and milked last or with a separate milking unit from those used on uninfected cows to minimize the risk of infection for other cows [24,52]. Spontaneous and complete recovery from M bovis mastitis has been reported [50–52]. Cows infected with other mycoplasmas may recover and stop shedding during the same lactation [23,24,28]. Slaughter of all infected cows is indicated when a few animals in the herd are infected. The exact mode of handling will vary from dairy to dairy based on the owner’s attitude, facilities, number of infected animals, the level of milk production and reproductive status of carrier animals, and the availability of replacements [23,24]. The use of rubber or plastic gloves and the disinfection of gloved hands between cows is advised when milking or treating cows in a herd infected with mycoplasmas [20,24,28,91]. Mycoplasmas have been isolated from bulk antibiotic treatment bottles [89]. Therefore, single-treatment devices are recommended if treatment for any type of mastitis is necessary. In large herds, culture of partial BTM samples collected after milking each production group may be used as a method to locate groups in which cows infected with mycoplasmas exist [46]. Switching the milking order of production groups at different milkings and days or using more than one bulk tank would speed up the sampling procedure. String sampling [45] or milk-line sampling [92]—the collection of milk samples from individual milk production groups by using appropriate devices installed in the milking system—is a very useful and inexpensive procedure to sample groups in this type of herd [92]. The procedure has greater sensitivity to monitor herds for contagious mastitis pathogens than bulk-tank sampling [45]. Milk residues from one string left in the pipeline, however, can act as a confounding factor for culture results when several groups are milked consecutively [45]. Recently, a study was undertaken in 21 herds to compare, among other parameters, bacteriologic culture results between milk-line and bulk-tank samples for milk collected from the same group of cows at the same milking [92]. Culture from 42 paired samples obtained with a sampling device (QMI Safe Septum Sani-Elbow, Quality Management, Oakdale, MN) installed in the milk line showed an agreement (herd culture positive or negative) for contagious Streptococcus agalactiae and Staphylococcus aureus of 100% and 75%, respectively [92]. Mycoplasmas were not included in this study. If a production group tests positive for Mycoplasma using partial BTM or string samples, then individual composite milk samples can be used to identify the infected cows in that group [46]. Cows should not be moved between production groups until laboratory results are provided and cows positive for Mycoplasma identified. Weekly monitoring of BTM to detect the presence of mycoplasmas should be encouraged in order to monitor the success of control procedures

R.N. Gonza´lez, D.J. Wilson / Vet Clin Food Anim 19 (2003) 199–221

213

following mycoplasmal mastitis outbreak. This monitoring should continue until pregnant heifers and all cows that were dry during the mycoplasmal mastitis outbreak have calved. Faulty milking machines and management practices influence the risk of mycoplasmal mastitis, the rapidity of spread in the herd, and the effectiveness of control procedures [20]. Proper milking procedures and sanitation including the use of premilking and postmilking teat dipping are of first importance. Only 70 colony-forming units of M bovis passing trough the teat canal into the mammary gland are needed to start an infection [17,75]; however, in large dairy herds, the authors have frequently observed that proper udder preparation is neglected in favor of moving cows rapidly through the milking parlor. Moreover, milkers trained to emphasize speed of milking do not usually practice thorough teat dipping. At Quality Milk Production Services, the authors favor the use of 1% iodine products to reduce the numbers of Mycoplasma on teat skin during mastitis outbreaks in herds. Five teat-dip classes were recently tested against challenge exposure to M bovis, M bovigenitalium, and M californicum using a modified excised-teat model [93]. Teats were collected from slaughtered dairy cows that were selected for the study because they did not have rough skin, chaps, or abrasions. All teat-dip formulations were evaluated as previously described [94] and showed to be efficacious against the three Mycoplasma species, achieving mycoplasmal logarithmic reductions greater than 4 on teat skin. The teat dips performed best against M bovigenitalium, with a mean logarithmic reaction of 6.29 [93]. Furthermore, the germicides accomplished complete killing of M bovis (0.5% iodine), M californicum (0.5% chlorhexidine gluconate), and M bovigenitalium (0.5% chlorhexidine gluconate and 0.64% sodium chlorite, 3% mandelic acid) [93]. The efficacy of teat-dip products against M bovis and possibly other mycoplasmas is reduced by the presence of biologic and organic materials [23,95]. Teat dipping should also be used before and after intramammary treatment of nonlactating or lactating cows for organisms different than Mycoplasma. Alcohol disinfection of teat ends just before intramammary treatment infusion is essential also. The use of backflushing for disinfection of milking units between cows has been stated to be important in control of mycoplasmal mastitis [23,57,96]. The installation of this system, however, is usually expensive and, in the authors’ experience, the system has minimal effect in reducing infections [50,52]. Alternative methods for disinfection of teat cups are spray washing or rinsing in buckets with sanitizer after milking each cow [20, 47,97,98] and cluster dunking [45]. The cluster dunking procedure includes backflushing the milking units with hose water until water runs clear and dunking (without air trapping) the milking units in a bucket with a germicidal solution [45]. Milking units are sanitized before milking hospital and recently calved cows and after milking known mastitic cows [45]. Although the aforementioned methods have been continuously recommended for

214

R.N. Gonza´lez, D.J. Wilson / Vet Clin Food Anim 19 (2003) 199–221

more than 30 years, the authors have not found measured evidence that those procedures—spraying or rinsing of clusters or cluster dunking—have an impact in reducing the number of mycoplasmas on teat-cup liners and new IMI rates. Great care should be used when purchasing cows and heifers. Milk from all replacements should be cultured for Mycoplasma as well as for Streptococcus agalactiae and Staphylococcus aureus before allowing replacements to commingle with the herd [24,49,56,81]. When herds are purchased, it is a good policy to culture all suspected mastitic cows and the bulk tank, but always keep in mind the low sensitivity of a single milk culture, as previously mentioned [56]. If animals are not cultured before or at the time of purchase, as in the case of dry cows, heifers, and calves, then they should be cultured as they calve to safeguard against mixing cows with contagious mastitis [50,56,99,100]. The practice of feeding mastitic waste milk to calves instead of commercial milk replacers increases the risk of spreading infection with several mastitis agents, principally contagious Mycoplasma and Streptococcus agalactiae, to calves [20,35]. Outbreaks of respiratory disease, polyarthritis, and ear infections have been described in calves fed discard milk from cows with mycoplasmal IMI [20,24,35,44,89,101]. In Florida, approximately 2 weeks after pasteurization failure occurred, dramatic increases in pneumonia and ear infection due to M bovis were observed in calves, most of them between 14 and 28 days of age [89]. Of the 50 calves that were observed from birth through 8 weeks old, only one new clinical case of respiratory disease was seen later than 35 days of age [89]. Although it has been recommended to avoid feeding milk infected with mycoplasmas to calves [35,45], pasteurization of mastitic waste milk decreases the risk of infection [89,101]. The pasteurization process, however, must be carefully monitored for quality assurance, and a regular schedule to sample and culture pasteurized milk for bacterial species content should be established [89,102]. As pasteurization failures occur, producers may decide to culture every batch of pasteurized milk for mycoplasmas [89]. Pasteurizers must be installed, maintained, and used consistently following manufacturers’ recommendations. If the holding tanks and the pasteurizer in continuous flow units are not properly maintained and cleaned, high bacteria regrowth rates can occur during the cool-down period [102]. When simpler batch pasteurizing units are used and waste milk is processed only once a day, strict sanitization procedures and immediate cooling and refrigeration of pasteurized milk is needed to avoid high bacteria regrowth rates [102]. For milk infected with mycoplasmas, heat treatment that results in the destruction of M canadense, which appears to be more heat resistant, should be used for the pasteurization of waste milk infected with mycoplasmas [101]. Researchers observed that M canadense remained viable for up to 3 minutes of exposure at 70C [101]. Unpublished observations at the University of California at Davis suggested that an exposure to 72.5C for at

R.N. Gonza´lez, D.J. Wilson / Vet Clin Food Anim 19 (2003) 199–221

215

least 5 minutes was necessary to kill M canadense in milk (Jon D. Dellinger, Davis, CA, personal communication to Rube´n N. Gonza´lez, 1987). Florida researchers recommend 74C to 76C for 15 to 20 seconds for flash pasteurization (high temperature, short duration) and 65.5C for 30 minutes for batch pasteurization (low temperature, long duration) of waste milk [89]. In summary, all actions to control and prevent mycoplasmal mastitis on dairy farms should be based on the understanding of the highly contagious nature, slow recovery rates, and the ineffectiveness of treatment of mycoplasmal infection [24]. Immunization Mastitis is the cow’s immune reaction to the invasion of the mammary gland by microorganisms such as mycoplasmas. Despite the fact that cows do not produce an anamnestic immune response following mastitis infection [103], the lack of an effective antimicrobial therapy and the serious economic threat that mycoplasmal mastitis can be for a dairy herd has made bacterins an attractive option for the control of mycoplasmal mastitis. Unfortunately, bacterins against mycoplasmal disease in animals have almost universally failed to prevent colonization, eliminate the organism, and prevent either disease outbreaks or the spread of the disease [5]. In the case of M bovis, results of experimental parenteral and intrammamary immunization with live and killed organisms did not indicate that the bovine udder became protected against infection, that clinical signs of the infection were reduced, or that existing infections were eliminated [91,104,105]. A M bovis bacterin absorbed in a propietary adjuvant is currently available in the United States (Biomune Co., Lenexa, KS). The patentpending product consists of multiple strains of M bovis and is recommended for first-calf heifers or multiparous cows to aid in the prevention of mycoplasmal mastitis. The product is labeled for three doses administered subcutaneously in the neck at 2-week to 4-week intervals prior to calving, with the third dose administered 2 to 3 weeks prior to calving. The manufacturer indicates that a booster dose may be administered whenever field conditions warrant, and that animals should not be vaccinated within 21 days of slaughter. The efficacy of this bacterin has received mixed anecdotal reviews electronically by veterinarian members of the American Association of Bovine Practitioners. The authors are not aware of any peerreviewed clinical trial undertaken to determine the efficacy of Biomune’s bacterin. The authors also know of the use of empirically developed autogenous M bovis bacterins to prevent this type of mastitis in the United States; however, none has proven efficacy for prevention, decreasing the incidence, or ameliorating the clinical signs of mycoplasmal bovine mastitis. The capability for rapid diversification of the cell surface antigens, as has been shown for M bovis [102], allow mycoplamas to successfully persist within their host environment and to evade the host immune system for long

216

R.N. Gonza´lez, D.J. Wilson / Vet Clin Food Anim 19 (2003) 199–221

periods [4,13]. It has already been mentioned that at least two different antigenic systems that are strikingly variable have been identified in M bovis. Furthermore, a study of 250 field isolates of M bovis, each representing a distinct herd from five European countries, revealed that Vsp-related DNA sequences occurred in all the isolates examined and that the number of Vsp genes varied among isolates [1]. Researchers also noticed that variations in expression occurred not only from one strain to another but also within the same lineage of clones from a single cell [106]. The lack of a true understanding of mycoplasmal pathogenesis and the cow immune response has hindered the development of an effective bacterin against mastitis and other diseases caused by Mycoplasma. The Vsp antigens of M bovis, however, have shown to be highly immunogenic [107] and may have immunoprophylatic value, despite their variation [106]. Meanwhile, it is critical that veterinary practitioners and dairy producers understand that traditional prevention and control programs, as described previously, are preferable to the use of bacterins developed without extensive knowledge of mycoplasmal pathogenic factors. Summary Mycoplasmal bovine mastitis is potentially a highly contagious disease that can cause severe economic problems in affected herds. The purchase of replacement heifers and cows are frequently the origin of mycoplasmal mastitis outbreaks in previously Mycoplasma-free herds. Purchased cows and heifers should be quarantined and tested for mycoplasmal mastitis before admission to the regular herd. Detection of Mycoplasma-infected cows by culture of milk is straightforward, although there are problems of sensitivity for its detection in milk samples that are inherent to the nature of the disease and laboratory procedures. After detection of infected cows, the best way to protect the herd is to culture all cows in the herd, cows with clinical mastitis, and all heifers and cows after calving and before entering the milking herd. Control of mycoplasmal mastitis requires test and culling from the herd of Mycoplasma-positive cows if possible. When a large number of cows are infected, strict segregation with adequate management is an option; however, animals in this group should never re-enter the Mycoplama-free herd. The functioning of the milking equipment and milking procedures should be evaluated carefully and any flaws corrected. There is no treatment for mycoplasmal mastitis, and vaccination has not proven to be efficacious to prevent, decrease the incidence, or ameliorate the clinical signs of mycoplasmal mastitis. Waste milk should not be fed to calves without pasteurization. M bovis may cause several other pathologies in animals of different ages on a farm, including pneumonia, arthritis, and ear infections. The survival of mycoplasmas in different farm microenvironments needs to be further investigated for its impact on the epidemiology of the disease.

R.N. Gonza´lez, D.J. Wilson / Vet Clin Food Anim 19 (2003) 199–221

217

References [1] Razin S. Mycoplasma taxonomy and ecology. In: Maniloff J, McElhaney RN, Finch LR, et al, editors. Mycoplasmas: molecular biology and pathogenesis. Washington, DC: American Society for Microbiology; 1992. p. 3–22. [2] Razin S, Yogev D, Naot Y. Molecular biology and pathogenicity of mycoplasmas. Microbiol Mol Biol Rev 1998;62:1094–156. [3] Rosenbusch RF. Biology and taxonomy of the Mycoplasmas. In: Whitford HW, Rosenbusch RF, Lauerman LH, editors. Mycoplasmosis in animals: laboratory diagnosis. Ames (IA): Iowa State University Press; 1994. p. 3–11. [4] Lysnyansky I, Sachse K, Rosenbusch R, Levisohn S, Yogev D. The vsp locus of Mycoplasma bovis: gene organization and structural features. J Bacteriol 1999;181:5734–41. [5] Minion FC. Molecular pathogenesis of mycoplasma animal respiratory pathogens. Front Biosci 2002;7:d1410–1422. [6] Razin S. Molecular biology and genetics of mycoplasmas (Mollicutes). Microbiol Rev 1985;49:419–55. [7] Gourlay RN, Howard CJ. Human and animal mycoplasmas. In: Tully JG, Whitcombs RF, editors. The mycoplasmas, vol 2. New York: Academic Press; 1979. p. 49–102. [8] Gourlay RN, Howard CJ. Recovery and identification of bovine mycoplasmas. In: Tully JG, Razin S, editors. Methods in mycoplasmology, vol. 2 New York: Academic Press; 1983. p. 81–9. [9] Citti C, Rosengarten E. Mycoplasma genetic variation and its implication for pathogenesis. Wien Klin Wochenschr 1997;109:562–8. [10] Rosengarten R, Behrens A, Stetefeld A, et al. Antigen heterogeneity among isolates of Mycoplasma bovis is generated by high frequency variation of diverse membrane surface proteins. Infect Immun 1994;62:5066–74. [11] Chambaud I, Wro´blewski H, Blanchard A. Interactions between mycoplasma lipoproteins and the host immune response. Trends Microbiol 1999;7:493–9. [12] Behrens A, Heller M, Kirchhoff H, et al. A family of phase- and size-variant membrane surface lipoprotein antigens (Vsps) of Mycoplasma bovis. Infect Immun 1994;62:5075–84. [13] Lysnyansky I, Ron Y, Sachse K, Yogev D. Intrachromosomal recombination within the vsp of Mycoplasma bovis generates a chimeric variable surface lipoprotein antigen. Infect Immun 2001;69:3703–12. [14] Rosenbusch RF, Peterson CA. Use of diagnostic accessions to evaluate the usefulness of variable surface proteins of Mycoplasma bovis as diagnostic test antigens. Abstract book of the 14th International Congress of the International Organization for Mycoplasmology, Vienna, Austria. Columbia (MO): International Organization for Mycoplasmology; 2002. p. 88. [15] Behrens A, Poumarat F, Le Grand D, et al. A newly identified immunodominant membrane protein (pMB67) involved in Mycoplasma bovis surface antigenic variation. Microbiology 1996;142(Pt 9):2463–70. [16] Nussbaum S, Lysnyansky I, Sachse K, Levisohn S, Yogev D. Extended repertoire of genes encoding variable lipoproteins in Mycoplasma bovis strains. Infect Immun 2002;70:2220–5. [17] Bennett RH, Jasper DE. Bovine mycoplasma mastitis from intramammary inoculation of small numbers of Mycoplasma bovis. I. Microbiology and pathology. Vet Microbiol 1978; 2:341–55. [18] Seffner W, Pfutzner H. Mycoplasma mastitis of cattle. 8. Pathological anatomy and histology of experimental Mycoplasma bovis mastitis. Arch Exp Veterinarmed 1980;34:817–26. [19] Van der Molen EJ, Grootenhuis G. An investigation of the pathology of mycoplasma mastitis in the cow. Tijdschr Diergeneeskd 1979;104(Suppl):126–33. [20] Jasper DE. Bovine mycoplasmal mastitis. In: Cornelius CE, Simpson BF, editors. Advances in veterinary sciences and comparative medicine. New York: Academic Press; 1981. p. 121–59.

218

R.N. Gonza´lez, D.J. Wilson / Vet Clin Food Anim 19 (2003) 199–221

[21] Jasper DE, Boothby JT, Thomas CB. Pathogenesis of bovine mycoplasma mastitis. Isr J Med Sci 1987;23:625–7. [22] Pfu¨tzner H, Sachse K. Mycoplasma bovis as an agent of mastitis, pneumonia, arthritis and genital disorders in cattle. Rev Sci Technol 1996;15:1477–94. [23] Bushnell RB. Mycoplasma mastitis. Vet Clin N Am 1984;6:301–12. [24] Jasper DE. Bovine mycoplasmal mastitis. J Am Vet Med Assoc 1979;175:1072–4. [25] Alexander PG, Slee KJ, McOrist S, et al. Mastitis in cows and polyarthritis and pneumonia in calves caused by Mycoplasma species bovine group 7. Aust Vet J 1985;62:135–6. [26] Hale HH, Helmboldt CF, Plastridge WN, et al. Bovine mastitis caused by a Mycoplasma species. Cornell Vet 1962;52:582–91. [27] Kumar A, Garg DN. Isolation of mycoplasma F-38 from the milk of mastitic cows. Vet Rec 1991;128:429. [28] Jasper DE. The role of Mycoplasma in bovine mastitis. J Am Vet Med Assoc 1982;181: 158–62. [29] Kirk JH, Glenn K, Ruiz L, et al. Epidemiologic analysis of Mycoplasma spp isolated from bulk-tank milk samples obtained from dairy herds that were members of a milk cooperative. J Am Vet Med Assoc 1997;211:1036–8. [30] Fish N, Rosendal S, Miller RB. The distribution of mycoplasmas in the genital tract of normal artificial insemination bulls. Can Vet J 1985;26:13–5. [31] Gonza´lez, RN, Jayarao, BM, Oliver, SP, et al. Pneumonia, arthritis and mastitis in dairy cows due to Mycoplasma bovis. Proceedings of the 32nd Annual Meeting of the National Mastitis Council. Kansas City, MO. Arlington (VA): National Mastitis Council; 1993. p. 178–86. [32] Kinde H, Daft BM, Walker RL, et al. Mycoplasma bovis associated with decubital abscesses in Holstein calves. J Vet Diagn Invest 1993;5:194–7. [33] O’Berry BA, Bryner JH, Frank AH. Isolation of mycoplasma from an aborted bovine fetus and vaginal mucus. Am J Vet Res 1966;27:677–80. [34] Rosendal S, Martin SW. The association between serological evidence of mycoplasma infection and respiratory diseases in feedlot calves. Can J Vet Res 1986;50:179–83. [35] Walz PH, Mullaney TP, Render JA, et al. Otitis media in preweaned Holstein dairy calves in Michigan due to Mycoplasma bovis. J Vet Diagn Invest 1997;9:250–4. [36] Stuart P, Davidson I, Slavin G, et al. Bovine mastitis caused by Mycoplasma. Vet Rec 1963;75:59–64. [37] Mackie DP, Ball HJ, Logan EF. Mycoplasma californicum mastitis in the dry dairy cow. Vet Rec 1986;119:350–1. [38] Gonza´lez RN, Sears PM, Merrill RA, et al. Mastitis due to Mycoplasma in the state of New York during the period 1972–1990. Cornell Vet 1992;82:29–40. [39] Broobanks EO, Carter ME, Holland JTS. Mycoplasma mastitis. N Z Vet J 1969;17:179–80. [40] Boughton E. Mycoplasma bovis mastitis. Vet Bull 1979;49:377–87. [41] Pan IJ, Ogata M. New sero-types of Mycoplasma laidlawii isolated from mastitic milk and urogenital tracts of cattle. I. Isolation of M laidlawii. Jap J Vet Sci 1969;31:83–93. [42] Counter DE. A severe outbreak of bovine mastitis associated with Mycoplasma bovigenitalium and Acholeplasma laidlawii. Vet Rec 1978;103:130–1. [43] Jasper DE, Al-Aubaidi JM, Fabricant J. Epidemiologic observations on mycoplasma mastitis. Cornell Vet 1974;64:407–15. [44] Bennett RH, Jasper DE. Nasal prevalence of Mycoplasma bovis and IHA titers in young dairy animals. Cornell Vet 1977;67:361–73. [45] Blackmer PE. Managing and monitoring for milk quality. Proceedings of the Northeast Dairy Producers Conference. Liverpool, NY. Alpine (NY): Northeast Dairy Producers Association; 2002. p. 63–80. [46] Gonza´lez RN. Mycoplasma mastitis in dairy cattle: if ignored, it can be a costly drain on the milk producer. Proceedings of the Regional Meeting of the National Mastitis Council. Quere´taro, Mexico; 1996. p. 37–45.

R.N. Gonza´lez, D.J. Wilson / Vet Clin Food Anim 19 (2003) 199–221

219

[47] Ruffo G, Socci A. La mastite micoplasmica. Folia Vet Lat 1972;3:750–67. [48] Jasper DE, Jain NC, Brazil LH. Clinical and laboratory observations on bovine mastitis due to Mycoplasma. J Am Vet Med Assoc 1966;148:1017–29. [49] Gonza´lez RN, Sears PM, Wilson DJ. Diagnosis of intramammary infections due to Mycoplasma bovis in dairy cattle. Proceedings of the 3rd IDF International Mastitis Seminar, Tel Aviv, Israel. Bruxelles, Belgium: International Dairy Federation; 1995. (S2) p. 23–7. [50] Gonza´lez RN. Mamitis micopla´smica en establecimientos lecheros: un enfoque holıstico a su diagno´stico y control. In: Marco JC, editors. Tratado de veterinaria pra´ctica. Mamitis bovina y calidad de leche, vol II. Madrid, Spain: Grupo Luza´n 5, S.A. de Ediciones; 1999. p. 77–91. [51] Gonza´lez RN, Sears PM, Wilson DJ, et al. Observations on Mycoplasma bovis mastitis and milk production. Abstract of Papers of the 74th Annual Meeting of the Conference of Research Workers in Animal Disease, Chicago. Fort Collins (CO): Conference of Research Workers in Animal Disease; 1993. p. 53. [52] Gonza´lez RN, Sears PM. Diagnosis, control, and effect on milk production of Mycoplasma bovis intramammary infections. Proceedings of the XVIII World Buiatrics Congress, Bologna, Italy; 1994. p. 681–4. [53] Sears PM, Gonza´lez RN, Wilson DJ, et al. Procedures for mastitis diagnosis and control. Vet Clin N Am 1993;9:445–68. [54] Gonza´lez RN, Jasper DE, Bushnell RB, et al. Relationship between mastitis pathogen numbers in bulk tank milk and bovine udder infections in California dairy herds. J Am Vet Med Assoc 1986;189:442–5. [55] Gonza´lez RN, Jasper DE, Farver TB, et al. Prevalence of udder infections and mastitis in 50 California dairy herds. J Am Vet Med Assoc 1988;193:323–8. [56] Gonza´lez RN, Wilson DJ. Realistic milk culture programs for herd expansion. Proceedings of the National Mastitis Council, Orlando FL. Madison (WI): National Mastitis Council; 2002. p. 118–24. [57] Thomas CB, Willeberg P, Jasper DE. Case-control study of bovine mycoplasmal mastitis in California. Am J Vet Res 1981;42:511–5. [58] Thurmond MC, Tyler JW, Luiz DM, et al. The effect of pre-enrichment on recovery of Streptococcus agalactiae, Staphylococcus aureus and mycoplasma from bovine milk. Epidemiol Infec 1989;103:465–72. [59] Clyde WA Jr, McCormack WM. Collection and transport of specimens. In: Razin S, Tully JG, editors. Methods in mycoplasmology, vol. 1. New York: Academic Press; 1983. p. 103–7. [60] Hogan JS, Gonza´lez RN, Harmon RJ, et al. Laboratory handbook on bovine mastitis Madison (WI): National Mastitis Council; 1999. [61] Freundt EA. Culture media for classic mycoplasmas. In: Razin S, Tully JG, editors. Methods in mycoplasmology, vol. 1. New York: Academic Press; 1983. p. 127–35. [62] Kenny GE. Mycoplasmas. In: Lennette EH, Balows A, Hausler WJ Jr, et al, editors. Manual of clinical microbiology. 4th edition. Washington, DC: American Society for Microbiology; 1985. p. 407–11. [63] Whitford HW. Isolation of mycoplasmas from clinical specimens. In: Whitford WW, Rosenbusch RF, Lauerman LH, editors. Mycoplasmosis in animals: laboratory diagnosis. Ames (IA): Iowa State University Press; 1994. p. 12–4. [64] Montgomerie JZ. L-forms of Pseudomonas aeruginosa. In: Madoff S, editors. The bacterial L-forms. New York: Marcel Dekker; 1986. p. 195–202. [65] Sears PM, Fettinger M, Marsh-Salin J. Isolation of L-form variants after antibiotic treatment in Staphylococcus aureus bovine mastitis. J Am Vet Med Assoc 1987;191:681–4. [66] Wilson CD, Little TWA, Roberts DH, et al. The isolation of L-forms of Streptococcus agalactiae from cases of bovine mastitis. Br Vet J 1971;127:253–63. [67] Clark HW, Bailey JS, Fowler RC, et al. Identification of Mycoplasmataceae by the fluorescent antibody method. J Bacteriol 1963;85:111–8.

220

R.N. Gonza´lez, D.J. Wilson / Vet Clin Food Anim 19 (2003) 199–221

[68] Imada Y, Uchida I, Hashimoto K. Rapid identification of mycoplasma by an indirect immunoperoxidase test using small square filter paper. J Clin Microbiol 1987;25: 17–21. [69] Infante Martı´ nez F, Herrera Putin˜o C, Lozaya Cha´vez F. Identificacio´n de micoplasmas en leche bovina mediante la prueba de Immuno UAT-INIFAP peroxidasa. In: Memoria premio Canifarma Industria Farmace´utica Veterinaria, vol. 1. Me´xico DF, Me´xico: Canifarma Industria Farmace´utica Veterinaria; 1992. p. 101–6. [70] Infante Martı´ nez F, Jasper DE, Stott JL, et al. Immunobinding assay for detection of Mycoplasma bovis in milk. Can J Vet Res 1990;54:251–5. [71] Thurmond MC, Holmberg CA, Luiz DM. Evaluation of a digitonin disk assay to discriminate between acholeplasma and mycoplasma from bovine milk. Cornell Vet 1989; 79:71–80. [72] Sachse K, Pfutzner H, Hotzel H, et al. Comparison of various diagnostic methods for the detection of Mycoplasma bovis. Rev Sci Tech Off Int Epiz 1993;12:571–80. [73] Pinnow CC, Butler JA, Sachse K, et al. Detection of Mycoplasma bovis in preservativetreated field milk samples. J Dairy Sci 2001;84:1640–5. [74] Pfu¨tzner H, Schimmel D. Mycoplasma bovis demonstration in offprings of cows affected with M bovis mastitis and its epidemiological significance. Zentralbl Vet Med 1985;32: 265–71. [75] Bennett RH, Jasper DE. Factor associated with differentiation of cattle resistant and susceptible to intramammary challenge exposure with Mycoplasma bovis. Am J Vet Res 1978;39:407–16. [76] Thomas LH, Howard CJ, Stott EJ, et al. Mycoplasma bovis infection in gnotobiotic calves and combined infection with respiratory syncytial virus. Vet Pathol 1986;23:571–6. [77] Jasper DE, Al-Aubaidi JM, Fabricant J. Isolation of mycoplasma from preputial washings of bulls. Cornell Vet 1974;64:296–302. [78] Kissi B, Juhosz S, Stipkovits L. Effect of mycoplasma contamination of bull semen on fertilization. Acta Vet Hung 1985;33:107–17. [79] Thomas LH, Howard CJ, Gourlay RN. Isolation of Mycoplasma agalactiae var bovis from a calf pneumonia outbreak in the south of England. Vet Rec 1975;95:55–6. [80] Jasper DE, Dellinger JD, Rollins MH, et al. Prevalence of mycoplasmal bovine mastitis in California. Am J Vet Res 1979;40:1043–7. [81] Gonza´lez RN, Sears PM, Wilson DJ, et al. Epidemiology of mycoplasmal bovine mastitis in the state of New York, USA. Proceedings of the 3rd IDF International Mastitis Seminar, Book 2. Tel Aviv, Israel; (S6) p. 68–9. [82] Pfuztner H. Epizootiology of the Mycoplasma bovis infection of cattle. In: Stanek G, Cassell GH, Tully JG, et al, editors. Recent advances in mycoplasmology. New York: Gustav Fischer Verlag; 1990. p. 394–9. [83] Hoblet K, Iqbal M. Mycoplasma mastitis: survey of Ohio herds. Proceedings of the 35th Annual Meeting of the National Mastitis Council, Nashville TN. Madison (WI): National Mastitis Council; 1996. p. 77–81. [84] Ruffo G, Nani S, Podesta A. Survival of Mycoplasma agalactiae var. bovis in several materials and at different temperatures. Arch Vet It 1969;20:459–64. [85] Ruffo G, Nani S, Astori E. Further investigations on the survival of Mycoplasma agalactiae var. bovis. Boll Ist Sieroter Milanese 1971;50:38–40. [86] Pfutzner H. Tenacity of Mycoplasma bovis. Zbl Bact Hyg 1984;A258:38–41. [87] Nagatomo H, Takegahara Y, Sonoda T, et al. Comparative studies of the persistence of animal mycoplasmas under different environmental conditions. Vet Microbiol 2001; 82:223–32. [88] Bray DR, Shearer JK, Donovan GA, et al. Approaches to achieving and maintaining a herd free of mycoplasma mastitis. Proceedings of the 36th Annual Meeting of the National Mastitis Council, Albuquerque NM. Madison (WI): National Mastitis Council; 1997. p. 132–7.

R.N. Gonza´lez, D.J. Wilson / Vet Clin Food Anim 19 (2003) 199–221

221

[89] Bray DR, Brown MB, Donovan GA. Mycoplasma again. Proceedings of the 38th Annual Florida Dairy Production Conference. Gainesville (FL); 2001. p. 52–60. [90] Jasper DE, Dellinger JD, Hakanson HD. Effectiveness of certain teat dips and sanitizers in vitro and on the skin against Mycoplasma agalactiae subsp. bovis. Cornell Vet 1976; 66:164–71. [91] Boothby JT, Jasper DE, Thomas CB. Experimental intramammary inoculation with Mycoplasma bovis in vaccinated and unvaccinated cows: effect on the mycoplasmal infection and cellular inflammatory response. Cornell Vet 1986;76:188–97. [92] Gooden S, Bey R, Farnworth R, et al. Field validation of a milk line sampling device for monitoring milk quality and udder health. J Dairy Sci 2002;85:1468–75. [93] Boddie RL, Owens WE, Ray CH, et al. Germicidal activities of representatives of five different teat dip classes against three bovine mycoplasma species using a modified excised teat model. J Dairy Sci 2002;85:1909–12. [94] Philpot WN, Boddie RL, Pankey JW. Hygiene in the prevention of udder infections. IV. Evaluation of teat dips with excised cows’ teats. J Dairy Sci 1978;61:950–5. [95] Pfutzner H, Scherwa B, Trubner S. Sensitivity of M bovis to desinfectants applied to udder region. Arch Exp Vet Med 1983;37:485–9. [96] Thomas CB. Outbreaks of mycoplasma mastitis do occur in Wisconsin. Focus (University of Wisconsin–Madison Veterinary Medical Teaching Hospital) 1993;10:4. [97] Bar-Moshe B. Control of a mycoplasma mastitis epizootic in a large dairy herd. Refuah Vet 1974;31:32–5. [98] Rinaldi A, Cervio G, Guallini L, et al. Osservazioni sul primo focolaio di mastite micoplasmica del bovino in Italia. Atti Soc Ital Sci Vet 1969;22:796–9. [99] Wilson DJ, Gonza´lez RN. Evaluation of milk culture, SCC, and CMT for screening herd additions. Proceedings of the 36th Annual Meeting of the National Mastitis Council Albuquerque NM; 1997. p. 127–31. [100] Wilson DJ. Mastitis biosecurity: Lessons from expansion in New York. Proceedings of the Regional Meeting of the National Mastitis Council. Waterloo, Canada; 1999. p. 10–7. [101] Butler JA, Sicles SA, Johanns CJ, et al. Pasteurization of discard mycoplasma mastitic milk used to feed calves: thermal effects on various mycoplasma. J Dairy Sci 2000; 83:2285–8. [102] Leadley S. Making use of waste milk. Northeast Dairy Business 2002;9:43. [103] Colditz IG, Watson DL. The immunophysiological basis for vaccinating ruminants against mastitis. Aust Vet J 1985;62:145–53. [104] Boothby JT, Jasper DE, Thomas CB. Experimental intramammary inoculation with Mycoplasma bovis in vaccinated and unvaccinated cows: effect on local and systemic antibody response. Can J Vet Res 1987;51:121–5. [105] Boothby JT, Schore CE, Jasper DE, et al. Immune responses to Mycoplasma bovis vaccination and experimental infection in the bovine mammary gland. Can J Vet Res 1988;52:355–9. [106] Poumarat F, Le Grand D, Solsona M, et al. Vsp antigens and vsp-related DNA sequences in field isolates of Mycoplasma bovis. FEMS Microbiol Lett 1999;173:103–10. [107] Sachse K, Helbig JH, Lysnyansky I, et al. Epitope mapping of immunogenic and adhesive structures in repetitive domains of Mycoplasma bovis variable surface lipoproteins. Infect Immun 2000;68:680–7. [108] Gonza´lez RN, Merrill RA, Sears PM. Shedding of Mycoplasma bovis from the udder of naturally infected cows and its importance for the diagnosis of bovine intramammary infections. J Dairy Sci 1992;75(Suppl 1):259. [109] Pfutzner H, Kielstein P, Martin J, et al. Studies into mycoplasma infection of calf. 2. Experimental infection of calf by Mycoplasma bovis. Arch Exp Vet Med 1983;37:445–51.