Experimental Mastitis Research Articles

Biochemical Alterations in the Blood, Milk and Tissues of Sheep Mammary Gland After Experimental Mycoplasmal Mastitis

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Experimental chlamydial mastitis in goats.

Experimental chlamydial mastitis in goats.

Selenium and Mammary Resistance to Infectious Disease

We designed an experimental mastitis trial to study, under more controlled conditions, the effect of Se on mammary resistance to disease. We divided twenty Holstein heifers into two groups of ten. One group was maintained on a Se deficient (Se-) diet with 0.04ppm Se on a total ration basis. The other group was fed a selenium adequate (Se+) diet that contained 0.14ppm Se. We fed both groups identical rations, except the Se+ group received an additional 2 mg of Se per head per day. All other nutrients, including Vitamin E, were fed at or above NRC recommended levels. We challenged the heifers at about 14 weeks post-partum with 30 colony forming units (cfu) of Escherichia coli in one quarter. Quarters selected for challenge had SCC <250,000 cells / ml and no prior infections.

Phenylbutazone and Flunixin Meglumine Fail to Show Beneficial Effects on Bovine Subclinical Mastitis

Inflammation is caused by the release of chemical mediators from tissues and migra-tory cells. These mediators include leuco-trienes and prostaglandins (PGs), which are generated from arachidonic acid (Vane & Botting 1987). Elevated concentrations of arachidonic acid metabolites have been found in experimental and spontaneous ma-stitis, which indicates that these substances may have a role in the pathophysiologic process of mastitis (Anderson et al. 1985, Atroshietal 1986).

Vaccination against experimental staphylococcal mastitis in ewes

Experiments were carried out in ewes using a new vaccine developed for the prevention of mastitis caused by Staphylococcus aureus. The vaccine comprised three major components: (i) killed S aureus cells which had been cultured to induce synthesis of pseudocapsule; (ii) toxoided staphylococcal beta haemolysin and (iii) the adjuvant dextran sulphate. Ewes systemically vaccinated twice during pregnancy developed significantly elevated circulating levels of IgG1 and IgG2 anti-pseudocapsule antibody, as well as increased serum titres of anti-beta haemolysin. Five different strains of S aureus were used to challenge both vaccinated and control ewes by the intramammary route during the ensuing lactation. The incidence of acute gangrenous mastitis and nonacute, clinical mastitis was significantly lower in vaccinated than in control groups after challenge with each strain. Vaccinated ewes produced significantly more milk than control ewes after challenge with four of the five strains of S aureus.

Biochemical Changes in Milk in Experimental Mycoplasmal Mastitis in Goats

Singh A., P. P. Gupta, S. P. Ahuja, H. S. Banga: Biochemical Changes in Milk in Experimental Acholeplasmal Mastitis in Goats. Acta vet. Brno, 60, 1991: 193-203. To study the biochemical changes in milk during unilateral acholeplasmal mastitis, 8 lactating goats were selected, out of which 7 animals were inoculated through the right teat canal with 2 ml of Acholeplasma laidlawii containing 109 colony forming units per 1 ml. The left udder halves of these 7 goats served as controls and one goat was kept as intact control. The effects of infection were monitored over a period of 19 days. During this period one goat was killed every 3rd day after infection, while 2 animals were killed on the 19th day, i.e. one experimentally infected and one intact control. All the goats infected with A. laidlawii developed clinical mastitis within 24 h, which persisted till the end of the experiment. The total protein, total immunoglo­ bulins (Ig), total phospholipids and free fatty acid contents of mastitic milk/mam­ mary secretions showed progressive increase, whereas total lipids and glycerides decreased substantially. However, there was a marginal increase in the total choleste­ rol concentration. The results indicated that Acholeplasma use lipolytic enzymes to degrade lipids so as to meet their needs of energy and for biosynthesis of membrane lipid bilayer during multiplication. The increase in milk protein especially Ig indicated humoral immune response to A. laidlawii and secretion of Ig into the mammary gland. Goats, milk/secretion, total proteins, lipids, cholesterol, FFA, phospholipids, glycerides, immunoglobulines

Dissemination of staphylococci in mice with experimental mastitis.

During the course of an acute, fatal mastitis in mice caused by intra-mammary injection of approximately 10(5) cfu of Staphylococcus aureus, small numbers of staphylococci were detected in liver, spleen, kidney and heart. Transport from the mammary gland was by the venous blood system with no evidence of lymph node involvement. Intravenous injection of 100 times more staphylococci (approximately 10(7) cfu) than were fatal by the intramammary route failed to kill the mice and small numbers of staphylococci were found in the visceral organs. However, intravenous injection of approximately 10(8) cfu of Staph. aureus was fatal and large numbers of staphylococci were found in the visceral organs. Treatment with cloxacillin (500 micrograms) by the intramammary or subcutaneous routes, or equally divided between these two routes, cleared the visceral organs of staphylococci; subcutaneous therapy had a limited and variable effect on numbers of staphylococci in the mammary gland while intramammary and divided treatments significantly reduced the numbers of staphylococci. Thus, staphylococci are disseminated to visceral organs during acute staphylococcal mastitis, but this makes a negligible contribution to the clinical disease.

Pathology of experimental ureaplasma mastitis in ewes.

One mammary gland of each of nine primum partum suckling ewes was inoculated with ureaplasma 8 weeks post-partum. Infected glands were swollen, hot, and painful from 16 hours post-infection. Subsequently there was agalactia and reduction in gland size. Histopathologic and ultrastructural examination indicated that the acute phase of the induced mastitis was characterized by necrosis of secretory epithelium with a neutrophil, eosinophil, and macrophage response. Subsequently there was lymphoid infiltration, acinar involution, and fibrosis. This report confirms the pathogenicity of ureaplasma for the ovine mammary gland, and is the first description of the pathologic features of ureaplasmal mastitis in sheep.

Inflammatory changes during experimental bovine mastitis induced by Staphylococcus aureus, Streptococcus dysgalactiae and Streptococcus uberis.

SummaryExperimental mastitis was induced in nine lactating dairy cows with Staphylococcus aureus, Streptococcus dysgalactiae and Streptococcus uberis (three cows in each group). One quarter of each cow was inoculated with organisms, the contralateral one remained untreated and served as a control. Inflammatory changes were monitored by measurements of somatic cell count, NAGase and antitrypsin. The whey was analyzed for its quality as a growth medium for E. coli.Clinical signs and the increase in the levels of the inflammatory parameters occurred 24 hours after inoculation; S. aureus caused more severe inflammatory changes than did streptococci. Infections by 5. aureus persisted longer than the others. The inflammation declined slowly after the infection had been cleared by the udder. The whey became an inhibitor for the growth of bacteria in vitro during the early inflammatory period in half of the infused quarters. Whey was found to stimulate bacterial growth later in the course of inflammation in all experimentally infected quarters.ZusammenfassungDurch Staphylococcus aureus, Streptococcus dysgalactiae und Streptococcus uberis während der experimentellen Mastitis hervorgerufene entzündliche Veränderungen: Bei neun laktierenden Kühen wurde mittels Staphylococcus aureus, Streptococcus dysgalactiae und Streptococcus uberis (drei Kühe pro Gruppe) experimentell eine Mastitis induziert. Jeweils in ein Euterviertel wurden die betreffenden Keime inoculiert, das contralaterale Viertel blieb unbehandelt und diente als Kontrolle. Die entzündlichen Veränderungen wurden mit Hilfe der Zellzahl, der NAGase und dem Antitrypsin der Milch verfolgt. Die Molke wurde auf ihre Eignung als Wachstumsmedium für E. coli geprüft. Klinische Symptome sowie ein Anstieg der Werte der Entzündungsparameter traten 24 h nach den Inoculation in Erscheinung. S. aureus verursachte deutlichere entzündliche Veränderungen als dies bei Streptococcen der Fall war. Die Infektionen mit S. aureus persistierten länger als die anderen Infektionen. Die Entzündung ging nach dem Verschwinden der Euterinfektion langsam zurück. Die Molke wirkte während des Anfangsstadiums der Entzündung bei der Hälfte der inoculierten Euterviertel hemmend auf das Bakterienwachstum. Im weiteren Verlauf der Infektion wirkte die Molke bei allen experimentell infizierten Vierteln stimulierend auf das Bakterienwachstum.

The effect of incorporation of cloxacillin in liposomes on treatment of experimental staphylococcal mastitis in mice.

The effect of incorporation of cloxacillin in liposomes on the treatment of staphylococcal mastitis was assessed bacteriologically 18 h after treatment of experimental infections in mice caused by two strains of Staphylococcus aureus. Intramammary treatments were cloxacillin incorporated in liposomes, cloxacillin in combination with liposomes, empty liposomes, cloxacillin in saline and saline alone. In none of the experiments did entrapment of cloxacillin within liposomes enhance its antibacterial effects. Electron microscopic studies demonstrated the presence of liposomes in neutrophils which also contained staphylococci. The results support the hypothesis that intracellular staphylococci are metabolically dormant and therefore not susceptible to the action of inhibitors of cell wall synthesis such as cloxacillin.

Flunixin fails to enhance cloxacillin in treatment of experimental staphylococcal mastitis in mice.

Flunixin fails to enhance cloxacillin in treatment of experimental staphylococcal mastitis in mice.

Morphological changes caused by experimental Streptococcus uberis mastitis in mice following intramammary infusion of pokeweed mitogen.

Mice were used as models for bovine mastitis in an attempt to modify the susceptibility of mammary glands to Streptococcus uberis infection. Murine mammary glands were injected with pokeweed mitogen (PWM) prior to experimental bacterial challenge to accelerate involution and enhance antimicrobial mechanisms. PWM injection reduced the numbers of streptococci recovered when compared to controls. Histological examination of tissues from PWM-treated mice revealed a reduction in secretory activity and advanced involution. PWM-treated tissues had considerably more leukocytes infiltrating the epithelium, lumen, and underlying connective tissue. Bacteria were observed within the epithelium and alveolar lumen and internalized within neutrophils and macrophages in both PWM-injected and control tissue. Results of this study suggest PWM injection provided some protection against S. uberis mastitis by accelerating mammary involution, enhancing antimicrobial defenses, and facilitating a marked cellular response prior to bacterial challenge.

Changes in immunoglobulin levels in whey during experimental Streptococcus agalactiae mastitis

Total protein and immunoglobulin levels in the wheys of eight first lactation heifers, four vaccinated and four unvaccinated, were measured during three consecutive experimental intramammary infections with Streptococcus agalactiae. There were no significant differences between infections 1, 2 and 3 in the protein or immunoglobulin content of the uninfected quarters. Peak whey total protein of the infected quarters came earlier with each infection, until by the third they were seen after eight hours. During this acute phase a reversal of the normal milk IgG1/IgG2 ratio in all infected quarters was measured. Increases in whey IgA and IgM in the infected quarters of the vaccinates were also noted. A similar response only occurred following the third infection of the unvaccinated animals. All whey immunoglobulin levels returned to normal by 48 hours after infection, after which only IgG1 levels increased in infected quarters.

Experimental Mycoplasma arginini mastitis in goats.

Experimental Mycoplasma arginini mastitis in goats.

Immunophysiological activity of supramammary lymph nodes of the ewe during the very early phase of staphylococcal mastitis

Efferent mammary lymph was collected from lactating ewes which were unimmunised (controls) or immunised during pregnancy with two doses of an attenuated live Staphylococcus aureus vaccine either in the hindlimb ("directly primed' supramammary nodes) or in the brisket ("indirectly primed' supramammary nodes). Mammary lymph was also collected from unimmunised animals in which the supramammary nodes had been extirpated several months before. Ewes in which the supramammary nodes had been directly primed by staphylococcal vaccination before challenge had a significantly greater output of IgM- and IgG2-containing cells in lymph and higher concentrations of IgG1 and IgG2 antibody against S aureus surface antigens than did other groups. Lymphadenectomised ewes had fewer leucocytes in mammary lymph but a much higher proportion of neutrophils than other ewes, indicating that afferent mammary lymph has an unusually high number of neutrophils and most of these cells are filtered out in the supramammary lymph nodes under normal circumstances. The results indicated that most of the leucocytes in efferent lymph were derived from the supramammary nodes. After induction of experimental staphylococcal mastitis there was a rapid drop in leucocyte output in lymph within one to four hours after infection; the data indicated that events within the supramammary nodes were responsible for this phenomenon. The output of immunoglobulin-containing cells was reduced during this phase. No significant increases in output of lymphoblasts, immunoglobulin-containing cells or specific antibody occurred during the six hours immediately following infection.

The pathogenesis of a high-virulence and a low-virulence strain of Corynebacterium bovis in the mammary gland of the mouse

A comparison was made of the pathogenesis of experimental mastitis in mice caused by a strain of Corynebacterium bovis (P3) isolated from clinical mastitis in the cow and a strain (NCDO 1930) isolated from the teat of a symptomless carrier cow. Strain NCDO 1930 elicited a neutrophil response which controlled the infection so that, after 6 to 8 days, 9 of 10 glands were sterile and one abscess was found. In contrast, the neutrophil response to strain P3 failed to control the infection and, by 6 to 8 days, 8 of 10 glands were infected and there were abscesses in 11 of 20 glands. The virulence of strain P3 was associated with its ability to colonize on and in milk fat globules, from which the organisms multiplied in the alveolar lumen irrespective of the neutrophil infiltration.

Experimental mastitis in mice induced by coagulase-negative staphylococci isolated from cases of mastitis in nursing women.

Coagulase-negative staphylococci are frequently isolated from the milk of women with signs of puerperal mastitis. In order to evaluate the pathogenicity of these bacteria, strains of S. epidermidis and S. saprophyticus isolated from cases of mastitis in nursing women were inoculated into the mammary glands of lactating mice. Although clinical signs of mastitis were absent, by histological examination, mastitis was demonstrated in 78-93% of the glands. Abscesses were found in a few cases only. The inoculated bacteria were re-isolated in 41-61% of the cases, and when inoculated in numbers of 10(2) to 10(4) c.f.u. the bacteria multiplied above input levels in several cases. It is concluded that coagulase-negative staphylococci isolated from the milk of women with puerperal mastitis can produce mastitis in mice and should be considered as a possible etiologic agent of mastitis in nursing women.

Efficacy of lincosaminide antibiotics in the treatment of experimental staphylococcal mastitis in lactating mice.

Staphylococcus aureus is a frequent cause of bovine mastitis worldwide. A model that may predict the efficacy of antimicrobial agents in the treatment of bovine mastitis induced by Staph. aureus was developed in lactating mice. Infection was established by the inoculation of lactating CF1 mice with Staph. aureus into the mammary gland via the teat duct. At the dose of bacteria used, 85-90% of the inoculated, untreated animals developed a nonlethal, acute mastitis within 48 h. Antibiotic treatment was administered subcutaneously or by the intramammary route. Lincosaminide antibiotics including lincomycin, clindamycin, and pirlimycin were evaluated in this system. Other compounds which have been used in therapy of bovine mastitis including novobiocin, penicillin G, ampicillin, cloxacillin and rifamycin-SV were used as reference antibiotics. Pirlimycin was the most effective of the antibiotics tested in this standardized system. Depending upon the route of administration, this novel lincosaminide was 15 to 95-fold more effective than clindamycin, three- to six-fold better than lincomycin, two- to ten-fold more effective than novobiocin, 13- to 17-times more effective than cloxacillin and 8- to 22-times better than rifamycin-SV on a weight-dose comparison. Penicillin G and ampicillin were the least effective drugs tested against mastitis induced by the beta-lactamase producing strain of Staph. aureus used in these assays. Pharmacokinetic experiments suggested that the greater effectiveness of pirlimycin compared to clindamycin and lincomycin was due to increased affinity for and prolonged retention in the mammary gland.

Immunological features of consecutive intramammary infections with Streptococcus agalactiae in vaccinated and non-vaccinated heifers

The antibody response of the bovine udder to experimental Streptococcus agalactiae mastitis was measured using an enzyme-linked immunosorbent assay (ELISA). Ten first-lactation heifers, four of which were rendered systemically hyperimmune to Str. agalactiae, were infected, via the teat canal, in the right front quarter on two or three consecutive occasions at 23 and 30-day intervals. Initially there was no specific antibody in the udder of the non-vaccinated heifers while the hyperimmune animals had evidence of specific whey antibody. During each infection the humoral response was essentially biphasic. In the non-vaccinated heifers the first phase coincided with the acute clinical reaction and specific antibody only appeared in the infected quarter. With the second phase, antibody appeared in both the infected and control quarters. By the third consecutive infection these changes were more marked and occurred more quickly and were very similar to those seen with the hyperimmune animals during their first infection. The results demonstrate that repeated intramammary infection with Str. agalactiae can produce high levels of serum and whey antibodies. However, the response in the udder was of short duration.

Experimental acute staphylococcal mastitis in the mouse: The influence of pathological changes on the kinetics and therapeutic action of cloxacillin

Acute mastitis was produced by inoculating 10 6 cfu Staphylococcus aureus into normal lactating-mouse mammary glands. Rapid multiplication of bacteria occurred, with the production of alpha toxin, liquefactive necrosis, and death. Intramammary administration of sodium cloxacillin simultaneously with, or soon after, infection prevented clinical disease and reduced bacterial numbers, whereas treatment of more advanced disease was unsuccessful. Higher concentrations of cloxacillin occurred in mastitic glands than in normal glands. The distribution of [ 35S]-labelled cloxacillin, as assessed by an autoradiographic technique, was good in normal but impaired in mastitic glands. Nevertheless, inhibitory concentrations were achieved throughout the diseased tissue. After treatment with cloxacillin many staphylococci were gram-negative; grampositive cocci were also seen, especially in association with neutrophils. Failure of treatment in the late stages of the disease was attributed to high bacterial numbers and decelerated multiplication.