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Infection of rabbit mammary glands with ovine mastitis bacterial strains
j. Comp. Path. I991 Vol. 104
I n f e c t i o n o f R a b b i t M a m m a r y Glands w i t h Ovine M a s t i t i s Bacterial Strains B. Amorena*, J. A. Garcia de Jal6nt, R. Baselga*, J. Ducha~, M. V. Latre t, L. M. Ferrer +, F. Sancho~, I. MAnsson§, K. Krovacek§ and A. Faris§ * EEAD ( CSIC) , Departmenl of Anirnal Production, SIA ( DGA ) , Ap. 727, 50080 garagoza, "~Deparlmem of Animal Pathology, Veterinary Faculty, 50013 ~aragoza, {.F.S. Limiled, San Marco, Zaragoza, Spain, and § Deparltnent of Veterinary Microbiology, College of Vaerinary Medicine, Swedish University of Agricultural Sciences, Biomedicum Box 583, Uppsala, Sweden
Summary An experimental model was developed in rabbits to study ovine mastitis. A total of 19 ovine mastitis bacterial strains (seven Slap/!j,lococcus aureus, four Slaph. chromogenes, four Slaph. hyicus and four Escherichia coli) were used for mammary gland infections. The histopathological results showed that the ovine mastitis types corresponded to experimental infections produced in the rabbit with the ovine strains. These results helped the grading of the bacterial species tested according to the severity of their effects on the mammary gland. The most pathogenic species was Slaph. aureus, [bllowed by E. coli, Slaph. hyicus and Slaph. chromogenes, in that order. There was, however, variation among strains within a given species (e.g. one out of seven Slaph. aureus strains gave rise to a mild infection in sheep and rabbits). The procedure was simple and consisted of introducing bacterial suspensions through alternate teat ducts of does with the help ofa cannula. It helped minimize the number of animals required in the experiments.
Introduction Cattle and sheep mastitis causes serious production losses. Mammary gland infection studies with cattle and sheep mastitis bacterial strains have been carried out in various animal species. The use of cattle (Plommet, 1959; Heald, 1979; Nickerson and Heald, 1981; Frost and Hill, 1982; Hill, Frost and Brooker, 1984) or sheep (Legall and Plommet, 1965; Fthenakis and Jones, 1990) as recipients has the inconvenience of a high individual cost. In studies of bovine mastitis, special mouse strains (BSVS) have been used as animal models (Chandler, 1970; Anderson, 1977), but the teat duct inoculation procedure applied in these cases requires expertise. The implied difficulties may be circumvented by the use of other procedures. Anderson (1972) and Anderson and Holmberg (1977), after cutting off the tip of the mouse teat, placed bacterial suspensions in contact with the gland to provoke infection. Adlam, Thorley, Ward, Collins, Lucken and Knight (1976) and Adlam, Ward, McCartney, Arbuthnott and Thorley (1977), after carrying out injections into 0021-9975/91/030289+ 14 $03.00/0
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rabbit m a m m a r y tissue, succeeded in producing gangrenous mastitis and chronic abscesses in some of the inoculated glands. When using the rabbit for experimental infections with ruminant mastitis bacteria, there are, however, several pathological and methodological aspects, not studied so far, that deserve investigation: (a) comparison of pathogenic effects caused by different bacterial species of a given genus (e.g. Staphylococcus) and of various strains within each bacterial species (E. coli, Slaph. aureus, etc.); (b) possibility of reproducing the various types of ruminant mastitis in the rabbit model; and (c) evaluation of the adequacy of a rabbit experimental model for ovine mastitis studies, using bacterial inoculations via the teat duct, as was done in mice (Chandler, 1970; Anderson, 1972, 1977), a procedure which may facilitate infection without teat damage. Rabbits are cheaper and have a greater number of teats than ruminants. Hence, they may help to reduce the cost and the number of animals involved in these studies. They are also larger than mice and may thus be easier to handle for i n t r a m a m m a r y infections via the teat duct. These considerations as well as the fact that sheep mastitis has been less extensively studied than cattle mastitis, motivated us to investigate the three aspects, (a) to (c), mentioned above. The study provides a convenient model for fundamental assays on mastitis, prior to final assessment in ruminant species. M a t e r i a l s and M e t h o d s
Bacteria usedfor Inoculation Nineteen ovine mastitis strains were obtained for inoculations (seven Staphylococcus aureus, four Staph. chromogenes, four Staph. hyicus and four Escherichia coli). Bacteria were grown in Todd-Hewitt broth (THB; 37°C, 24 h). Subsequently, they were washed twice in phosphate buffered saline (PBS; pH 7"2). The concentration was adjusted to 107 viable bacteria per ml PBS (bacteria were plated at this stage for viable count).
Animals Ten Rasa Aragonesa ewes (Table 1) with various types of clinical mastitis, mammary abscesses or apparently normal mammary glands according to clinical criteria, were used for histopathological studies and as source of naturally produced ovine mastitis strains. For experimental infections, four healthy multiparous Rasa Aragonesa ewes were inoculated 24 to 48 h post-parturition. Seven females of the Spanish White Giant rabbit breed were also used for inoculations 24 to 48 h post-parturition. This breed did not exhibit any special mammary gland characteristics and was randomly chosen among rabbit breeds for this study. The mothers were separated from their offspring in all cases. On the day before inoculation (always after parturition), each animal was examined clinically (special attention being paid to the mammary glands). Milk was c~ltured this day and the day of infection to insure that the animals were free of bacteria.
Selection of Bacterial Dosefor inoculation To select the bacterial dose to be used in the rabbit infections, a titration was carried out in a female rabbit with strain 5A (Staph. aureus), by introducing 0"5 ml of suspension containing 5 x 102, 5 x 104 and 5 x 106 viable bacteria through the teat ducts of alternate glands with a cannula. The latter inoculum was the only one that produced mastitis, based on observations at 24 and 48 h. Hence, 5 x 106 viable bacteria in 0'5 ml PBS per rabbit per teat was considered an appropriate dose for rabbit
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infection experiments, intentionally designed for the animals to develop infection in a short time. Considering the relative size and physiology of sheep and rabbit mammary glands, an inoculum of 10 7 bacteria per sheep per teat was chosen for the sheep con trois used in the study.
Inoculation Technique Prior to infection, females were exhaustively milked. Subsequently, in the case of" rabbits, 0'5 ml of the bacterial suspension (5 x 106 cfu) was introduced through each teat duct, into alternate glands, through a 27G eannula. The same volume of sterile PBS was introduced as control through the remaining teat ducts (i.e. each rabbit was used for three strains and three PBS controls; in general, the remaining two teats were too small and thus not suitable for inoculations). The glands were gently massaged while pressing the teat to avoid leakage. For sheep inoculations, a similar procedure was applied, except that in this case, 1 ml of bacterial suspension (10~cfia) was inoculated into one gland of the animal (through a 21G eannula), leaving the other gland as a control in which 1 ml PBS was introduced. The specific bacterial strains used for the sheep inoculations were 1A (Staph. aureus), 8B (Staph. chrornogenes), 6A (Slaph. tzyicus) and 7B (E. coli).
Pathology of Experimentally-infected Glands Swelling and induration of inoculated mammary glands were evaluated 24 and 48 h after infection. The reactions were scored from . . . . . (lack of reaction) or "( + )" (very weak reaction) to " + + + + " (very strong reaction). Following the second evaluation (48 h), the animals were killed and the mammary glands were removed and stored in 10 per cent buffered formalin. These tissues were embedded in paraffin wax, sectioned 5 gm thick and stained with haematoxylin and eosin (HE) and Gram's method for histopathological examination. Comparative histological grading of mammary lesions (generalized or focal necrosis, oedema, haemorrhage, thrombosis etc.), originated by the different strains, ranged from " - " to " + + + " according to the severity of the reaction.
Bacteriological Assays Bacterial culture filtrates were prepared according to the procedure ofKrovacek, Faris and M~msson (1987) with a modification (incubation of bacteria in a rotary incubator was done in THB at 37°C for 24 h). These filtrates were used for rabbit skin vascular permeability factor assays (rabbit skin tests) and cytotoxicity assays on Chinese hamster ovary (CHO) cells, carried out as described by Krovacek et al. (1987). The production of alpha- and beta-haemolysins, coagulase, protease, DNAase, protein A and clumping factor was determined according to standard procedures (Larpent and Larpent-Gourgaud, 1985).
Results
Natural Ovine Mastitis and Rabbit Experimental Infections As shown in T a b l e 1, the 10 n a t u r a l cases o f o v i n e mastitis were classified at the time o f d e a t h according to histopathological criteria and subsequently comp a r e d with experimentally infected rabbits. Both sides of the ovine gland (left and right) were e x a m i n e d independently. Similarly, when rabbits were infected with the ,bacterial strains isolated from these 10 sheep, each teat was e x a m i n e d independently. Mastitis was reproduced in all cases (no infection
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failures were observed), and external observations could be evaluated as early as 24 h after inoculation, Following infection with Slaph. aureus strains, the rabbit mammary glands inoculated with strains 1A, 2A, 4A, 5A and 7C showed at 24 and 48 h after inoculation, intense tumescence and induration, being warmer than normal glands and painful upon palpation (Table 1, Fig. 1). The histopathological study of this material revealed the presence of gangrenous mastitis, characterized by a massive necrosis of the mammary parenchyma (Fig. 2). The processes of congestion, oedema, haemorrhage and vascular thrombosis were active in all cases. Inflammation, indicated by the presence of neutrophils, was mild in infections with strains 1A, 4A and 7C and intense in those with strains 2A and 5A (Table 2). In all cases, large bacterial aggregates, intensely basophilic, were found throughout the m a m m a r y tissue (Fig. 2). Strain 4A showed a tendency to adhere to the epithelial cells of excretory ducts and alveoli. Infection With strain 8C, isolated from a case of acute catarrhal mastitis, gave rise to an intense tumescence of the rabbit gland at 24 and 48 h after inoculation. Microscopically, a severe degeneration of alveolar epithelial cells, a mild necrosis and an intense inflammatory infiltration of neutrophils were observed (Table 2). Finally, strain 9B obtained from a non-mastitic gland with an occluded teat (9R; Table 1), caused mild macroscopic alterations and the microscopic examination revealed very mild vascular and inflammatory alterations. Infections with this strain and with 8C resulted in the presence of few bacteria and absence of bacterial aggregates (Table 2). Rabbit infections with Staph. chromogenes caused a slight or moderate external reaction at 24 and 48 h after inoculation in the case of strains 3A and 5B, but no external alterations were observed with strains 7D or 8B. There were slight histopathological differences between these strains (Table 2), but in general~ they produced a mild or medium catarrhal mastitis, localized in the excretory ducts and surrounding glandular alveoli. The most characteristic lesions consisted of intra-alveolar neutrophil infiltration, neutrophil lysis, degeneration of epithelial cells and destruction of some alveoli (Fig. 3). Strain 3A was more aggressive than the others, causing hyperaemia and intense haemorrhage (Table 2). Staph. hyicus strains 6A, 6B, 10B and 10C gave rise, in rabbits, to a mild (or nonexistent) macroscopic reaction in the inoculated rabbits (Table 1). However, from histopathological examination, all strains were associated with the presence of coagulation, necrotic loci and destruction of epithelial cells of ducts and alveoli. There was also destruction of many alveoli and massive intra- and extra-alveolar neutrophil infiltrations. In the alveoli and excretory duct lumens, there was abundant epithelial and neutrophil cell debris and a small number of moderately basophilic bacteria, which did not form aggregates (Fig. 4). Infection with E. coli strains 7A, 7B, 9A and 10A caused a mild to moderate external modification of the rabbit glands (Tables 1 and 2) which was histopathologically characterized by wide areas of coagulation necrosis, having destroyed alveoli in the centre, forming a homogenous and eosinophilic mass together with epithelial and inflammatory cell lysates and debris. These
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Fig.
Comparative images illustrating m a m m a r y glands fi'om a healthy doe in the lactation period (top) and from a doe infected with Slaph. attreus strain 2A (bottom). Pronounced tumescence, oedema and haemorrhage at the infection site are observed in the latter, as well as a size o['approximately twice the normal.
Fig. 2.
Histological [)'atures of a rabbit m a m m a r y gland inoculated with Slaph. aureus (IA): Extensive necrosis o[" m a m m a r y alveoli with clumps oF basophilic bacteria (arrows). HE x 150.
Fig. 3.
Histologieal [ieatures of a rabbit m a m m a r y gland inoculated with Slaph, chromogenes (3A): alveolar destruction with neutrophils in alveolar lumen. HE x 150.
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necrotic areas were surrounded mainly by healthy gland tissue (Fig. 5). A finding, characteristic of these strains, was the presence of blood vessel thrombosis. The number of bacteria found in these cases was reduced. Bacteria were often seen adherent to epithelial cells of ducts and alveoli in Gram-stained preparations.
Sheep Experimental Infections The macroscopic and microscopic study of the mammary gland of the sheep used in experimental infections (Table 1) revealed different types of mastitis: the ewe inoculated with strain 1A (Staph. aureus) developed a v e r y severe acute gangrenous mastitis; the ewe inoculated with strain 8B (Staph. chromogenes) suffered from a mild acute catarrhal mastitis; the ewe inoculated with strain 6A (Slaph. @icus) developed a catarrhal acute mastitis with small necrotic loci next to excretory ducts and alveoli and the one infected with strain 7B (E. colt) suffered from a definite acute catarrhal mastitis with large areas of coagulation necrosis. These histopathological findings were similar to those described in natural infections in sheep and/or in rabbit experimental infections.
Bacleriological assays The results obtained from the bacteriological assays showed that of the seven Siaph. aureus strains studied, three (1A, 2A and 5A) produced protein A , ['our (1A, 2A, 5A and 7C) produced alpha- and beta-haemolysins, two showed only a weak beta-haemolysin production (4A and 8C) and one (9B) did not produce alpha- or beta-haemolysins (Table 3). The latter strain showed (like strain 7C) protease activity, but in contrast with the remaining staphylococcal strains used in these tests, it did not show any clumping factor, coagulase or DNAase activities. Furthermore, the culture supernatant of this strain did not elicit a congestion or necrosis reaction in the rabbit skin test; in contrast with supernatants from other Slaph. aureus strains (1A, 2A, 4A, 5A and 7C, ill that order of strength; strain 8C was not tested), it was non-cytotoxic to CHO cells. It was found that the strains belonging to other bacterial species (coagulase negative staphylococci and E. colt), with the exception of Staph. chromogenes strain 8B and E. colt strain 9A, showed protease activity. DNAase activity was found for Slaph. chromogenes strains 3A, 5B and 7D (low activity) and for E. colt strains 10B and 10C (high activity). Only culture supernatants of one Slaph. chromogenes strain (7D) and two E. colt strains (7A and 7B) were cytotoxic for CHO. The culture supernatant of E. colt strain 9A produced a rabbit skin reaction (induration and necrosis), but no evidence of skin reaction was obtained for culture supernatants of the remaining non-Staph, aureus strains. Discussion
The bacterial species studied in this work represent the major bacterial agents involved in ovine mastitis, with the exception of Pasleurella haemolytica (Kimberling, 1988; Watson, Franklin, Davies, Kettlewell and Frost, 1990), which
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Fig. 4.
Histological ti:atures of a rabbit mammary gland inoculated with Slaph. Io,icus (6A): massive neutrophilic infiltration in interstitial tissue (I), in lumen of excretory ducts (left) arid ill alveoli (right). HE x 60.
Fig. 5.
Rabbit mammary gland inoculated with E. coli (IOA): An inflammatory cell bomldary (arrows) separates the area of coagulation necrosis (N) from the healthier glandular paeenchyn'm (rap), HE x 60.
Fig, 6.
Rabbit mamnmry gland inoculated with E. coli ( 10A): Blood vessel thrombosis ('I') and ueutrophilic infiltration in the interstitial spaces aad alveolar lumen (left). HE x 150.
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was not included in the study. All five Slaph. azzreus strains obtained from severe acute gangrenous, chronic purulent or abscess mastitis, reproduced extensive necrotic areas in the rabbit m a m m a r y gland. These findings are similar to those observed in experimental infections of cattle and sheep (Legall and PloNmet, 1965; Nickerson and Heald, 1981). The Slaph. aureus strain producing catarrhal mastitis in sheep (8C) also gave rise to a catarrhal mastitis in rabbits. The reason why this strain was pathogenically milder than other strains of this species is unknown. According to Loeffler, Greasy, Norcross and Paape (1988), Staph. aureus produces up to 30 extracellular products, primarily toxins and enzymes. The role of the products in the pathogenesis of ruminant staphylococcal mastitis is not well defined, with exceptions such as alpha-haemdysin, which is thought to play a key role in acute gangrenous staphylococcal mastitis. As may be gathered from Table 3, where the various classical bacteriological properties of each strain are illustrated, only a weak beta-haemolysin activity was associated with strain 8C. This weak haemolytic activity m a y help to explain why strain 8C is associated with a non-gangrenous mastltis. Strain 4A (also with a weak beta-haemolysin production), produced cistern abscesses in natural ovine infections and showed adhesiveness to the m a m m a r y gland epithelial tissue. Other cases of in vivo Staph. aureus adherence to ruminant epithelia have been described. Gudding, McDonald and Cheville (1984) observed attachment of Slaph. aureus strains to epithelial cells in bovine experimental infections. A question arises concerning whether adherence might have played a role in the pathogenesis of this particular ewe's m a m m a r y gland. Examining the history of this ewe (from which strain 4A was isolated), it was [bund that this animal had repeatedly suffered from mastitis and lack of milk production during five successive lambings. It is possible that adherence of bacteria to the m a m m a r y gland epithelial tissue was responsible for chronicity and that this affliction was not due to inefficacy of antibiotic treatment (strain 4A was sensitive to the majority of the common antibiotics tested in the antibiogram analysis carried out; data not shown). It might also be possible that leucocytes migrated to the places where adherent bacterial aggregates were present, resulting in the production of abscesses in the gland. Strain 9B, the least pathogenic of the Sl@h. aureus strains tested in this work (in rabbits and in sheep), did not show any haemolytic activity nor cytotoxic effects on C H O when culture filtrates were tested (Table 3). These filtrates were unstable when submitted to a freeze-thaw cycle and produced induration but not congestion or necrosis in the rabbit skin test. This strain was also clumping factor- and DNAase-negative. These characteristics were unique among the Slaph. aureus strains tested and may help understanding the strain's low pathogenicity. Jonsson, Lindberg, Haraldsson and WadstrSm (1985) suggested that, in mice, alpha-haemolysin and coagulase are virulence determinants of Slaph. aureus and that protein A is not an important virulence determinant of this species. Our results in rabbits are in agreement with these observations. It is tempting to draw conclusions on associations between each of the bacterial properties of the strains under study and the lesions produced. However, the number of sheep and rabbits used were low for association
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studies involving the high number of properties under study. Nonetheless, it appears that the role of protease production in mastitis is questionable, whereas the production of alpha- and beta-haemolysins, clumping factor, coagulase, DNAase or bacterial culture supernants which are positive in the rabbit skin tests and in CHO cell cytotoxicity tests, cannot be discarded as a possible virulence factor, when considering the data obtained. Staph. chromogenes and Slaph. hyicus strains, obtained from chronic mastitis or apparently healthy ovine glands were able to reproduce mild or moderate mastitis in rabbits. These findings help to determine the role of coagulasenegative staphylococci in subclinical mastitis of sheep (Fthenakis and Jones, 1990). The lesions caused by E. coli strains in the rabbit mammary gland, characterized by large areas of coagulation necrosis, are very similar to those produced by this bacterial species in cattle under natural and experimental conditions (Frost and Hill, 1982; Hazlett, Little, Maxie and Barnum, 1984; Hill et al., 1984). Although there is little information on ovine mastitis E. coli pathogenicity, in our opinion, this bacterial species could play several roles: (a) agent accompanying other bacteria (e.g. Slaph. aureus); (b) agent producing subclinical mastitis; and (c) primary major mastitis agent. The strong pathogenic effect of most Slaph. aureus and some of E. coli strains in both the experimental rabbit infections and the ovine natural inl~ctions, would be expected, knowing that in sheep these species are relatively major pathogens when compared to the other bacterial species of Table 1 (Jones, 1985; Hueston, Hartwing and Judy, 1986). The above considerations lead to the conclusion that the rabbit experimental model for infection with ovine mastitis strains helps to evaluate the degree of pathogenicity of different agents. In our case, we can classify the bacterial species from a higher to a lower mastitis pathogenic power, as follows: Staph. aureus, E. coli, Slaph. hyicus and Staph. chromogenes, in that order. These observations are in agreement with those of Bramley, King, Higgs and Neave (1979) with regard to the first two species. They found that, in cattle, for establishment of experimental infections, the number of cfu was higher for E. coli, when compared with Slaph. aureus. With respect to the types of mastitis found in experimental infections, we have classified mastitis into various categories, with terms that define the pathological status in each particular case: acute, chronic, mild, severe, catarrhal, gangrenous, purulent, with abscesses, etc. This classification could be carried out in natural and experimental sheep mastitis infections as well as in their rabbit experimental infection counterparts. Several authors have established sheep mastitis types. Plommet in 1959, using experimental sheep infections classified the types ofmastitis into three groups: type I, mild mastitis; type II, severe mastitis; and type III, gangrenous mastitis. These groups may well include the mastitis categories described in this work for the rabbit and sheep infections. When studying the similarity of results obtained in rabbits and in sheep, it was noticed that strains 5B and 8B ofSlaph, chromogenes and strain 7B orE. coli were isolated from the same ovine mammary gland sides (5L, 8L and 7L, respectively, Table 1) as the corresponding Slaph. aureus strains 5A, 8C and 7C.
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Hence, as expected, it was observed that the rabbit infection reactions with the purified strains 5B, 8B or 7B were milder than the natural infection reactions observed in sheep with the mentioned strain admixtures, containing Slaph. aureus strains in all cases. Altogether, these results show a clear correspondence between the various types of ovine natural or induced mastitis and the types of rabbit mastitis experimentally induced by the different ovine strains. Thus, rabbit infections m a y help predict the outcome of sheep infections. This makes the rabbit experimental model very convenient for ovine mastitis strain studies. The model has several additional advantages: (a) the inoculation technique is secure and easy, it does not require magnifying lenses; (b) the inflammation spread between glands is hardly detectable; and (c) three alternate glands can be infected in a given animal, which helps to minimize the number of animals used for experimentation. Although, with the proposed model, many aspects ofovine pathogenesis can be studied, there are several points that ought to be considered before extrapolating the findings in rabbits (with the single dose bacterial infections described) to sheep. For example, in natural infections, the dose of infecting bacteria may vary and different effects may accordingly be observed (Bramley el al., 1979). On the other hand, the number of somatic cells present in m a m m a r y gland secretions at the time of infection may be crucial for establishment of the infection (Bramley, 1976), but this information is not provided by the rabbit model. Finally, mammalian species-specific differences (sheep vs. rabbit) and individual genetic and physiological differences within these mammalian species cannot be evaluated when using the rabbit model alone, without carrying out concomitant infections in the ruminant species u n d e r study. For these reasons, in many cases, the rabbit experimental model proposed ought to be considered very helpful for preliminary studies of experiments on strain pathogenesis, to reduce costs and to avoid unnecessary waste of large animals, which could be used later for final assessment. Acknowledgments
This work was funded by Comisi6n Asesora de Investigaci6n Cientifica y T~cnica (Project No. PB86-0217), Consejo Asesor de Investigaci6n (DGA, Project No. PCA-6/87) and Diputaci6n General de Arag6n (Livestock Production Section). References
Adlam, C., Thorley, C. M., Ward, P. D., Collins, M., Lucken, R. N. and Knight, P. A. (1976). Natural and experimental mastitis in rabbits. Journal of Comparalive Pathology, 86, 581-593. Adlam, C., Ward, P. D., McCartney, C., Arbuthnott, J. P. and Thorley, C. M. (1977). Effect of immunization with highly purified alpha and beta toxins on staphylococcal mastitis in rabbits. Infection and Immunity, 17, 250-256. Anderson, J. C. (1972). The effect of teat damage on the incidence of experimental mastitis in the mouse. Research in Veterinary Science, 13, 390-392. Anderson, J.C. (1977). Experimental staphylococcal mastitis in the mouse: the induction of chronic mastitis and its response to antibiotic therapy. Journal of Comparative Pathology, 87, 611-621. Anderson, J. C. and Holmberg, O. (1977). The invasiveness of Staphylococcus aureus and
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Staphylococcus epidermidis for the mammary gland of the mouse. Acta Veterinaria Scandinavica, 18, 129-137. Bramley, A.J. (1976). Variations in the susceptibility of lactating and non-lactating bovine udders to infection when infused with Escherichia coll. Journal of Dairy Research, 43, 205-211. Bramley, A.J., King, J. S., Higgs, T. M. and Neave, F. K. (1979). Colonization of the bovine teat duct following inoculation with Staphylococcus aureus and Escherichia coll. British Veterinary Journal, 135, 149-161. Chandler, R.L. (1970). Experimental bacterial mastitis in the .mouse. Journal of Medical Microbiology, 3, 273-282. Frost, J. and Hill, A.W. (1982). Pathogenesis of experimental bovine mastitis following a small inoculum of Eseherichia coll. Research in Veterinary Science, 33, 105-112. Fthenakis, G.C. and Jones, J . E . T . (1990). The eft~et of inoculation of coagulase-negative staphylococci into the ovine mammary gland. Journal of Comparative Pathology, 102, 211-219. Gudding, R., McDonald, J.S. and Cheville, N.F. (1984). Pathogenesis of Staphylococcus aureus mastitis: bacteriologic, histologic and ultrastructural pathologic findings. American Journal of Veterinary Research, 45, 2525-2531. Hazlett, M.J., Little, P. B., Maxie, M. G. and Barnum, D. A. (1984). Fatal mastitis of dairy cows: A retrospective study. Canadian Journal of Comparative Medicine, 48, 125-129. Heald, C.W. (1979). Morphometric study of experimentally induced Staphylococcus aureus mastitis in the cow. American Journal of Veterinary Research, 40, 1294-1298. Hill, A.W., Frost, A.J. and Brooker, B.E. (1984). Progressive pathology of severe Escherichia coil mastitis in dairy cows. Research in Veterinary Science, 37, 179-187. Hueston, W.D., Hartwing, N.R. and Judy, J.K. (1986). Patterns of nonclinical intramammary infection in a ewe flock. Journal of American Veterinary Medical Association, 188, 170-172. Jones, J. E.T. (1985). An investigation of mastitis in sheep: preliminary phase. Proceedings of the Sheep Veterinary Society, 48-51. Jonsson, P., Lindberg, M., Haraldsson, I. and Wadstr6m, T. (1985). Virulence of Staphylococcus aureus in a mouse mastitis model: studies of alpha haemolysin, coagulase and protein A as possible virulence determinants with protoplast fusion and gene cloning. Infection and Immunity, 49, 765-769. Kimberling, C. V. (1988). Mastitis. In Diseases of sheep, 3rd Edit.Jensen and Swift, Eds, Lea and Febiger, Philadelphia, pp. 34-38. Krovacek, K., Faris, A. and MAnsson, !. (1987). Cytotoxic and skin permeability factors produced by Vibrio anguillarum. Aquaculture, 67, 87-91. Larpent, J.-P. and Larpent-Gourgaud, M. (1985). Manuel pratique de microbiologie. Collection Mdthodes. Hermann. Paris. 230 pp. Legall, A. and Plommet, M. (1965). Experimental mastitis in ewes. Annales de Biologic Animale, Biochimie et Biophysique, 5, 113-130. Loefller, D.A., Greasy, M.T., Norcross, N.L. and Paape, M.L. (1988). Enzyme-linked immunosorbent assay for detection of leukocidin toxin from Staphylococcus aureus in bovine milk samples. Journal of Clinical Microbiology, 27, 1331-1334. Nickerson, S.C. and Heald, C.W. (1981). Histopathologic response of the bovine mammary gland to experimentally induced S. aureus infection. American Journal of Veterinary Research, 42, 1351-1355. Plommet, M. (1959). Mammite staphylococcique de la brebis: Infection exp~rimentale. Annales de l'Institut Pasteur, 97, 440--441. Watson, D. L., Franklin, N. A., Davies, H. I., Kettlewell, P. and Frost, A.J. (1990). Survey of intrammary infections in ewes on the New England Tableland of New South Wales. Australian Veterinary Journal, 67, 6-8. I Received, August 16th, 1990 Accepted, November 14th, 1990_J
I n f e c t i o n o f R a b b i t M a m m a r y Glands w i t h Ovine M a s t i t i s Bacterial Strains B. Amorena*, J. A. Garcia de Jal6nt, R. Baselga*, J. Ducha~, M. V. Latre t, L. M. Ferrer +, F. Sancho~, I. MAnsson§, K. Krovacek§ and A. Faris§ * EEAD ( CSIC) , Departmenl of Anirnal Production, SIA ( DGA ) , Ap. 727, 50080 garagoza, "~Deparlmem of Animal Pathology, Veterinary Faculty, 50013 ~aragoza, {.F.S. Limiled, San Marco, Zaragoza, Spain, and § Deparltnent of Veterinary Microbiology, College of Vaerinary Medicine, Swedish University of Agricultural Sciences, Biomedicum Box 583, Uppsala, Sweden
Summary An experimental model was developed in rabbits to study ovine mastitis. A total of 19 ovine mastitis bacterial strains (seven Slap/!j,lococcus aureus, four Slaph. chromogenes, four Slaph. hyicus and four Escherichia coli) were used for mammary gland infections. The histopathological results showed that the ovine mastitis types corresponded to experimental infections produced in the rabbit with the ovine strains. These results helped the grading of the bacterial species tested according to the severity of their effects on the mammary gland. The most pathogenic species was Slaph. aureus, [bllowed by E. coli, Slaph. hyicus and Slaph. chromogenes, in that order. There was, however, variation among strains within a given species (e.g. one out of seven Slaph. aureus strains gave rise to a mild infection in sheep and rabbits). The procedure was simple and consisted of introducing bacterial suspensions through alternate teat ducts of does with the help ofa cannula. It helped minimize the number of animals required in the experiments.
Introduction Cattle and sheep mastitis causes serious production losses. Mammary gland infection studies with cattle and sheep mastitis bacterial strains have been carried out in various animal species. The use of cattle (Plommet, 1959; Heald, 1979; Nickerson and Heald, 1981; Frost and Hill, 1982; Hill, Frost and Brooker, 1984) or sheep (Legall and Plommet, 1965; Fthenakis and Jones, 1990) as recipients has the inconvenience of a high individual cost. In studies of bovine mastitis, special mouse strains (BSVS) have been used as animal models (Chandler, 1970; Anderson, 1977), but the teat duct inoculation procedure applied in these cases requires expertise. The implied difficulties may be circumvented by the use of other procedures. Anderson (1972) and Anderson and Holmberg (1977), after cutting off the tip of the mouse teat, placed bacterial suspensions in contact with the gland to provoke infection. Adlam, Thorley, Ward, Collins, Lucken and Knight (1976) and Adlam, Ward, McCartney, Arbuthnott and Thorley (1977), after carrying out injections into 0021-9975/91/030289+ 14 $03.00/0
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rabbit m a m m a r y tissue, succeeded in producing gangrenous mastitis and chronic abscesses in some of the inoculated glands. When using the rabbit for experimental infections with ruminant mastitis bacteria, there are, however, several pathological and methodological aspects, not studied so far, that deserve investigation: (a) comparison of pathogenic effects caused by different bacterial species of a given genus (e.g. Staphylococcus) and of various strains within each bacterial species (E. coli, Slaph. aureus, etc.); (b) possibility of reproducing the various types of ruminant mastitis in the rabbit model; and (c) evaluation of the adequacy of a rabbit experimental model for ovine mastitis studies, using bacterial inoculations via the teat duct, as was done in mice (Chandler, 1970; Anderson, 1972, 1977), a procedure which may facilitate infection without teat damage. Rabbits are cheaper and have a greater number of teats than ruminants. Hence, they may help to reduce the cost and the number of animals involved in these studies. They are also larger than mice and may thus be easier to handle for i n t r a m a m m a r y infections via the teat duct. These considerations as well as the fact that sheep mastitis has been less extensively studied than cattle mastitis, motivated us to investigate the three aspects, (a) to (c), mentioned above. The study provides a convenient model for fundamental assays on mastitis, prior to final assessment in ruminant species. M a t e r i a l s and M e t h o d s
Bacteria usedfor Inoculation Nineteen ovine mastitis strains were obtained for inoculations (seven Staphylococcus aureus, four Staph. chromogenes, four Staph. hyicus and four Escherichia coli). Bacteria were grown in Todd-Hewitt broth (THB; 37°C, 24 h). Subsequently, they were washed twice in phosphate buffered saline (PBS; pH 7"2). The concentration was adjusted to 107 viable bacteria per ml PBS (bacteria were plated at this stage for viable count).
Animals Ten Rasa Aragonesa ewes (Table 1) with various types of clinical mastitis, mammary abscesses or apparently normal mammary glands according to clinical criteria, were used for histopathological studies and as source of naturally produced ovine mastitis strains. For experimental infections, four healthy multiparous Rasa Aragonesa ewes were inoculated 24 to 48 h post-parturition. Seven females of the Spanish White Giant rabbit breed were also used for inoculations 24 to 48 h post-parturition. This breed did not exhibit any special mammary gland characteristics and was randomly chosen among rabbit breeds for this study. The mothers were separated from their offspring in all cases. On the day before inoculation (always after parturition), each animal was examined clinically (special attention being paid to the mammary glands). Milk was c~ltured this day and the day of infection to insure that the animals were free of bacteria.
Selection of Bacterial Dosefor inoculation To select the bacterial dose to be used in the rabbit infections, a titration was carried out in a female rabbit with strain 5A (Staph. aureus), by introducing 0"5 ml of suspension containing 5 x 102, 5 x 104 and 5 x 106 viable bacteria through the teat ducts of alternate glands with a cannula. The latter inoculum was the only one that produced mastitis, based on observations at 24 and 48 h. Hence, 5 x 106 viable bacteria in 0'5 ml PBS per rabbit per teat was considered an appropriate dose for rabbit
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infection experiments, intentionally designed for the animals to develop infection in a short time. Considering the relative size and physiology of sheep and rabbit mammary glands, an inoculum of 10 7 bacteria per sheep per teat was chosen for the sheep con trois used in the study.
Inoculation Technique Prior to infection, females were exhaustively milked. Subsequently, in the case of" rabbits, 0'5 ml of the bacterial suspension (5 x 106 cfu) was introduced through each teat duct, into alternate glands, through a 27G eannula. The same volume of sterile PBS was introduced as control through the remaining teat ducts (i.e. each rabbit was used for three strains and three PBS controls; in general, the remaining two teats were too small and thus not suitable for inoculations). The glands were gently massaged while pressing the teat to avoid leakage. For sheep inoculations, a similar procedure was applied, except that in this case, 1 ml of bacterial suspension (10~cfia) was inoculated into one gland of the animal (through a 21G eannula), leaving the other gland as a control in which 1 ml PBS was introduced. The specific bacterial strains used for the sheep inoculations were 1A (Staph. aureus), 8B (Staph. chrornogenes), 6A (Slaph. tzyicus) and 7B (E. coli).
Pathology of Experimentally-infected Glands Swelling and induration of inoculated mammary glands were evaluated 24 and 48 h after infection. The reactions were scored from . . . . . (lack of reaction) or "( + )" (very weak reaction) to " + + + + " (very strong reaction). Following the second evaluation (48 h), the animals were killed and the mammary glands were removed and stored in 10 per cent buffered formalin. These tissues were embedded in paraffin wax, sectioned 5 gm thick and stained with haematoxylin and eosin (HE) and Gram's method for histopathological examination. Comparative histological grading of mammary lesions (generalized or focal necrosis, oedema, haemorrhage, thrombosis etc.), originated by the different strains, ranged from " - " to " + + + " according to the severity of the reaction.
Bacteriological Assays Bacterial culture filtrates were prepared according to the procedure ofKrovacek, Faris and M~msson (1987) with a modification (incubation of bacteria in a rotary incubator was done in THB at 37°C for 24 h). These filtrates were used for rabbit skin vascular permeability factor assays (rabbit skin tests) and cytotoxicity assays on Chinese hamster ovary (CHO) cells, carried out as described by Krovacek et al. (1987). The production of alpha- and beta-haemolysins, coagulase, protease, DNAase, protein A and clumping factor was determined according to standard procedures (Larpent and Larpent-Gourgaud, 1985).
Results
Natural Ovine Mastitis and Rabbit Experimental Infections As shown in T a b l e 1, the 10 n a t u r a l cases o f o v i n e mastitis were classified at the time o f d e a t h according to histopathological criteria and subsequently comp a r e d with experimentally infected rabbits. Both sides of the ovine gland (left and right) were e x a m i n e d independently. Similarly, when rabbits were infected with the ,bacterial strains isolated from these 10 sheep, each teat was e x a m i n e d independently. Mastitis was reproduced in all cases (no infection
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failures were observed), and external observations could be evaluated as early as 24 h after inoculation, Following infection with Slaph. aureus strains, the rabbit mammary glands inoculated with strains 1A, 2A, 4A, 5A and 7C showed at 24 and 48 h after inoculation, intense tumescence and induration, being warmer than normal glands and painful upon palpation (Table 1, Fig. 1). The histopathological study of this material revealed the presence of gangrenous mastitis, characterized by a massive necrosis of the mammary parenchyma (Fig. 2). The processes of congestion, oedema, haemorrhage and vascular thrombosis were active in all cases. Inflammation, indicated by the presence of neutrophils, was mild in infections with strains 1A, 4A and 7C and intense in those with strains 2A and 5A (Table 2). In all cases, large bacterial aggregates, intensely basophilic, were found throughout the m a m m a r y tissue (Fig. 2). Strain 4A showed a tendency to adhere to the epithelial cells of excretory ducts and alveoli. Infection With strain 8C, isolated from a case of acute catarrhal mastitis, gave rise to an intense tumescence of the rabbit gland at 24 and 48 h after inoculation. Microscopically, a severe degeneration of alveolar epithelial cells, a mild necrosis and an intense inflammatory infiltration of neutrophils were observed (Table 2). Finally, strain 9B obtained from a non-mastitic gland with an occluded teat (9R; Table 1), caused mild macroscopic alterations and the microscopic examination revealed very mild vascular and inflammatory alterations. Infections with this strain and with 8C resulted in the presence of few bacteria and absence of bacterial aggregates (Table 2). Rabbit infections with Staph. chromogenes caused a slight or moderate external reaction at 24 and 48 h after inoculation in the case of strains 3A and 5B, but no external alterations were observed with strains 7D or 8B. There were slight histopathological differences between these strains (Table 2), but in general~ they produced a mild or medium catarrhal mastitis, localized in the excretory ducts and surrounding glandular alveoli. The most characteristic lesions consisted of intra-alveolar neutrophil infiltration, neutrophil lysis, degeneration of epithelial cells and destruction of some alveoli (Fig. 3). Strain 3A was more aggressive than the others, causing hyperaemia and intense haemorrhage (Table 2). Staph. hyicus strains 6A, 6B, 10B and 10C gave rise, in rabbits, to a mild (or nonexistent) macroscopic reaction in the inoculated rabbits (Table 1). However, from histopathological examination, all strains were associated with the presence of coagulation, necrotic loci and destruction of epithelial cells of ducts and alveoli. There was also destruction of many alveoli and massive intra- and extra-alveolar neutrophil infiltrations. In the alveoli and excretory duct lumens, there was abundant epithelial and neutrophil cell debris and a small number of moderately basophilic bacteria, which did not form aggregates (Fig. 4). Infection with E. coli strains 7A, 7B, 9A and 10A caused a mild to moderate external modification of the rabbit glands (Tables 1 and 2) which was histopathologically characterized by wide areas of coagulation necrosis, having destroyed alveoli in the centre, forming a homogenous and eosinophilic mass together with epithelial and inflammatory cell lysates and debris. These
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Fig.
Comparative images illustrating m a m m a r y glands fi'om a healthy doe in the lactation period (top) and from a doe infected with Slaph. attreus strain 2A (bottom). Pronounced tumescence, oedema and haemorrhage at the infection site are observed in the latter, as well as a size o['approximately twice the normal.
Fig. 2.
Histological [)'atures of a rabbit m a m m a r y gland inoculated with Slaph. aureus (IA): Extensive necrosis o[" m a m m a r y alveoli with clumps oF basophilic bacteria (arrows). HE x 150.
Fig. 3.
Histologieal [ieatures of a rabbit m a m m a r y gland inoculated with Slaph, chromogenes (3A): alveolar destruction with neutrophils in alveolar lumen. HE x 150.
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necrotic areas were surrounded mainly by healthy gland tissue (Fig. 5). A finding, characteristic of these strains, was the presence of blood vessel thrombosis. The number of bacteria found in these cases was reduced. Bacteria were often seen adherent to epithelial cells of ducts and alveoli in Gram-stained preparations.
Sheep Experimental Infections The macroscopic and microscopic study of the mammary gland of the sheep used in experimental infections (Table 1) revealed different types of mastitis: the ewe inoculated with strain 1A (Staph. aureus) developed a v e r y severe acute gangrenous mastitis; the ewe inoculated with strain 8B (Staph. chromogenes) suffered from a mild acute catarrhal mastitis; the ewe inoculated with strain 6A (Slaph. @icus) developed a catarrhal acute mastitis with small necrotic loci next to excretory ducts and alveoli and the one infected with strain 7B (E. colt) suffered from a definite acute catarrhal mastitis with large areas of coagulation necrosis. These histopathological findings were similar to those described in natural infections in sheep and/or in rabbit experimental infections.
Bacleriological assays The results obtained from the bacteriological assays showed that of the seven Siaph. aureus strains studied, three (1A, 2A and 5A) produced protein A , ['our (1A, 2A, 5A and 7C) produced alpha- and beta-haemolysins, two showed only a weak beta-haemolysin production (4A and 8C) and one (9B) did not produce alpha- or beta-haemolysins (Table 3). The latter strain showed (like strain 7C) protease activity, but in contrast with the remaining staphylococcal strains used in these tests, it did not show any clumping factor, coagulase or DNAase activities. Furthermore, the culture supernatant of this strain did not elicit a congestion or necrosis reaction in the rabbit skin test; in contrast with supernatants from other Slaph. aureus strains (1A, 2A, 4A, 5A and 7C, ill that order of strength; strain 8C was not tested), it was non-cytotoxic to CHO cells. It was found that the strains belonging to other bacterial species (coagulase negative staphylococci and E. colt), with the exception of Staph. chromogenes strain 8B and E. colt strain 9A, showed protease activity. DNAase activity was found for Slaph. chromogenes strains 3A, 5B and 7D (low activity) and for E. colt strains 10B and 10C (high activity). Only culture supernatants of one Slaph. chromogenes strain (7D) and two E. colt strains (7A and 7B) were cytotoxic for CHO. The culture supernatant of E. colt strain 9A produced a rabbit skin reaction (induration and necrosis), but no evidence of skin reaction was obtained for culture supernatants of the remaining non-Staph, aureus strains. Discussion
The bacterial species studied in this work represent the major bacterial agents involved in ovine mastitis, with the exception of Pasleurella haemolytica (Kimberling, 1988; Watson, Franklin, Davies, Kettlewell and Frost, 1990), which
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Fig. 4.
Histological ti:atures of a rabbit mammary gland inoculated with Slaph. Io,icus (6A): massive neutrophilic infiltration in interstitial tissue (I), in lumen of excretory ducts (left) arid ill alveoli (right). HE x 60.
Fig. 5.
Rabbit mammary gland inoculated with E. coli (IOA): An inflammatory cell bomldary (arrows) separates the area of coagulation necrosis (N) from the healthier glandular paeenchyn'm (rap), HE x 60.
Fig, 6.
Rabbit mamnmry gland inoculated with E. coli ( 10A): Blood vessel thrombosis ('I') and ueutrophilic infiltration in the interstitial spaces aad alveolar lumen (left). HE x 150.
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was not included in the study. All five Slaph. azzreus strains obtained from severe acute gangrenous, chronic purulent or abscess mastitis, reproduced extensive necrotic areas in the rabbit m a m m a r y gland. These findings are similar to those observed in experimental infections of cattle and sheep (Legall and PloNmet, 1965; Nickerson and Heald, 1981). The Slaph. aureus strain producing catarrhal mastitis in sheep (8C) also gave rise to a catarrhal mastitis in rabbits. The reason why this strain was pathogenically milder than other strains of this species is unknown. According to Loeffler, Greasy, Norcross and Paape (1988), Staph. aureus produces up to 30 extracellular products, primarily toxins and enzymes. The role of the products in the pathogenesis of ruminant staphylococcal mastitis is not well defined, with exceptions such as alpha-haemdysin, which is thought to play a key role in acute gangrenous staphylococcal mastitis. As may be gathered from Table 3, where the various classical bacteriological properties of each strain are illustrated, only a weak beta-haemolysin activity was associated with strain 8C. This weak haemolytic activity m a y help to explain why strain 8C is associated with a non-gangrenous mastltis. Strain 4A (also with a weak beta-haemolysin production), produced cistern abscesses in natural ovine infections and showed adhesiveness to the m a m m a r y gland epithelial tissue. Other cases of in vivo Staph. aureus adherence to ruminant epithelia have been described. Gudding, McDonald and Cheville (1984) observed attachment of Slaph. aureus strains to epithelial cells in bovine experimental infections. A question arises concerning whether adherence might have played a role in the pathogenesis of this particular ewe's m a m m a r y gland. Examining the history of this ewe (from which strain 4A was isolated), it was [bund that this animal had repeatedly suffered from mastitis and lack of milk production during five successive lambings. It is possible that adherence of bacteria to the m a m m a r y gland epithelial tissue was responsible for chronicity and that this affliction was not due to inefficacy of antibiotic treatment (strain 4A was sensitive to the majority of the common antibiotics tested in the antibiogram analysis carried out; data not shown). It might also be possible that leucocytes migrated to the places where adherent bacterial aggregates were present, resulting in the production of abscesses in the gland. Strain 9B, the least pathogenic of the Sl@h. aureus strains tested in this work (in rabbits and in sheep), did not show any haemolytic activity nor cytotoxic effects on C H O when culture filtrates were tested (Table 3). These filtrates were unstable when submitted to a freeze-thaw cycle and produced induration but not congestion or necrosis in the rabbit skin test. This strain was also clumping factor- and DNAase-negative. These characteristics were unique among the Slaph. aureus strains tested and may help understanding the strain's low pathogenicity. Jonsson, Lindberg, Haraldsson and WadstrSm (1985) suggested that, in mice, alpha-haemolysin and coagulase are virulence determinants of Slaph. aureus and that protein A is not an important virulence determinant of this species. Our results in rabbits are in agreement with these observations. It is tempting to draw conclusions on associations between each of the bacterial properties of the strains under study and the lesions produced. However, the number of sheep and rabbits used were low for association
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studies involving the high number of properties under study. Nonetheless, it appears that the role of protease production in mastitis is questionable, whereas the production of alpha- and beta-haemolysins, clumping factor, coagulase, DNAase or bacterial culture supernants which are positive in the rabbit skin tests and in CHO cell cytotoxicity tests, cannot be discarded as a possible virulence factor, when considering the data obtained. Staph. chromogenes and Slaph. hyicus strains, obtained from chronic mastitis or apparently healthy ovine glands were able to reproduce mild or moderate mastitis in rabbits. These findings help to determine the role of coagulasenegative staphylococci in subclinical mastitis of sheep (Fthenakis and Jones, 1990). The lesions caused by E. coli strains in the rabbit mammary gland, characterized by large areas of coagulation necrosis, are very similar to those produced by this bacterial species in cattle under natural and experimental conditions (Frost and Hill, 1982; Hazlett, Little, Maxie and Barnum, 1984; Hill et al., 1984). Although there is little information on ovine mastitis E. coli pathogenicity, in our opinion, this bacterial species could play several roles: (a) agent accompanying other bacteria (e.g. Slaph. aureus); (b) agent producing subclinical mastitis; and (c) primary major mastitis agent. The strong pathogenic effect of most Slaph. aureus and some of E. coli strains in both the experimental rabbit infections and the ovine natural inl~ctions, would be expected, knowing that in sheep these species are relatively major pathogens when compared to the other bacterial species of Table 1 (Jones, 1985; Hueston, Hartwing and Judy, 1986). The above considerations lead to the conclusion that the rabbit experimental model for infection with ovine mastitis strains helps to evaluate the degree of pathogenicity of different agents. In our case, we can classify the bacterial species from a higher to a lower mastitis pathogenic power, as follows: Staph. aureus, E. coli, Slaph. hyicus and Staph. chromogenes, in that order. These observations are in agreement with those of Bramley, King, Higgs and Neave (1979) with regard to the first two species. They found that, in cattle, for establishment of experimental infections, the number of cfu was higher for E. coli, when compared with Slaph. aureus. With respect to the types of mastitis found in experimental infections, we have classified mastitis into various categories, with terms that define the pathological status in each particular case: acute, chronic, mild, severe, catarrhal, gangrenous, purulent, with abscesses, etc. This classification could be carried out in natural and experimental sheep mastitis infections as well as in their rabbit experimental infection counterparts. Several authors have established sheep mastitis types. Plommet in 1959, using experimental sheep infections classified the types ofmastitis into three groups: type I, mild mastitis; type II, severe mastitis; and type III, gangrenous mastitis. These groups may well include the mastitis categories described in this work for the rabbit and sheep infections. When studying the similarity of results obtained in rabbits and in sheep, it was noticed that strains 5B and 8B ofSlaph, chromogenes and strain 7B orE. coli were isolated from the same ovine mammary gland sides (5L, 8L and 7L, respectively, Table 1) as the corresponding Slaph. aureus strains 5A, 8C and 7C.
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Hence, as expected, it was observed that the rabbit infection reactions with the purified strains 5B, 8B or 7B were milder than the natural infection reactions observed in sheep with the mentioned strain admixtures, containing Slaph. aureus strains in all cases. Altogether, these results show a clear correspondence between the various types of ovine natural or induced mastitis and the types of rabbit mastitis experimentally induced by the different ovine strains. Thus, rabbit infections m a y help predict the outcome of sheep infections. This makes the rabbit experimental model very convenient for ovine mastitis strain studies. The model has several additional advantages: (a) the inoculation technique is secure and easy, it does not require magnifying lenses; (b) the inflammation spread between glands is hardly detectable; and (c) three alternate glands can be infected in a given animal, which helps to minimize the number of animals used for experimentation. Although, with the proposed model, many aspects ofovine pathogenesis can be studied, there are several points that ought to be considered before extrapolating the findings in rabbits (with the single dose bacterial infections described) to sheep. For example, in natural infections, the dose of infecting bacteria may vary and different effects may accordingly be observed (Bramley el al., 1979). On the other hand, the number of somatic cells present in m a m m a r y gland secretions at the time of infection may be crucial for establishment of the infection (Bramley, 1976), but this information is not provided by the rabbit model. Finally, mammalian species-specific differences (sheep vs. rabbit) and individual genetic and physiological differences within these mammalian species cannot be evaluated when using the rabbit model alone, without carrying out concomitant infections in the ruminant species u n d e r study. For these reasons, in many cases, the rabbit experimental model proposed ought to be considered very helpful for preliminary studies of experiments on strain pathogenesis, to reduce costs and to avoid unnecessary waste of large animals, which could be used later for final assessment. Acknowledgments
This work was funded by Comisi6n Asesora de Investigaci6n Cientifica y T~cnica (Project No. PB86-0217), Consejo Asesor de Investigaci6n (DGA, Project No. PCA-6/87) and Diputaci6n General de Arag6n (Livestock Production Section). References
Adlam, C., Thorley, C. M., Ward, P. D., Collins, M., Lucken, R. N. and Knight, P. A. (1976). Natural and experimental mastitis in rabbits. Journal of Comparalive Pathology, 86, 581-593. Adlam, C., Ward, P. D., McCartney, C., Arbuthnott, J. P. and Thorley, C. M. (1977). Effect of immunization with highly purified alpha and beta toxins on staphylococcal mastitis in rabbits. Infection and Immunity, 17, 250-256. Anderson, J. C. (1972). The effect of teat damage on the incidence of experimental mastitis in the mouse. Research in Veterinary Science, 13, 390-392. Anderson, J.C. (1977). Experimental staphylococcal mastitis in the mouse: the induction of chronic mastitis and its response to antibiotic therapy. Journal of Comparative Pathology, 87, 611-621. Anderson, J. C. and Holmberg, O. (1977). The invasiveness of Staphylococcus aureus and
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