Experimental Staphylococcal Mastitis Research Articles

Experimental staphylococcal mastitis in bitches: Clinical, bacteriological, cytological, haematological and pathological features

The objectives of the work were to study the features of experimentally induced canine mastitis and to present hypotheses regarding the pathogenesis of the disease. The right caudal abdominal mammary gland of six bitches was inoculated on day 8 after whelping with Staphylococcus intermedius to induce mastitis; adjacent mammary glands were used as controls. Clinical examination, bacteriological and cytological (whiteside test, Giemsa) examination of mammary secretion, as well as haematological tests were performed from 5 days before until 34 days after challenge. Mastectomy was sequentially performed 1, 2, 4, 18, 26 and 34 days after challenge in each of the bitches, in order to carry out a pathological examination of mammary glands. All animals developed clinical mastitis: challenged glands became painful, hot, enlarged and oedematous; secretion was brownish, purulent, with flakes or clots, subsequently becoming yellowish and thick. Staphylococci were isolated from all inoculated glands (up to 22 days). WST was positive in 41/46 samples from inoculated glands and 66/138 samples from control glands; neutrophils predominated during the acute stage. Blood leukocyte counts increased, whilst platelet counts decreased. Gross pathological findings initially included congestion, purulent discharge and subcutaneous oedema; then abscesses, brownish areas and size decrease were seen. Salient histopathological features were initially neutrophilic infiltration, haemorrhages, destruction of mammary epithelial cells and alveoli, and then infiltration by lymphocytes, shrunken alveoli, loss of glandular architecture and fibrous tissue proliferation. We conclude that in bitches, intrammamary inoculation of Staphylococcus intermedius can induce clinical mastitis, followed by subclinical disease. The disorder is characterized by bacterial isolation and leukocyte influx in challenged glands, by leukocyte presence in adjacent mammary glands, by increased blood leukocyte counts and by destruction of mammary parenchyma.

Vaccination against experimental staphylococcal mastitis in dairy heifers

Vaccination-challenge experiments were carried out with dairy heifers using new, killed cell-toxoid-adjuvant Staphylococcus aureus vaccines. The organisms in the vaccines were cultured under conditions which simulated in vivo growth and induced expression of a pseudocapsule. Dextran sulphate which promotes synthesis of IgG 2 antibody was included in the vaccines as the primary adjuvant. Vaccinated heifers developed very high levels of both IgG 1 and IgG 2 anti-pseudocapsule antibody in serum, however, titres of neutrallsing antibody against toxoided haemolysins were generally low. Vaccinated and unvaccinated control heifers were challenged by intramammary infusion of three virulent strains of S aureus in four experiments. Vaccinated heifers were more resistant to clinical mastitis following challenge than were controls, and the vaccinates had significantly greater milk production than controls following challenge. The most promising vaccine had dextran sulphate combined with mineral oil as the adjuvant injected intramuscularly.

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.

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.

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.

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.

Therapy of experimental staphylococcal mastitis in the mouse with cloxacillin and rifampicin, alone and in combination

Cloxacillin effectively killed Staphylococcus aureus in vitro and in experimental acute mastitis in the mouse, but was ineffective in reducing bacterial counts in those with experimental chronic mastitis. Rifampicin was similarly bactericidal for staphylococci in vitro and in acute mastitis but it also significantly reduced numbers of viable bacteria in chronic mastitis. Exposure of multiplying staphylococci in vitro and in vivo to rifampicin alone led to the emergence of resistant bacteria, but this was prevented by the use of a combination of rifampicin with cloxacillin. This combination showed neither antagonism nor synergy in vitro, was more effective than either antibiotic alone in acute mastitis and killed S aureus in the chronic infection as effectively as rifampicin alone.

The Effect of Suckling on the Course of Experimental Staphylococcal Mastitis in the Mouse

The effect of suckling on the course of experimental staphylococcal mastitis in the mouse was to induce a spectrum of histopathological changes. These changes extended from complete liquefactive necrosis of the gland to the involvement of isolated areas of the gland in a chronic response. The reactions in suckling mice were compared with the acute reaction to intramammary inoculation of staphylococci in non-suckling mice and with the chronic reaction to intramammary inoculation of staphylococci in mice pre-inoculated with endotoxin by the intramammary route. By inoculating suckling mice with organisms suspended in an irritant (endotoxin) or an anti-inflammatory agent (dexamethosone) the response was predominately chronic or acute respectively. The value of the study was to reveal the various pathways along which pathological changes in the mammary gland may proceed.

A histopathological study of the effects of highly purified staphylococcal alpha and beta toxins on the lactating mammary gland and skin of the rabbit

Injection of highly purified staphylococcal alpha toxin into lactating rabbit mammary tissue produced dose-dependent areas of haemorrhagic necrosis and subsequent ischaemia. Injection of beta toxin produced transient dose-dependent erythema. High doses of alpha toxin destroyed the tissue architecture but infiltration with polymorphonuclear leukocytes did not occur. Injections of beta toxin produced intense erythema and infiltration with polymorphonuclear leukocytes. Mixtures of these toxins produced lesions which corresponded to the alpha toxin administered; the erythematous effect of beta toxin was inhibited. Comparison of these findings with those from sections obtained from experimental staphylococcal mastitis in rabbits suggested that the haemorrhagic necrotic syndrome known as “blue breast” was directly attributable to alpha toxin. The role of beta toxin, however, remains unclear.

Experimental staphylococcal mastitis in the mouse:the persistence of chronic infection from one lactation to the next

Chronic staphylococcal mastitis was established in 50 mammary glands of 25 mice. The mice were mated and in the subsequent lactation there was alveolar mastitis in one of 50 glands. A further five glands had abscesses that contained staphylococci but there was no alveolar mastitis. The results are discussed in relation to the large number of infections that persist from one lactation to the next in cows not subjected to mastitis control measures.

The Epidemiology and Pathogenesis of Experimental Staphylococcal and Coliform Mastitis in the Mouse

The invasiveness of Staphylococcus aureus and Escherichia coli for the mammary gland of the mouse was compared by depositing the organism on the tip of damaged or intact teats in the presence or absence of suckling, and sampling the mammary glands for the deposited organism 48 h later. 83% of glands became infected following exposure to S. aureus in suckling mice with teat damage; under the same conditions 3% of glands exposed to E. coli became infected. 17% of glands became infected following exposure to S. aureus in non-suckling mice with damaged teats; under the same conditions 20% of glands exposed to E. coli became infected. Pregnant mice with damaged teats were susceptible to infection with E. coli (20% became infected) immediately prior to parturition and this level of infection could be achieved in suckling mice if damaged teats were repeatedly exposed to E. coli . Infections arising from exposure of teats to E. coli were histologically and clinically identical to those induced by inoculating approximately 20 organisms of E. coli directly into the mammary gland. The inoculation of 20 organisms of S. aureus directly into the mammary gland produced an acute reaction whereas infections resulting from exposure of teats to S. aureus were chronic. Colonization of the teat by S. aureus preceded the establishment of chronic mastitis, but E. coli infections resulted from the chance entry into the mammary gland of a few contaminating organisms. The difference in epidemiology between staphylococcal and coliform mastitis is emphasized.

Experimental staphylococcal mastitis in the mouse: The induction of chronic mastitis and its response to antibiotic therapy

Two strains of Staphylococcus aureus derived from chronic bovine mastitis caused an acute reaction when inoculated into the mammary glands of mice. The response in mice was converted to a chronic mastitis if a neutrophil population was elicited in the alveolar lumen by intramammary inoculation of endotoxin 6 h before staphylococcal challenge. Chronic mastitis was established in this way with each of the strains of S. aureus and in each case the infection failed to respond to intramammary therapy with sodium cloxacillin. The results are discussed in relation to bovine mastitis.

Immunology of experimental staphylococcal mastitis in mice

Summary Anti-staphylococcal serum, inoculated intramammarily, significantly reduced the number of bacteria recovered from mammary glands in which staphylococcal mastitis had been induced. In addition, there was an alleviation of some of the pathological clinical signs following the immune-serum treatment. Intramammary inoculated normal serum and systemically inoculated immune serum failed to elicit any protection. The role of cell-mediated immunity in relation to staphylococcal mastitis was not clearly established.

Natural and experimental staphylococcal mastitis in rabbits

Two natural outbreaks of rabbit mastitis caused by a single phage-type of coagulase-positive staphylococcus were studied. The disease was characterized by abscess formation from which animals slowly recovered without development of immunity to subsequent infection. A vaccine containing the causal organism and its products failed to protect against the disease. When lactating does maintained under laboratory conditions were artificially infected either by injection or by suckling nasally colonized young, the disease produced was not always of the abscess type. Experimentally infected animals frequently died within 48 h. from a gangrenous spreading condition. Both abscess and gangrenous types of disease could be established with very low numbers of organisms. It may be concluded that staphylococcal mastitis in rabbits takes one of two clinical forms. One form is typified by abscess formation and is rarely fatal; the other appears as a gangrenous speading lesion which is frequently rapidly fatal. Since cows may contract both chronic and gangrenous staphylococcal mastitis, the rabbit appears to be a useful and inexpensive alternative test animal.

The increased resistance of mice to experimental staphylococcal mastitis following inoculation of endotoxin

Intramammary inoculation of endotoxin in mice three days before intramammary challenge with staphylococci induced resistance to the staphylococci. Endotoxin given by the intraperitoneal route also induced resistance when inoculated three days before challenge. Resistance was not induced, however, if the mice were allowed to continue lactating during the induction period. Histological examination of mammary glands following different combinations of lactation/involution and endotoxin treatment indicated that endotoxin given by either the intramammary or the intraperitoneal route caused an accelerated involution of the mammary gland and suggested that the involuted gland was inherently more resistant. This was confirmed by demonstrating the same resistance in mice after five days of natural involution when the mammary glands had reached the degree of involution produced by endotoxin in three days.

Experimental staphylococcal mastitis in the mouse: A morphometric study of early changes in mammary gland structure

The changes occurring in mouse mammary gland structure 6 and 12 h. after intramammary inoculation of a virulent strain of Staphylococcus aureus are described using morphometric analysis. At 12 h. after inoculation there was an increase in interacinar tissue, and a decrease in epithelial area and secretory cell area compared with the involution controls. There were also changes in the organelles of the secretory cell in inoculated mice which were consistent with accelerated involution.

Experimental staphylococcal mastitis in the mouse: Histological, ultrastructural and bacteriological changes caused by a virulent strain of Staphylococcus aureus

The pathogenesis of experimental mastitis in the mouse was determined by histopathological, ultrastructural and bacteriological examination of the mammary glands of mice at various times after the intramammary inoculation of a strain of Staphylococcus aureus of high virulence. In this system the critical point was the interaction of staphylococci and neutrophils between 12 and 18 h. after inoculation of staphylococci. In the majority of mice the neutrophils did not succeed in containing the staphylococci, there was rapid extracellular multiplication in the alveolar lumen, toxin production, coagulative necrosis and some mortality 24 to 48 h. after inoculation.

Experimental staphylococcal mastitis in the mouse: The effect of inoculating different strains into separate glands of the same mouse

The effect of inoculation of different strains of staphylococcus, one into each of two mammary glands of the same mouse, was investigated using the mouse mastitis model. The effect of the combination of two different strains was compared with the effect of the homologous combinations. The results show that a strain that normally exhibits a low virulence is phenotypically enhanced when inoculated in combination with a strain of high virulence, and that there is no such effect when the strains combined are equal in virulence.

The effect of allogeneic leucocytes in the bovine mammary gland.

Abstract Extract There are marked similarities between de- scriptions of chronic bovine mastitis (Helmboldt et al., 1953 Helmboldt, C. G., Jungherr, E. J. and Plastridge, W. N. 1953. The histopathology of bovine mastitis. Storrs agric. Exp. Stn Bull., 305: 1–90. [Google Scholar]; Derbyshire, 1958 Derbyshire, J. B. 1958. The pathology of experimental staphylococcal mastitis in the goat. J. comp. Path., 68: 449–454. [Google Scholar]) the appearance of tissue allografts undergoing rejection (Waksman, 1962 Waksman, B. H. 1962. “Tissue damage in the “delayed” (cellular) type of hypersensitivity”. In 2nd International Symposium on Immunopathology, Edited by: Grabar, P. and Miescher, P. 146–146. Stuttgart: Schwabe. In [Google Scholar]) and reactions of the graft versus host (GVH) type in various situations (Jones et al., 1969 Gey, G. O. and Gey, M. K. 1936. The maintenance of human normal cells and tumour cells in continuous culture. Am. J. Cancer, 27: 45–76. [Google Scholar]). It nas been shown that bovine milk, which can contain large numbers of leucocytes, may be transferred from one cow's udder into the empty udder of another cow via a “pipeline” milking machine (Whittlestone and Fell, 1969 Whittlestone, W. G. and Fell, L. R. 1969. “Machine milking and mastitis”. In Proc. N.Z. School and Conf. on Machine Milking, Vol. 2, 145–149. SAuckland: University of Auckland. [Google Scholar]). It is possible that allogeneic leucocytes transferred in this wav could either produce changes typical of chronic bovine mastitis or predispose the recipient udder to infection with the organisms involved in clinical mastitis. Accordingly, the following experiments were undertaken to examine the response of recipient udders to the intramammary injection of allogcneic leucocytes.