Monitoring of basophil sensitization to antigens of the cat flea (Ctenocephalides felis felis): a new tool for the diagnosis of feline flea bite hypersensitivity?

Suitability of blood basophils for in vitro diagnosis of flea allergy dermatitis (FAD) or flea bite hypersensitivity was studied in cats. A functional in vitro test (FIT) for sensitized type I allergic effector cells was used to evaluate the degree and kinetics of in vivo basophil sensitization against flea antigens in cats under long-term flea exposure. FIT results were compared with intradermal (IDT) and serological testing. Before, during, and after weekly repeated exposure to Ctenocephalides felis; 14 cats were repetitively FIT-assessed for general and flea-specific sensitization. In three cats, flea-specific sensitization was seen before and throughout flea exposure. Five cats, although generally sensitized, never developed a flea-specific sensitization. Six cats initially FIT-negative became sensitized for flea antigen during flea infestation. Induction, upregulation, and binding of C. felis-specific sensitizing antibodies to basophils during flea challenge may explain the developing sensitization in these cats. Strong discrepancies between the levels of flea-specific circulating IgE and basophil sensitization contrasted comparable results for basophil and mast cell sensitization using FIT and IDT, respectively. Hence, the FIT might provide an immunological supplement to the clinical diagnosis of FAD in cats by elucidating the state of basophil-sensitization to flea antigens. And it may be a comfortable alternative to IDT.

Dexamethasone depresses the expression of L-selectin but not the in vivo migration of bovine neutrophils into the uterus.

There are several indications that periparturient depression of functional properties of polymorphonuclear neutrophilic granulocytes (PMN) may be of great importance for the pathogenesis of genital infections after calving. As the periparturient period is characterized by high plasma levels of corticosteroids, the hypothesis to be tested was that high concentrations of glucocorticoids during the periparturient period suppress uterine PMN in number and functionality. An in vivo endometritis model was applied to examine uterine PMN. Recombinant human interleukin-8 (rhIL-8) was infused into the uterus of estrous cows and heifers 24 h after pretreatment with dexamethasone (0.07 mg/kg i.m.). Six hours after rhIL-8 infusion high numbers of uterine PMN were isolated and characterized as to their immunophenotype and function. Animals treated with dexamethasone animals showed leucocytosis due to neutrophilia in peripheral blood. Despite a downregulation of expressed L-selectin, cattle treated with dexamethasone showed more uterine PMN than those treated with placebo. Dexamethasone decreased plasma concentrations of the immunomodulatory steroidal hormones cortisol and estrogen. Dexamethasone directly reduced the generation of reactive oxygen species (ROS) by uterine PMN. This may be a useful mechanism since it would protect the endometrium from tissue damage by excessive extracellular ROS. However, it is not known if the net effect is in fact a reduction in ROS, as the number of uterine cells increases. Our study shows that glucocorticoids may not be considered immunosuppressive in all cases and may play an important role in the regulation of post partum uterine defense mechanisms.

Alpha-1-acid glycoprotein inhibits phorbol ester-induced but not Fc-receptor-induced generation of reactive oxygen species in bovine peripheral blood neutrophils.

Alpha-1-acid glycoprotein (AGP) is an acute-phase protein with anti-inflammatory and immunomodulating properties. AGP is described as a potent inhibitor of the production of reactive oxygen species (ROS) in human neutrophils. However, published reports about the mechanism of inhibition are conflicting. The influence of bovine AGP on the production of ROS by bovine peripheral blood polymorphonuclear leucocytes (PMN) was studied using a highly sensitive method approaching its inhibitory mechanism. ROS production in PMN was induced with phorbol 12-myristate 13-acetate (PMA) or opsonized Staphylococcus aureus bacteria. ROS generation was quantified and evaluated by flow cytometry. AGP efficiently suppressed PMA, but did not opsonize bacteria-induced ROS generation in vitro. The suppressive effect was concentration-dependent and adversely proportional to PMA concentration. The selective inhibitory potential of AGP in comparison with ovalbumin (OVA) and bovine serum albumin (BSA) showed that ROS inhibition was not a mere protein effect. ROS production was suppressed only if AGP and PMA were simultaneously present with PMN. Pre-incubation of PMN with AGP did not alter the PMN response to PMA. Moreover, AGP could not suppress ROS production after pre-stimulation of PMN with PMA. Human and bovine AGP did not differ in their inhibitory potential to the PMA-induced ROS production in bovine, human and equine PMN. The results show that AGP does not modulate bovine neutrophil functions directly, but acts as a scavenger of PMA.

Some immune parameters in carp cyprinus Carpio susceptible and resistant to the haemoflagellate Trypanoplasma borreli.

The present study addresses aspects of the (specific) immune response of carp to the haemoflagellate Trypanoplasma borreli. Sera of resistant carp contained antibodies, which agglutinated the flagellates in vitro. When flagellates were incubated in fish sera from resistant carp, binding of antibodies to flagellates could be demonstrated by flow cytometry, and T. borreli were effectively killed. Heat-treatment of the sera prevented killing, indicating that complement activation is important for the control of a T. borreli infection. Sera of carp that were highly susceptible to infection with T. borreli contained no antibodies capable of binding to or killing the parasite. Furthermore, specific antibodies were not generated after experimental infection. This lack of antibody production in susceptible carp is not due to a general unresponsiveness of lymphoid cells, since peripheral blood leukocytes (PBL) from susceptible and resistant carp responded to mitogenic stimuli in vitro with lymphocyte proliferation in a similar manner. However, viable flagellates were significantly less able to stimulate proliferation of PBL from susceptible carp. In vitro-produced culture supernatants of freshly isolated PBL from both carp lines (but not those of head kidney cells) differentially modulated the mitogen-induced proliferation of PBL from susceptible and resistant carp. The supernatants enhanced the proliferation of leukocytes obtained from individuals from the same carp line, but suppressed the mitogen-induced proliferation of PBL from the other line tested. This indicates that lymphoid cells from susceptible and resistant carp differ in their spectrum of spontaneously produced immunomodulatory mediators. Whether this is decisive for a T. borreli-specific and successful immune response is discussed.

Degenerative endometrial changes do not change the functional capacity of immigrating uterine neutrophils in mares.

An endometritis model was used to investigate the influence of degenerative endometrial changes (endometrosis) on functional parameters of uterine neutrophils in the horse. Six hours after intrauterine application of recombinant human interleukin-8 (rhIL-8), the uteri of 15 mares were flushed with phosphate-buffered saline. Quantitative and qualitative flow cytometric assays were then made to determine the absolute numbers, viability, phenotype, generation of reactive oxygen species (ROS), and phagocytic activity of immigrated polymorphonuclear neutrophilic granulocytes (PMN). Recombinant hIL-8 attracted similarly high numbers of similarly viable PMN into the uteri of mares with or without degenerative endometrial changes. Compared with blood PMN, immigrated uterine neutrophils displayed significantly upregulated expression of CD11a/CD18 (LFA-1) on uterine PMN whereas major histocompatibility complex class I molecules were expressed at lower densities. The ability to phagocytose opsonized streptococci did not differ between uterine and blood PMN. However, uterine PMN displayed a higher capacity to generate ROS. On average, uterine PMN of mares with degenerative endometrial changes showed phenotypical and functional characteristics similar to those of mares with a histologically healthy endometrium. Therefore, degenerative endometrial changes per se did not reduce the functional capacity of equine uterine neutrophils in mares.

[The prevalence of Actinobaculum suis in boars of breeding herds in the Omsk region (Russian Federation) by indirect immunofluorescence technique]. / Untersuchung zur Verbreitung von Actinobaculum suis unter den Ebern in den Schweinezuchtbetrieben des Gebiets Omsk (Russische Föderation) unter Verwendung der indirekten Immunofluoreszenz-Technik.

Actinobaculum suis (Corynebacterium suis, Eubacterium suis, Actinomyces suis) was detected in the preputial diverticulum of 64,8% of 162 boars investigated in 8 districts of the region Omsk (Russian Federation) by indirect immunofluorescent technique. Until yet no informations were available about the prevalence of Actinobaculum (A.) suis in swine herds of the Russian Federation. The study shows that A. suis, as a main aetiological factor of cystitis and pyelonephritis in sows, is widely spread among the boars of the region Omsk. Prevalence of A. suis was not influenced by housing conditions, age or breed of investigated boars. Indirect immunofluorescent technique for detection of A. suis provides a good method for screening investigations with high numbers of samples.

Isolation and cryopreservation of functionally competent equine leucocytes.

Sufficient numbers of functionally competent polymorphonuclear neutrophil granulocytes (PMN) seem to be of major importance during the course of equine endometritis. In this study, we wanted to establish a method for cryopreservation of functionally competent neutrophils for an intended local endometritis therapy in mares. The separation of leucocytes by hypotonic lysis of whole blood from clinically healthy mares was superior to the separation by dextrose sedimentation. After suspension of the cells in the cryoprotective solution [equine plasma with 5% (v/v) dimethyl sulphoxide (DMSO)], the leucocytes were frozen in liquid nitrogen. A temperature gradient with low cooling velocity (1 degree C/min between 4 and -70 degrees C) resulted in highest numbers of viable cells after thawing. Thawed PMN had a high phagocytic capacity for opsonized streptococci. Their ability to generate reactive oxygen species (ROS) after stimulation with a phorbol ester was even higher than that of freshly isolated PMN and was preserved up to 6 h after thawing. The results of this study indicate that cryopreservation of PMN may provide viable and functionally competent neutrophils for therapeutic use in mares susceptible to endometritis.

Development and comparison of in vivo and in vitro models for endometritis in cows and mares.

In order to investigate pathogenic mechanisms of acute endometritis in cows and mares, we established an in vivo model in both species. Based on the results of an in vitro transmigration system, human recombinant interleukin-8 (rhIL-8; 1.25 microg per mare and 5 microg per cow in 50 ml phosphate-buffered saline) was used to attract polymorphonuclear neutrophil granulocytes (PMNs) into the uteri. Peak numbers of uterine neutrophils were attracted after 6h, in both cows and mares. On average, mares responded more sensitively than cows, with 15 times higher numbers of rhIL-8-attracted uterine neutrophils (72+/-8 x 10(7)cells). In contrast to in vitro studies, in vivo migrated neutrophils (uterine neutrophils) of both species displayed a significantly reduced MHC class I expression. Expression of the CD11a molecule was significantly enhanced on equine uterine neutrophils but downregulated on bovine cells. Compared with untreated autologous peripheral neutrophils, both uterine and in vitro migrated neutrophils showed no alteration of phagocytic capacity. The ability to generate reactive oxygen species (ROS) was significantly upregulated in bovine and equine uterine neutrophils. This was also observed after in vitro migration of equine neutrophils, whereas ROS generation by bovine neutrophils was significantly depressed. In summary, the concept of inducing endometritis directly by local application of human interleukin-8 has been reliably successful in cows and mares. The model permits the analysis of PMN migration into the uterus under defined and controlled conditions. The observed differences between cows and mares with respect to phenotypical and functional characteristics of in vivo attracted uterine cells point to species-related features of neutrophil migration. In vitro transmigrated bovine and equine cells partially differ in phenotype and function from uterine neutrophils. Therefore, the in vitro transmigration assay cannot completely represent the in vivo endometritis model described here.

The haemoflagellate Trypanoplasma borreli induces the production of nitric oxide, which is associated with modulation of carp (Cyprinus carpio L.) leucocyte functions.

In an attempt to characterise the role of nitric oxide (NO) in immune responses of carp, carp leucocytes obtained during an acute T. borreli infection were examined, for their capacity to generate NO. In a second set of experiments the impact NO on viability of the parasite and on the modulation of functional carp leucocyte responses were tested in vitro. Both in carp head-kidneys and in the peripheral blood, the fractions of lymphoblasts among separated leucocytes were increased. However, the relative proportions of granulocytes among head-kidney leucocytes (HKL) significantly decreased during infection, whereas granulocytes appeared among peripheral blood leucocytes (PBL). The cellular dynamics of HKL and PBL of infected carp were paralleled by an enhanced spontaneous NO release in vitro. NO production was further increased after addition of viable parasites to these cultures. The hypothesis that NO had a possible role in granulocyte activation and lymphocyte proliferation in carp was supported by the reduction of mitogen-induced proliferative responses of PBL from healthy carp in the presence of NO donor substances. The negative effects of NO on lymphocyte proliferation were contrasted by enhancing effects on granulocyte functions: the inhibition of NO generation in T. borreli-stimulated HKL cultures by the l-arginine analogue L-NMMA reduced the viability of granulocytes and their phagocytic activity. Even massive amounts of nitric oxide produced by donor substances (up to 600 micromol l(-1) NO(-)(2)) caused no reduction in the numbers of viable T. borreli flagellates in vitro. Thus, in carp, T. borreli seems to induce high amounts of NO in vivo which are apparently not harmful for the parasite but which may interfere with co-ordinated interactions of activated cells aiming at the defence of the parasite.

Head kidney neutrophils of carp (Cyprinus carpio L.) are functionally modulated by the haemoflagellateTrypanoplasma borreli.

In the present work responses of carp (Cyprinus carpio) head kidney-derived neutrophils to the blood parasite T. borreli, and the consequences of these responses for parasite survival and other host response mechanisms, were studied. In co-cultures of head kidney leucocytes (HKL) with viable and lysed T. borreli a prominent shape change of neutrophilic granulocytes towards increased size and complexity was observed. In addition, the longevity of neutrophils in vitro was prolonged in the presence of T. borreli antigens. In these cultures, neutrophils also exhibited an increased phagocytosis activity. An up regulation of the production of nitric oxide (NO) and reactive oxygen species (ROS) was observed in T. borreli- and mitogen-stimulated HKL cultures. However, addition of live, fluorescence-labelledT. borreli to previously stimulated HKL cultures, revealed neither killing nor phagocytosis of the parasite by activated neutrophils. Moreover, viable T. borreli, when added to HKL cultures of infected carp, reduced their phagocytosis activity and NO production. Supernatants of co-cultures between T. borreli and HKL also contained mediators, which suppressed a mitogen-induced proliferative response of peripheral blood leucocytes (PBL) in vitro. Thus, while T. borreli itself appeared not to be sensitive to responses of activated neutrophils, the flagellates interferes with the production of immunomodulatory signals of these cells, probably resulting in a partial immunosuppression, which may favour the parasite development in vivo.

Expression of a 4-(hydroxy-3-nitro-phenyl) acetyl (NP) specific equi-murine IgE antibody that mediates histamine release in vitro and a type I skin reaction in vivo.

Due to characteristic clinical signs, immunoglobulins of isotype E (IgE) are believed to be involved in several allergic diseases of the horse. To date, closer investigations have been hampered by the fact that neither purified equine IgE nor anti-equine IgE monoclonal antibodies were available for IgE isotype determination. As an approach to solve this problem, we constructed a stable cell line (EqE6) that expresses recombinant equi-murine IgE specific for 4-(hydroxy-3-nitro-phenyl) acetyl (NP). Biochemical analysis of the purified protein revealed a highly glycosilated IgE monomer of approximately 230,000 Da. The biological ability of the NP-IgE to mediate histamine release after crosslinking with antigen was demonstrated in vitro using equine blood leucocytes. In vivo, the intradermal application of NP-IgE followed by antigen crosslinking induced a type I hypersensitivity skin reaction in horses. Both results indicate that the recombinant NP-IgE contains an intact and functional Fc(epsilon) RI binding site and mediates effector functions in equine basophils and cutaneous mast cells. This equi-murine IgE can be used for the production of IgE-specific polyclonal and monoclonal antibodies. In addition, the NP specificity allows the antigen-specific activation of equine Fc(epsilon)-receptor-expressing cells, such as mast cells and basophils. This property could be used to investigate IgE-mediated mechanisms for a better understanding of equine type I allergic diseases.

Flow cytometric testing of immunological effects of a phytomedicinal combination (equimun) and its compounds on bovine leucocytes.

One of the major goals of this study was to establish fast, reliable and sensitive assays for the quality control of immunomodulating phytopreparations and to determine whether pharmacological compounds or phytopreparations have effects on bovine immune cells. Flow cytometric methods were chosen because they are very sensitive in the detection of even subtle effects on cells. In this study, we addressed the question of whether these methods are useful in monitoring the effects of EquiMun and its compounds on bovine leucocytes in vitro. EquiMun is a fixed combination of Echinacea purpurea (Ec), Thuja occidentalis (Th) and elemental phosphorus (Ph) in different starting concentrations. Separated blood mononuclear cells (MNC) and polymorphonuclear cells (PMN, mainly neutrophils) were cultured for up to 44 h in vitro in the presence or absence of the tested substances. Whereas MNC were not affected by any of the compounds, EquiMun, Ec, Th and Ph significantly reduced the forward scatter (size) of cultured PMN without affecting their side scatter (granularity). The size effects were paralleled by a significantly enhanced viability of PMN after 20 h in culture. The observed effects were constant over wide concentration ranges and indicate a very similar reaction of leucocytes from individual cows. Whereas spontaneous generation of reactive oxygen species (ROS) by neutrophils was up-regulated by Ph and EquiMun, EquiMun down-regulated the phorbol ester-stimulated ROS production. However, ROS generation by neutrophils displayed a large inter-individual variation with less apparent, down-regulatory effects of EquiMun. The ability of PMN to kill target cells via antibody-independent cellular cytotoxicity showed small inter-individual variations and was enhanced by Ec and Th but not by Ph and EquiMun, probably due to dose-dependent effects. In summary, the flow cytometric characterization of cellular viability and shape changes of neutrophils seem to be a suitable and reliable approach for the quality test of immunomodulating phytomedicines based on bioassays.

Lochial secretions of Escherichia coli- or Arcanobacterium pyogenes-infected bovine uteri modulate the phenotype and the functional capacity of neutrophilic granulocytes.

It has been suggested that in cases of puerperal endometritis of cattle infected with Escherichia coli and Arcanobacterium pyogenes, the neutrophils are compromised in their defense capacity or downregulated functionally. In addition to direct bacterial effects, contents of lochial secretions and secreted products of locally activated polymorphonuclear neutrophilic granulocytes (PMNs) may also account for changes in function of freshly immigrating neutrophils. In this study, lochial secretions were obtained from healthy cows and from cows infected by E. coli or A. pyogenes. Separated uterine PMN of infected cows displayed an altered phenotype and function which correlated with the degree of bacterial contamination. Concurrently tested circulating PMN showed no such changes. Infected lochial secretions sterilized by filtration also changed the phenotype of blood PMN. Lochial secretions of healthy cows displayed only minor effects. The effects on PMN function in infected cows varied: ingestion was less affected, whereas generation of reactive oxygen species (ROS) was severely depressed. Concurrently tested purified bacterial products (solubles and fragments) of E. coli and A. pyogenes did not induce the phenotypical and functional changes observed in blood PMN. Since infected lochia also contained high numbers of immigrated and probably activated PMN, the influence of supernatants from phorbol myristate acetate-activated PMN were tested on freshly isolated blood PMN. Such supernatants also increased the expression of certain surface molecules and inhibited the ROS generation. Thus, reduced function and altered phenotypes of PMN which immigrate into the uteri of cows with bacterial endometritis is due not only to interactions with bacteria or bacterial products, but is also to the uterine milieu.

Products of fourth-stage larvae of Oesophagostomum dentatum induce proliferation in naïve porcine mononuclear cells.

Infection of pigs with Oesophagostomum dentatum is a major cause of economic losses in pig productions. Whether infection with this nematode results in a protective immunity is still in debate and information about immune-modulating properties of O. dentatum are lacking. The present study investigated the question whether products of O. dentatum larvae modulate the proliferative response of porcine blood mononuclear cells (poMNC) in vitro. The poMNC of naïve and O. dentatum-infected pigs were cultured for 72 h in the presence of products (total homogenates and culture supernates) derived from third- (L3) and fourth-stage larvae (L4) of O. dentatum. Numbers of vital cells and blast-transformed cells were determined flow cytometrically. No larvae product induced an accelerated death of poMNC in vitro. In contrast, products of L4 (but not L3) significantly increased the numbers of vital poMNC in vitro (up to 187%). In addition, L4 products (homogenates and supernates, 0.1-10 microg/ml) but not those of L3 induced significant blastogenesis of poMNC. This was seen with poMNC from naïve and from O. dentatum-infected animals. In spite of these effects, the larvae products were not able to modulate the mitogen-induced (Concanavalin A) poMNC proliferation of naïve and infected animals. In summary, larvae of O. dentatum contain and secrete products with potential immunomodulatory capacity for porcine peripheral blood mononuclear cells. The differential effects of L3 and indicate that the parasite alters its set immunomodulatory substances during its development. This has to be considered in further studies and may help to identify the mediators involved.

Nucleotide sequence and restriction fragment length polymorphisms of the equine Cvarepsilon gene.

IgE is the dominant immunoglobulin isotype involved in type I hypersensitivities in mammals. The heavy chain constant region domains of equine IgE are encoded by a single gene, the Cvarepsilon gene. By restriction analysis of cDNA from 15 unrelated horses, we have now identified two Cvarepsilon alleles, characterised by a Sma I restriction fragment length polymorphism, which we designated Cvarepsilon(a) and Cvarepsilon(b). Sequence analysis of both, Cvarepsilon(a) and Cvarepsilon(b) cDNA, showed in addition two single base exchanges resulting in two amino acid substitutions. Both sequences have only 95.9% homology of the coding region sequence with the published equine Cvarepsilon sequence, which could represent a third haplotype. Polymorphism of the IgE heavy chain constant region gene, as described here, might well impose genetic variability on the effector functions of equine IgE predisposition to allergic diseases in horses.

Flow cytometric analysis of mitogen-induced activation of rainbow trout (Oncorhynchus mykiss) peripheral blood leucocytes.

Proliferation of rainbow trout peripheral blood leucocytes in vitro is usually assessed by measuring incorporated tritiated thymidine. In this report we monitored the in vitro proliferative response to the mitogen Concanavalin A (Con A) by means of flow cytometry (FCM) and 3H-thymidine incorporation. When analysed by FCM, blood leucocytes displayed two main cell populations with distinct forward and side scatter (FSC/SSC) characteristics: lymphocytes with low FSC/SSC values and non-lymphoid leucocytes (NLL) with increased FSC/SSC values. The nature of these cell types were confirmed by microscopy. Interestingly, the FSC/SSC pattern of lymphocytes remained unchanged after in vitro stimulation with Con A, whereas cells from the NLL population showed a marked shift towards increased FSC values. In stimulated cultures, the increase of FSC values of the NLL population significantly correlated with contemporarily measured 3H-thymidine incorporation (r = 0.7, P < 0.001). The mitogenic response of blood leucocytes originating from different individual fish varied over wide ranges. It was found to be related to the numbers of NLL present in the leucocyte sample. The present results show that qualitative and quantitative FCM analysis of morphological parameters (FSC/SSC) of blood leucocytes makes it possible to discriminate between leucocyte populations of the rainbow trout and to monitor cell proliferation experiments.

Characterization of leukocytotoxic and superantigen-like factors produced by Staphylococcus aureus isolates from milk of cows with mastitis.

Staphylococcus aureus is a major pathogen for cattle, causing various forms of subclinical and clinical mastitis. Two groups of virulence factors (leukotoxins and superantigens) are supposed to play an important role in the initiation and/or the exacerbation of this disease. In order to detect all known and putative members of leukotoxins and SAgs (superantigens), we tested secreted factors of different S. aureus isolates in flow cytometry-based assays. Isolates were sampled from 68 cows of different farms and cultured for 24h in vitro. Supernatants were then coincubated with purified polymorphonuclear granulocytes (PMN) or combinations of blood mononuclear cells (MNC) and PMN. Viable PMN and MNC were determined by quantitative flow cytometry. In addition, we recorded the proliferation-inducing potential of isolate supernatants for bovine MNC. Based on these criteria, the supernatants of S. aureus isolates fell in three groups. The first group (n=32), termed LT-SNs (leukotoxin-containing supernatants), killed purified granulocytes (neutrophils and eosinophils) in vitro. The second group of supernatants (n=20), termed SAg-SN (superantigen-containing supernatants), induced activation and proliferation of mononuclear cells (MNC) and, only in the presence of MNC, resulted in a selective depletion of neutrophils after 24h in vitro. The third group of supernatants (n=16) contained neither LTs or SAgs. Functionally, SAg-SNs behaved like purified staphylococcal enterotoxin A (SEA) or SEB tested in parallel. The absence of SAg-like activity in LT-SNs was confirmed by heat treatment of LT-SNs, which destroyed the leukocytotoxic activity, but did not reveal any MNC-activating potential. This study, therefore, suggests, that pathogenic S. aureus isolates either produce leukotoxins or superantigens and that both groups of virulence factors can easily be differentiated by the functional assays described. The prevalence of leukotoxin- or superantigen-producing isolates was comparable among cattle with subclinical (LT=41%; SAg=30.8%) mastitis. The higher frequency of LT-producing isolates in cases of clinical mastitis (LT=55.2%; SAg=27.6%) was not significant. At least, these findings argue against the dominant role of superantigens or leukotoxins in S. aureus-induced bovine mastitis.