Mycoplasmosis: experimental mastitis. Demonstration of antibody in milk

According to several authors specific antibodies can be demonstrated in serum of cows with mycoplasma mastitis. For practical reasons, and for pathogenetic studies, it would be of interest if presence of antibody in milk could be established. To elucidate this and to examine the pathogenicity of the type strain, PG 11, of M. bovigenitalium, the experiment described below was conducted.     

Mycoplasmosis: experimental inoculation of calves with a Danish strain of Mycoplasma bovigenitalium

During the fall of 1966 several strains of Mycoplasma were isolated from semen samples of bulls. A strain, “K”, isolated from a bull with vesiculitis was selected for a series of experiments and proved to be pathogenic for the mammary gland of cows and for the genital tract of bulls (Ernø 1967, Blom & Εrnø 1967). The “K” strain has now been examined serologically by the author, and it must be considered as belonging to the species M. bovigenitalium as determined by complement fixation and indirect haemagglutination tests. This communication reports the results of experimental inoculation of calves.     

Experimental mastitis caused by Mycoplasma bovigenitalium and M. canadense in the ewe

Twelve strains of M. bovigenitalium and two of three strains of M. canadense caused an infection resulting in a pathogenic effect when experimentally inoculated into the ovine mammary gland. Differences in the pathogenesis were quantified by the duration of continuous mycoplasma excretion and the duration of high milk cell levels, but variation in the susceptability of the experimental animals prevented the establishment of firm conclusions on the relative virulence of the strains. Seven M. bovigenitalium and two M. canadense strains were eventually eliminated naturally from the infected glands, but four M. bovigenitalium strain infections ultimately became sub-clinical with intermittent mycoplasma excretion and low milk cell levels.     

Mycoplasmosis: cervical and uterine inoculation of heifers with a Danish strain of Mycoplasma bovigenitalium

The “Κ” strain of Mycoplasma bovigenitalium has proved to be pathogenic for the mammary gland of cows, for the genital tract of bulls, and for calves (Εrnø 1967, Blom & Εrnø 1967, Εrnø 1969). Antibodies can be demonstrated in the blood following infection of the mammary gland, and after intravenous and intraperitoneal inoculation of calves with or without clinical signs of infection.     

Mastitis in a 7-week old calf caused by Mycoplasma bovigenitalium

This is the first report of an intramammary infection caused by Mycoplasma bovigenitalium in a 7-week old Holstein calf. The calf was initially presented for a non-weight bearing lameness in the left hind limb. The clinical examination revealed not only a septic arthritis of the tarsus but also an infection affecting the right rear mammary gland. The source of M. bovigenitalium was not found but the most likely explanation is spread from the infected left tarsus. This case report demonstrates that mycoplasma mastitis can occur in pre-weaned calves, which could play a role in the epidemiology of mycoplasma mastitis in dairy herds.     

Bovine mycoplasma mastitis from intramammary inoculation of small numbers of Mycoplasma bovis. I. Microbiology and pathology

Intrammary inoculation of 70 colony forming units (cfu) of Mycoplasma bovis into one quarter of four previously non-infected cows resulted in severe mastitis in inoculated and uninoculated quarters. Hematogenous spread of the infection was most likely, as mycoplasma was isolated from the peripheral blood of three of four cows and the milking machine was designed to prevent quarter to quarter communication of milk and air. The presence of large numbers of mycoplasma exceeding 10⁶ cfu/ml of milk preceded the onset of overt sero-purulent mastitis by 1–3 days. In general, the severity, duration of the infection and within cow spread of mastitis to adjacent quarters after the inoculation of 70 cfu was indistinguishable from naturally occurring mycoplasma mastitis.The pathology of the chronically infected quarters consisted of alveolar involution and moderate to severe mononuclear infiltration and an increase in interalveolar and loose connective tissue. The quarters of one cow resolving the infections at the time of slaughter were not as severely affected and contained numerous milk-producing alveoli and many alveoli with hyperplasia of the alveolar cells.     

Studies of bovine mycoplasma mastitis. 1. Review of literature on the occurrence of bovine mastitis with Mycoplasma involvement]

The clinical pattern as well as the pathologico-anatomic or histological changes due to mycoplasma mastitis are neither specific nor pathognomic. Mastitis pathogens so far described included M. bovis, M. bovigenitalium, A. laidlawii, A. axanthum, M. alkalescens, M. canadense, M. dispar, M. bovirhinis, strains of Group 7 according to Leach, strain ST 6, and ureaplasma strains. In the GDR, enzootic mastitis has been confined to A. laidlawii and A. axanthum.     

The indirect hemagglutination test for the detection of antibodies in cattle naturally infected mycoplasmas.

Stable mycoplasma antigens for the indirect hemagglutination test (IHA) were prepared employing glutaraldehyde treated sheep erythrocytes sensitized with Mycoplasma agalactiae subsp. bovis and Mycoplasma bovigenitalium antigens. Employing these antigens mycoplasma antibodies were detected in sera from cattle which had mastitic symptoms due to natural infection with either M. agalactiae subsp. bovis or M. bovigenitalium. A total of 200 cows from four herds were examined at varying intervals for the presence of M. agalactiae subsp. bovis and for the detection of antibody using growth inhibition and IHA tests. Mycoplasmas were isolated from 37 animals. Growth inhibiting antibody was detected from 56 of the 200 animals. In the IHA tests, antibody titer greater than or equal to 1:80 were detected in 148 animals, 76 of these having antibody titers greater than or equal to 1:160, while sera of 116 normal control animals had no growth inhibiting antibody and none had IHA antibody titers greater than 1:40. M. bovigenitalium was isolated from the milk of three of 26 animals in a fifth herd during an outbreak of mastitis. Growth inhibiting antibodies were demonstrated in the sera of ten of the 26 animals. However, the IHA test detected antibody titers of greater than or equal to 1:160 in 13 animals and of 1:80 in one of the 26 animals. To determine the specificity of the IHA tests, M. agalactiae subsp. bovis and M. bovigenitalium antigens were reacted with rabbit hyperimmune typing sera produced against 12 species of bovine mycoplasmatales. Homologous antisera showed IHA antibody titers of 1:1280 and 1:2560 against M. agalactiae subsp. bovis and M. bovigenitalium respectively, whereas heterologous antisera showed IHA antibody titers of less than or equal to 1:20. Also eight type-specific bovine antisera were reacted with M agalactiae subsp. bovis and M. bovigenitalium antigens in homologous and heterologous tests. Homoogous reactions showed IHA antibody titers greater than or equal to 1:320, whereas heterologous reactions showed IHA titers of less than or equal to 1:20. This IHA test promises to be useful for the detection of bovine mycoplasma antibodies in sera from cattle infected with M. agalactiae subsp. bovis or M. bovigenitalium. Thes test is sensitive, reproducible and specific and the technique is relatively simple and rapid. The antigens were stable for at least seven months.     

Experimental infection of bulls and cows with bluetongue virus serotype 20

Bluetongue virus serotype 20 (BTV20) was inoculated intradermally and subcutaneously in 4 bulls and by the intrauterine route in 8 nulliparous cows after insemination at oestrus. Viraemia was detected intermittently between 8 and 21 days after inoculation. Virus was isolated from tissue samples of 2 cows and a bull after slaughter at 14 days and from one bull at 28 days. Group reactive and type specific antibodies to BTV20 were demonstrated from 17 to 27 days after infection. No antibodies were detected in the animals slaughtered at 14 days. No clinical signs of disease were seen during the experiment and no gross or histopathological changes referable to BTV20 infection were observed post-mortem. Because of the viraemia and the production of detectable serum antibodies, gametes from these cattle would be excluded from export.     

A mild outbreak of bovine mastitis associated with Mycoplasma bovigenitalium.

Mycoplasma bovigenitalium was isolated from milk samples from 16 of 99 cows on one farm during a 15-week period in the summer of 1986. One cow was severely affected, four cows had relatively mild signs of mastitis, and three had only altered dry-cow udder secretions. Eleven of the infected cows were dry and three had been calved less than 48 hours. The abrupt method of drying-off and improvements in cleaning of the milking equipment were introduced, but no other control measures were instituted to eradicate the mycoplasma infections. After this mild outbreak of mastitis the herd was monitored for the next 17 months. In total 19 cows had a mycoplasma isolated from udder secretions. Acholeplasmas were isolated from 14 cows but were not associated with clinical mastitis. The udder infections with mycoplasmas apparently resolved without resorting to the segregation and culling of infected animals.     

The kinetics of inflammation and phagocytosis during bovine mastitis induced by Streptococcus agalactiae bearing the protein X

The protein X of Streptococcus agalactiae is a surface antigen borne by a high proportion of strains isolated from bovine mastitis. We have tested the capacity of two strains of X-bearing Streptococcus agalactiae to induce mastitis in dairy cows. The reference X-strain (411.07) produced an intramammary infection with local clinical signs in the three inoculated quarters. Another X-bearing strain (443.31) of bovine origin produced infection in all 11 quarters inoculated with only 25 or 85 colony-forming units. In naive cows, strain 433.31 induced less exudation of plasma into the milk, shedding of bacteria, macroscopic alteration, and a lower somatic cell count (SCC) than did the reference strain. Only one quarter spontaneously eliminated the infection before antibiotic treatment 9 days after inoculation.The serum of all the cows contained naturally acquired or induced antibodies to the challenge strain (443.31) and possessed opsonic activity. Before inflammation occurred, the milk was almost devoid of antibody or opsonic activities. The early phase of infection was characterized by rapid multiplication of streptococci in the milk, followed by a sharp drop in bacterial counts concomitant with the onset of inflammation.Three cows immunized with protein X displayed higher SCC and bactericidal activity in milk from the inoculated quarter at the onset of inflammation than non-immunized cows. Two of the three immunized cows underwent an early and transient febrile episode and eliminated the infection.     

Mycoplasmosis: experimental and spontaneous infections of the genital tract of bulls

A strain of M. bovigenitalium was isolated from semen of a bull (K) with chronic seminal vesiculitis. Using this strain a vesiculitis of the same type as found in bull K, characterized by simultaneous acute and chronic lesions, was induced experimentally in 2 bulls by direct inoculation into the vesicular glands. In the acute phase a marked infiltration of eosinophils was found in the interstitial tissues and alveoli whereas in the chronic phase fibrosis, lymphoid and epithelial hyperplasia were seen. Degeneration of vascular walls and connective tissue was common.On inoculation into the testis of 3 bulls a chronic epididymitis and ampullitis were produced. The histological changes were of the same type as found in the vesicular glands of the 3 bulls with vesiculitis.By indirect hemagglutination a specific and significant increase in serum antibody could be demonstrated. As the titers were low and the maximum titers were reached early, it will probably not often be possible to make an etiological diagnosis on the basis of serological evidence.A comparative experiment employing the type strain (PG 11) of M. bovigenitalium was performed. A rise in antibody titer was seen, but neither clinical nor histological changes could be demonstrated.     

Infertility in heifers caused by pathogenic strain of Mycoplasma bovigenitalium

Mycoplasma bovigenitalium (M. bovigenitalium, strain AL) was inoculated by insemination during estrous into the uterus or the cervix of 12 heifers. The inoculum consisted of a mixture of M. bovigenitalium (strain AL) and diluted semen taken from a highly fertile bull free of mycoplasma infection. Mycoplasma organisms were recovered 3 days postinoculation (PI) from the vaginal mucous of eight of 12 inoculated heifers, and at weekly intervals thereafter until the time of necropsy. All inoculated heifers had granular vulvovaginitis; some also had mucopurulent vaginal discharges. Six of the 12 infected heifers were inseminated more than once, yet none became pregnant. Macroscopic changes observed at necropsy in the genital tracts, in addition to granular vulvovaginitis, consisted of mucopurulent discharges emananting from the uterus, cervix, and vagina. All ovaries had corpora lutea. Mycoplasmas were recovered at necropsy from eight of the 12 heifers. Isolations were made from the vaginal wall, cervix, uterus, right and left oviducts, and the ovaries. All recovered mycoplasms were identified as M. bovigenitalium. It was concluded that M. bovigenitalium (strain AL) can cause inflammatory changes and infertility in heifers.     

Mastitis severity induced by two Streptococcus uberis strains is reflected by the mammary immune response in vitro

Streptococcus uberis is the most common environmental mastitis pathogen causing udder inflammations of different severities in dairy cows. The aim of the study was to investigate if the different clinical outcome of mastitis induced by different strains of S. uberis can be reflected in the mammary immune response. Mammary epithelial cells and somatic milk cells were treated with heat inactivated and living S. uberis of strain A and strain B in vitro. Strain A was repeatedly isolated from a chronically infected quarter during 8 months, and persisted in the quarter despite antibiotic treatment. Strain B caused an acute clinical mastitis and was not further isolated after a single antibiotic treatment. Treatment with Strain B induced a more pronounced increase of mRNA-expression of various immune factors (interleukin-8, interleukin-1beta, RANTES, and lactoferrin) in mammary epithelial cells than strain A. In contrast to mammary epithelial cells the response of removed somatic milk cells showed no differences between the stimulation with two S. uberis strains. Tumor necrosis factor-alpha mRNA expression was not differently induced by the two strains. In conclusion, the characteristics of different severities of mastitis that are induced by different S. uberis strains in vivo can also be reflected at the level of the immune response of the mammary gland in vitro.     

Studies of Mycoplasma mastitis in cattle. 6. Testing of Mycoplasma isolated from milk for udder pathogenicity

Strains of M. bovis, A. laidlawii, A. axanthum, and one unidentified strain of the family of mycoplasmataceae as well as altered secretion obtained from cows with mastitis were intracisternally applied in experiments and proved to be pathogenic to cattle udder. The results are likely to suggest the importance of mycoplasma isolated from the milk of mastitis cows to the aetiology of enzootic mastitis in three large dairy cattle stocks. Intravenous application of M. bovis and A. laidlawii caused neither mastitis nor mycoplasma secretion in the milk.     

Detection of mycoplasma in mastitic milk by PCR analysis and culture method.

Mycoplasma alkalescens, M. bovigenitalium, M. bovirhinis and M. bovis were directly detected from milk specimens by a polymerase chain reaction (PCR) when milk specimens were centrifuged and treated with mycoplasmal lysis buffer. The sensitivity of this PCR method was 110 to 1,400 colony forming units (CFU). This method was useful for the detection of mycoplasmas in milk specimens from cows at an early stage of mycoplasmal mastitis since a small amount of mycoplasma could be detect in milk without culture. The results were available within 12 hr, which is faster than conventional culture techniques. M. bovirhinis was detected in more than 70% of mastitic milk specimens when mycoplasmas were detected in milk specimens from 30 cows with mastitis by this PCR method.     

Isolation of mycoplasmas from the canine genital tract: a survey of 108 healthy dogs

Cultivation of preputial or vaginal swab samples from 108 apparently healthy dogs gave presumptive mycoplasma growth in 72 cases. Specific identification of 58 mycoplasma isolates based on biochemical studies, indirect immunofluorescence and growth inhibition tests showed association with eight different species or groups, only four of which had previously yielded genital isolates. Fourteen strains were related to M bovigenitalium.     

Characteristics of an unusual mycoplasma isolated from a case of caprine mastitis and arthritis with possible systemic manifestations

A mycoplasma designated strain GM790A was isolated from milk and internal organs of 2 lactating goats showing mastitis and arthritis. The isolate was not related serologically to any of the currently known ovine-caprine mycoplasmas, except an isolate designated Mycoplasma sp. G, first recorded from the external ear canal of clinically normal goats in Australia. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and restriction enzyme DNA studies of strain GM790A and Mycoplasma sp. G revealed similar but not identical patterns. The inoculation of strain GM790A into the teat canal of 2 lactating goats resulted in an abrupt diminution of lactation leading to mastitis and agalactia in about 3 days. A maximum of 1 x 10(7) colony-forming units (CFU) of the mycoplasma were shed per ml of mammary secretion. Milk production partially resumed at a low level 3 weeks postinoculation, the longest period tested, but the milk still contained 1 x 10(2) CFU of the agent. The results of this study indicate that strain GM790A possesses pathogenic potential for the goat and most probably represents a new species of the genus Mycoplasma.     

Binding of a surface protein of Staphylococcus aureus to cultured ovine mammary gland epithelial cells.

Staphylococcus aureus is the most persistent pathogen causing ovine mastitis. This study investigated S. aureus binding to cultured epithelial cells obtained from the mammary gland. A staphylococcal 145kDa cell wall adhesin, originally isolated from a bovine mastitis strain, was detected in lysostaphin-solubilized ovine mastitis strains and in the encapsulated strain A. This adhesin was able to bind to cultured ovine mammary gland epithelial cells (MGEC) and to a rat intestinal epithelial cell line (RIE-1), exhibiting different electrophoretic mobilities that could be attributable to protein polymorphism. Inhibition assays using antibodies against 145kDa adhesin and against whole bacteria showed the specificity of the binding to cells. The role of this protein in adherence was assessed by adherence inhibition tests carried out in vitro with radiolabeled bacteria and cultured epithelial cells. Preincubation of bacteria with antibodies against adhesin 145kDa or against strain c195 resulted in a statistically significant decrease of adherence. These experiments suggest that adherence of S. aureus to MGEC may be critical for colonization.     

Experimental infections of bulls with Mycoplasma (M.) bovis and M. bovigenitalium

M. bovis or M. bovigenitalium species experimentally used in intrapreputial infection were re-isolated from 7 of 8 bulls. Clinically manifest diseases did not develop at all, though slight inflammatory lesions were recorded from the genital tract of 2 animals. Mycoplasma findings are discussed together with results obtained from spermatological and hematological investigations.