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The experimental production of mastitis in sheep by intramammary inoculation of Pasteurella haemolytica
J. Comp. Path, 1991 Vol. 105
The Experimental Production of Mastitis in Sheep by Intramammary Inoculation of Pasteurella haemolytiea E. T. S. E1-Masannat, J. E. T. Jones and M. J. Scott Department of Animal Health, The Royal VeterinaryCollege,Boltons Park, PottersBar, EN6 1NB, U.K.
Summary Acute mastitis was consistently produced in primiparous ewes by inoculation of the mammary gland, via the teat canal, with an isolate of Pasteurella haemolylica, serotype A9, originating from a field case of ovine mastitis. Mastitis developed following the inoculation of as few as 10 colony forming units of this isolate, suggesting that only a small number of organisms would be required to initiate the disease under natural conditions provided they were already beyond the teat canal. Clinical signs and macroscopic lesions were well developed within 24 h of inoculation and were similar to those found in the naturally-occurring disease. The ability to reproduce mastitis consistently will facilitate studies of the pathogenesis of the disease and the comparison of different isolates of P. haemolyticawith respect to virulence determinants.
Introduction Ovine mastitis associated with pasteurella infection has been reported sporadically from several countries since the beginning of the century, hut, in some of the early reports, it is not always clear whether the organism described is Pasteurella haemolytica or P. multocida. In the United Kingdom, the first record of ovine mastitis caused by an organism which, almost certainly, was P. haemolytica is that of Leyshon (1929). He examined 38 cases of mastitis in ewes and found that four were caused by organisms of the "Pasteurella group"; the characteristics of the bacteria he described are those of P. haernolytica. There are few reports of experimental infection of the ovine mammary gland with bacteria that are indisputably P. haemolytica. It seems highly likely that the organism referred to as P. mastitidis by Simmons and Ryley (1954) was P. haemolytica and was used by them in the form of a 24-h broth culture to produce mastitis in a ewe. Krzyzanowski and Lakomy (1972) reported that severe mastitis developed in ewes that were inoculated by the intramammary route with P. haemolytica isolated from a field case of the disease; isolates from the brain and lung of lambs produced only mild mastitis. P. haemolytica and Staphylococcus aureus are the principal causes of ovine mastitis in lowland flocks in England and Wales (Jones, 1985). Much experimental work has been done on staphylococcal mastitis in ruminants, but very little on pasteurella mastitis. 0021-9975/91/080455+ 11 $03.00/0
© 1991 Academic Press Limited
456
Induction of Mastitis in Sheep
In this paper we report the establishment o f infection in the ovine m a m m a r y gland with a small n u m b e r o f P . haernolytica, leading to the d e v e l o p m e n t o f an acute necrotizing mastitis. T h e aims of our investigations were to d e t e r m i n e how soon after inoculation mastitis might develop and to d e m o n s t r a t e w h e t h e r the clinical and pathological features o f e x p e r i m e n t a l l y i n d u c e d mastitis were similar to those o f the naturally occurring disease. Preliminary studies had shown that a strain o f P . haemolytica, ES26L, in our collection was highly pathogenic for the m a m m a r y gland. It was, therefore, used in these experiments. M a t e r i a l s and M e t h o d s
Animals All ewes were 2 years old, primiparous and were of the Welsh Mountain (19) and Poll Dorset (19) breeds. Each ewe, together with its lamb(s) was penned individually on straw in a covered yard. The ewes were fed hay and concentrates. After parturition, the ewes were examined daily, particular attention being paid to the udder and its secretions and to rectal temperature. Five ewes of each of the two breeds, from the same population and maintained under similar conditions, served as controls for clinical evaluation.
Laboratory Procedures (i) Before Inoculation. Samples of milk were inoculated on Columbia agar enriched with 5 per cent defibrinated sheep blood. Two plates of media were used; one was incubated aerobically and one anaerobically, in the "Gas-pak" system (Becton Dickinson U.K. Ltd) at 37"C for 2 days. Plates were examined daily. Only those ewes with clinically healthy udders, from which no bacteria had been isolated were used in the experiment. (ii) After Inoculation. Milk samples were cultured as described above. Duplicate films from individual milk samples were allowed to dry for 1 h and immersed for 7 min in a defatting fixative made of 52 per cent absolute alcohol, 44 per cent xylol and 4 per cent glacial acetic acid. One film was stained by Gram's method and the other by Giemsa and examined microscopically for the presence of bacteria and cells. The remainder of each milk sample was preserved by the addition of two drops of formalin and sent to the Milk Marketing Board laboratory at Worcester where cells were counted by an electronic particle counter (Coulter Counter, Model MCC). At 3, 6, 9, 12, 18, 24, 48, 72 and 96 h after inoculation the ewes were examined clinically and milk samples were collected for bacteriological examination and, if the physical nature of the milk was suitable, for cell counts. Lambs were removed from their dams for 1 h before milk collection in order to obtain a sufficient volume of mammary secretion.
Bacterial Inoculum A P. haemolytica isolate, ES26L~ serotype A9, originating from a fatal field case of acute ovine mastltis and maintained in 5-h glycerol blood-broth cultures at - 20°C was used in all experiments. On the day before inoculation, the organism was subcultured from the broth onto Columbia sheep blood agar and, after 18 h incubation at 37°C, colonies were inoculated into 20ml of pre-warmed Todd-Hewitt broth and incubated aerobically for 5 h at 37°C. Several dilutions of the 5-h broth cultures in phosphate buffered saline (PBS), pH 7.3, were prepared so that each inoculum contained approximately 1000 to 2000, 100
457
I n d u c t i o n o f M a s t i t i s i n Sheep
to 200 or 10 colony forming units (cfu) of P. haemolytica in 2"0 ml as required in each of three experiments. Bacterial counts were determined by the method of Miles, Misra and Irwin (1938). Three replicate counts were made and the mean taken as the estimate of the dose given. The inocula were used immediately after preparation. Control inocula consisted of 2 ml sterile PBS.
Inoculation Procedure Ewes were inoculated 2 to 4 days after lambing. Lambs were removed from their dams 1 h before inoculation. Both teats &each ewe were cleansed with cotton soaked with 70 per cent ethanol and left for a few seconds to dry. The first streams of milk expressed from the glands were discarded and then 8 to 10 ml of milk were collected from each gland into a sterile container for subsequent laboratory examination. The bacterial inoculum was deposited in the cistern of the left gland via the teat canal by means of a 21 gauge, 25 ram, stainless steel cannula attached to a 2'0 ml syringe. PBS was injected into the right, control, gland. Two hours later the lambs were returned to their dams.
Experimental Design Three experiments were conducted. In the first, each of 11 Welsh Mountain ewes was inoculated with 1000 to 2000 cfu, In the second, 16 Poll Dorset ewes were inoculated with 100 to 200 cfu and in the third experiment, eight Welsh Mountain and three Poll Dorset ewes were inoculated with approximately 10 cfu. O f the 38 ewes, thirty were killed at predetermined intervals after inoculation, four died and four were allowed to survive (Table 1).
Necropsy Procedures The ewes that died were necropsied within 3 h of death and those killed at predetermined times post-inoculation (3, 6, 9, 12, 18, 24, 48, 72 and 96h) were necropsied immediately. Ewes were killed by intravenous injection of pentobarbitone
Table 1 The experimental production of mastitis in Welsh Mountain and Poll Dorset ewes by i n t r a m a m m a r y inoculation o£ Pasteurella haernolytica (A9) at one of three infective doses
Number of ewes killed (or that died*) after receiving the stated dose ( c.fit) in experiment Interval between inoculation and
death ( h)
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2 (D) (100 to 200)
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et al.
E1-Masannat
sodium. The mammary gland and internal organs of each animal were examined. The presence, severity and extent of lesions were assessed by visual examination. Tissue samples from the right and left mammary glands were fixed in 10 per cent buffered formalin and processed by standard methods for histopathological examination. Samples from right and left mammary glands, supramammary lymph nodes, heart blood, liver and spleen of each ewe were cultured on Columbia blood agar and incubated aerobically and anaerobically for 48 h at 37°C. In the second experiment, on the Dorset ewes, samples of mammary secretions were collected for bacterial counts at 3, 6, 9, 12 and 18 h post-inoculation and immediately before killing. Cubes (1 cm3) &mammary tissue from the distal third of the gland were taken from the same animals, at the time of killing. The number of bacteria in 1 ml of secretion and I g of tissue was estimated by the method of Miles, Misra and Irwin
(1938).
Results Acute mastiffs developed in all animals inoculated. Neither the number of bacteria inoculated nor the breed of the ewe influenced the time of onset or the severity of mastiffs.
Clinical Findings An increase in rectal temperature (Fig. 1) and slight enlargement and hardness of the inoculated glands, accompanied by erythema, were evident within 12 h of inoculation. In most ewes, the skin over the affected glands became hot and discoloured 24 to 48 h after inoculation (Fig. 2). Signs of acute mastitis were maximal on day 3 or 4 after inoculation (Figs 4 and 6) and most of the skin
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Induction of Mastitis in Sheep
459
over the inoculated glands became blue or black with a clear line of demarcation, separating areas of necrotic tissue from the adjoining living tissue. The gland became soft, cold and insensitive and the overlying subcutaneous tissue was oedematous. The nature of the m a m m a r y secretions varied greatly depending on the stage of the disease. Among the early changes observed were increased viscosity and the presence of clots or flakes; in more advanced stages, secretions were watery, straw-coloured or brown with thick clots and, later, blood-stained. Eventually, only a few drops of fluid could be expressed from the teats of affected glands. In a few ewes, the uninoculated control glands were also swollen and the milk was reduced in quantity but was of normal appearance. However, in three severely affected ewes (one died at 5 days) there was clinical and pathological evidence of mastitis in the control gland. Systemic signs of illness appeared on day 1 after inoculation and included loss of appetite, listlessness, rapid breathing and fever. The mean rectal temperature of ewes during the acute phase ofmastitis is shown in Fig. 1. Some ewes became very ill 3 to 4 days after inoculation and had diarrhoea and more pronounced dyspnoea and pyrexia. They became depressed and did not allow the lambs to suck; they remained standing but were reluctant to walk. Four ewes died between days 4 and 6 post-inoculation. In two of four ewes which survived the acute phase, sloughing of necrotic tissue began about a week after inoculation and was complete within two weeks. Healing of the skin took approximately 8 weeks. During the period following sloughing, appetite was restored. In the other two ewes, suppuration of necrotic tissue developed about a week after inoculation. Sloughing took place over the next few weeks and necrotic tissue (Fig. 7) was gradually replaced by fibrous tissue and healing was complete in 8 to 9 weeks. The uninfected control glands in all four ewes involuted and were restored to normal function during the subsequent lactation.
Necropsy Findings (i) Mammary Glands. At 12 h post-inoculation, glands were slightly enlarged; petechiae and ecchymoses were evident on the cut surface. At and beyond 18 h, the glands were markedly swollen and the affected parenchyma was intensely reddened as the result ofhyperaemia and haemorrhage (Fig. 3). The red areas were either multifocal, often giving the gland a mottled appearance, or confluent and sharply demarcated from adjacent tissue. Such lesions were extensive 3 to 4 days after inoculation and often involved the whole gland (Fig. 5). In many affected glands, there was marked distension of veins and, in some, thrombosis was commonly recognized by the presence of firm, grey clots adhering to the intima. At 1 to 2 days post-inoculation, ducts in the affected glands were plugged by threads of fibrin and clots of milk (Fig. 3). A serous, sero-sanguinous, or purulent exudate filled and distended the acini and the interlobular septae were clearly demarcated. The milk ducts and cistern were filled with brown
460
E . T . S . E l - M a s a n n a t e t al .
Induction of Mastitis in Sheep
461
masses of" inflammatory exudate containing threads of fibrin and necrotic debris. In the three ewes in which the uninoculated gland was clinically affected, there were haemorrhagic lesions. In many ewes, there was much oedema in the subcutaneous tissue overlying the glands, in the inguinal region and, occasionally, in the ventral abdomen, flank, perineum and thighs. Supramammary lymph nodes were oedematous and swollen; some were intensely reddened and haemorrhagic. (ii) Other Organs and Tissues. There was no evidence of any lesions which might have signified generalized infection.
Hislopathological Findings The earliest change detected was infiltration of many neutrophils into the alveoli. Twelve hours after inoculation, this infiltration was widespread; small areas of haemorrhage and hyperaemia were evident at this stage. There was degeneration of alveolar epithelium and many desquamated epithelial cells, neutrophils and fluid were present in the alveoli. Within 24 h of inoculation, necrotic areas had developed within the parenchyma and there was considerable haemorrhage. Venous thrombosis was frequently seen. In the course of 3 to 4 days, the entire parenchyma was involved in inflammatory and degenerative reactions characterized by a dense infiltration of'neutrophils, hyperaemia, haemorrhage and necrosis.
Bacteriological Findings (i) Microscopic Examination of Mammary Secretion. Films of secretion from inoculated glands collected up to 9 h post-inoculation revealed bacteria only. At a n d beyond 12 h post-inoculation, P. haemolytica and leucocytes, chiefly
Fig. 2.
Udder o[' ewe, 1 clay after inoculation of left gland with 1000 cfu of P. tzaemolytica; the gland is enlarged and there is discolouration of the skin.
Fig, 3.
Cut surface of udder illustrated in Fig. 2. The left gland is enlarged, clots of milk are present and there is a hyperaemic reaction anteriorly.
Fig. 4.
Udder of ewe, 3 clays after inoculation of left gland with 100 cfu of P. haemogytica. There is enlargement of both glands and a pronounced cyanotic reaction of the skin.
Fig. 5.
Cut surface of the udder illustrated in Fig. 4. There is a confluent hyperaemic, haemorrhaglc and necrotic reaction involving the whole of the left gland. The parenchyma has separated from surrounding connective tissue and there is subcutaneous oedema ventrally.
Fig, 6,
Uclder o['ewe, 3 days al'tcr inoculation of left gland with 100 cth o['P. haemolytica; the gland atld teat are enlarged but there is no discolouradon of the skin.
Fig. 7.
Udder of ewe, 21 clays after inoculation of the right gland with l0 cfu ofP. haemo!ylica. Much of the gland has been slonghed. There is a purulent cxudate from the l'emainlng uecrotie tissue. At tile periphery of the lesion there is evidence ~[' healing.
462
E. T. S. E 1 - M a s a n n a t e t al.
neutrophils, were present in substantial numbers. Some leucocytes contained engulfed P. haemo~tica but, in most cases, large masses of P. haem@tica were free in the altered secretion. Leucocytes showed degenerative changes. (ii) Cultural Examination. At 3, 6, 9, 12, 18, 24, 48, 72 and 9 6 h postinoculation, P. haemolylica (A9) was isolated in pure culture from the secretion of all inoculated glands except, at 72 h, in two ewes in which it was isolated together with Streptococcus acidominimus in one and with Strep. boris in the other. At necropsy, P. haemolytica (A9) was isolated from the secretions and tissues of all inoculated glands. In three ewes, killed at 12, 24 and 48 h, respectively, after inoculation, P. haemolytica was isolated from the control glands. Macroscopic lesions were not seen in these glands at necropsy, but there was a marked increase in the number of cells in the milk ( > 14x l0 s cells per ml) of two of these ewes sampled immediately before killing at 24 and 48 h, respectively. In three ewes in which the uninoculated half developed mastitis, P. haemolytica was isolated in large numbers from the haemorrhagic lesions at the time of necropsy 3, 4 and 5 days, respectively, after inoculation. Whenever there was well established mastitis, P. haemolytica was usually isolated from supramammary lymph nodes. P. haemolytica was not isolated from the ipsilateral lymph node (ipln) of any of the eight ewes killed within 9 h of intramammary inoculation, but it was isolated from the contralateral lymph node (cln) of one of two ewes killed at 9 h. In 26 ewes that were killed or died between 12 h and 6 days after inoculation, in which the m a m m a r y lymph nodes were examined bacteriologically, P. haemolytica was isolated from the ipln of 20 ewes and from the cln of 16 of these; in two ewes it was isolated from the cln only and in four ewes from neither ipln nor cln. In three of the ewes from which P. haemolytica was isolated from the cln, the control glands were infected and had signs of mastitis. In one ewe from which P. haemolytica was isolated from the cln, the uninoculated gland was infected but there were no signs of mastitis. Y. haemolytica was not isolated from heart blood, liver or spleen of any ewe. In all experiments, every isolate of P. haemolytica grown from various tissues and secretions was examined serologically and found to belong to serotype A9. (iii) Bacterial Counts in Infected Mammary Secretions and Tissues. In each of five pairs of ewes in which the numbers of bacteria in m a m m a r y secretions and tissues were counted at intervals of 3, 6, 9, 12 and 18 h, respectively, after inoculation, maximal numbers were detected at 12 and 18 h (Fig. 8).
Cell Counts of Milk in Uninfected and Infected Glands A 10-fold increase in the cell count of milk from inoculated glands occurred at approximately 12 h post-inoculation. In the period when milk samples were suitable for examination, this increase in cell count was maintained at about 107 per ml. Generally, samples taken from inoculated glands later than 24 h after inoculation were physically unsuitable for counting cells.
463
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Discussion Inoculation of the mammary gland with P. haernolytica, ES26L, invariably resulted in acute mastitis of varying severity. The production of acute mastitis with as few as 10 cfu, inoculated via the teat canal, is perhaps the most significant finding of this experiment. It allows the reasonable speculation that only a few organisms would be required to produce the disease under natural conditions once they were beyond the teat canal. It seems that adult sheep are much more susceptible to pure P. haemolytica biotype A infection by the intramammary route than by any other route tested in the past. For example, the experimental production of pneumonic pasteurellosis requires the intrabronchial administration of huge doses of the organism or the utilization of the synergic effects of viruses or mycoplasmas (Smith, 1990). The rapidity with which mammary lesions developed was evident 12 h after inoculation by the presence of hyperaemia and multifocal haemorrhages. The clinical signs and the nature of well developed lesions were similar to those of acute mastitis described by Leyshon (1929) and seen by us during the course of our field work. The development of infection of the mammary gland with isolate ES26L was manifested in three phases: (i) a lag phase of less than 3 h duration, (ii) a
464
E. T. S. E1-Musunnat e t al.
phase of bacterial multiplication, peaking at approximately 7 x 106 organisms per ml milk and 4 x 106 organisms per g tissue, 12 h post-inoculation (Fig. 8), (iii) a reactive phase ofleucoeytic infiltration, hyperaemia and haemorrhage at about 12 h post-inoculation when the number of bacteria was maximal. This phase was manifested macroscopically by intense red mottling of the mammary parenchyma. After 25 h, infected glands developed severe, irreversible pathological changes (Fig. 5). The onset of inflammation was marked by an increase in the number of somatic cells in milk, evident 12 h post-inoculation, at which time the mean cell counts of secretion from inoculated glands were 10-fold higher than those from uninoculated glands. A delay of approximately 12 h occurred between inoculation and the appearance of large numbers of leucocytes in milk, a time lapse allowing sufficient opportunity for the bacteria to become established. In three ewes, the control glands developed haemorrhagic necrotizing lesions from which P. haernolytica was isolated. In another three ewes, P. haemolytica was isolated from the control gland in which there were no lesions. It may be that, in some ewes, infection was transmitted to the control gland by cross sucking between inoculated and control glands. Since there was no evidence of generalized infection in any ewe, haematogenous spread of infection to uninoculated glands seems unlikely. Some control glands were swollen but the milk appeared normal and no bacteria were isolated from it. In the majority of ewes, P. haemolSica was isolated from both the supramammary ipln and cln but, in some ewes, P. haemolytica was isolated only from supramammary lymph nodes of the control uninoculated glands. This may be explained by a cross-over of the lymph vessels from each of the two glands of the udders, as described by Vollmerhaus
(1981). Cultural examination of heart blood, liver and spleen in all inoculated animals failed to demonstrate generalized infection. It seems probable that severe systemic illness and, in some ewes, death, was brought about by toxin(s) produced by P. haemolytica during its multiplication in the mammary gland; it is possible that endotoxin played a part in the genesis of clinical signs and pathological changes. In ewes that survived the acute disease, recovery was characterized by complete loss of mammary tissue of affected glands and therefore permanent loss of function. In the unaffected glands of ewes that recovered, lactation was normal in the following lambing season. This consistently reproducible experimental model will enable various aspects of the pathogenesis of pasteurella mastitis to be studied and will facilitate comparison of different strains of P. haemolytica especially in relation to virulence determinants. Acknowledgments
We are grateful to Mr R. Goldsmith for care of the sheep. This work was supported by the Overseas Development Administration of the Foreign Office.
Induction of Mastitis in Sheep
465
References
Jones, J. E. T. (1985). An investigation of mastitis in sheep: preliminary phase. Proceedings Sheep Veterinary Society, 10, 48-51. Krzyzanowski, J. and Lakomy, M. (1972). [Clinical course and experimental treatment of mastitis in sheep produced by the administration of Pasteurella haemolytica]. Polskie Archiwum Wele~ynaryjne 15, Fasc. 2, 287-305. Abstract in Veterinary Bulletin (1973), 43, 2458. Leyshon, W . J . (1929). An examination of a number &cases of ovine mastitis. The Velerinary Journal, 85, 286-300 and 331-344,. Miles, A. A., Misra, S. S. and Irwin, J. O. (1938). The estimation of the bactericidal power of the blood. Journal of IIygiene, Cambridge, 38, 732-739. Simmons,.G.C. and Ryley, J. W. (1954). Ovine mas~tis with special reference to mastitis caused by Pasleurella mastitidis. O.jleenslandJournal of Agricullural and Animal Sciences, 11, 29-35. Smith, G. R. (1990). Pasteurella infections. In Topley and Wilson's Principles of Baeleriology, Virology and Immunily Vol. 3. G.R. Smith and C.S. Easmon, Eds, Edward Arnold, London, pp. 382-388. Vollmerhaus, B. ( 1981). Lymphatic system. In The Anatomy of the Domestic Animals Vol. 3. A. Schummer, H. Wilkens, B. Volmerhaus and K. H. Habermehl, Eds, Paul Parey, Berlin, p. 4.17.
I Received, June 8lh, 1991 ~ Accepted, July 17th, 1991_]
The Experimental Production of Mastitis in Sheep by Intramammary Inoculation of Pasteurella haemolytiea E. T. S. E1-Masannat, J. E. T. Jones and M. J. Scott Department of Animal Health, The Royal VeterinaryCollege,Boltons Park, PottersBar, EN6 1NB, U.K.
Summary Acute mastitis was consistently produced in primiparous ewes by inoculation of the mammary gland, via the teat canal, with an isolate of Pasteurella haemolylica, serotype A9, originating from a field case of ovine mastitis. Mastitis developed following the inoculation of as few as 10 colony forming units of this isolate, suggesting that only a small number of organisms would be required to initiate the disease under natural conditions provided they were already beyond the teat canal. Clinical signs and macroscopic lesions were well developed within 24 h of inoculation and were similar to those found in the naturally-occurring disease. The ability to reproduce mastitis consistently will facilitate studies of the pathogenesis of the disease and the comparison of different isolates of P. haemolyticawith respect to virulence determinants.
Introduction Ovine mastitis associated with pasteurella infection has been reported sporadically from several countries since the beginning of the century, hut, in some of the early reports, it is not always clear whether the organism described is Pasteurella haemolytica or P. multocida. In the United Kingdom, the first record of ovine mastitis caused by an organism which, almost certainly, was P. haemolytica is that of Leyshon (1929). He examined 38 cases of mastitis in ewes and found that four were caused by organisms of the "Pasteurella group"; the characteristics of the bacteria he described are those of P. haernolytica. There are few reports of experimental infection of the ovine mammary gland with bacteria that are indisputably P. haemolytica. It seems highly likely that the organism referred to as P. mastitidis by Simmons and Ryley (1954) was P. haemolytica and was used by them in the form of a 24-h broth culture to produce mastitis in a ewe. Krzyzanowski and Lakomy (1972) reported that severe mastitis developed in ewes that were inoculated by the intramammary route with P. haemolytica isolated from a field case of the disease; isolates from the brain and lung of lambs produced only mild mastitis. P. haemolytica and Staphylococcus aureus are the principal causes of ovine mastitis in lowland flocks in England and Wales (Jones, 1985). Much experimental work has been done on staphylococcal mastitis in ruminants, but very little on pasteurella mastitis. 0021-9975/91/080455+ 11 $03.00/0
© 1991 Academic Press Limited
456
Induction of Mastitis in Sheep
In this paper we report the establishment o f infection in the ovine m a m m a r y gland with a small n u m b e r o f P . haernolytica, leading to the d e v e l o p m e n t o f an acute necrotizing mastitis. T h e aims of our investigations were to d e t e r m i n e how soon after inoculation mastitis might develop and to d e m o n s t r a t e w h e t h e r the clinical and pathological features o f e x p e r i m e n t a l l y i n d u c e d mastitis were similar to those o f the naturally occurring disease. Preliminary studies had shown that a strain o f P . haemolytica, ES26L, in our collection was highly pathogenic for the m a m m a r y gland. It was, therefore, used in these experiments. M a t e r i a l s and M e t h o d s
Animals All ewes were 2 years old, primiparous and were of the Welsh Mountain (19) and Poll Dorset (19) breeds. Each ewe, together with its lamb(s) was penned individually on straw in a covered yard. The ewes were fed hay and concentrates. After parturition, the ewes were examined daily, particular attention being paid to the udder and its secretions and to rectal temperature. Five ewes of each of the two breeds, from the same population and maintained under similar conditions, served as controls for clinical evaluation.
Laboratory Procedures (i) Before Inoculation. Samples of milk were inoculated on Columbia agar enriched with 5 per cent defibrinated sheep blood. Two plates of media were used; one was incubated aerobically and one anaerobically, in the "Gas-pak" system (Becton Dickinson U.K. Ltd) at 37"C for 2 days. Plates were examined daily. Only those ewes with clinically healthy udders, from which no bacteria had been isolated were used in the experiment. (ii) After Inoculation. Milk samples were cultured as described above. Duplicate films from individual milk samples were allowed to dry for 1 h and immersed for 7 min in a defatting fixative made of 52 per cent absolute alcohol, 44 per cent xylol and 4 per cent glacial acetic acid. One film was stained by Gram's method and the other by Giemsa and examined microscopically for the presence of bacteria and cells. The remainder of each milk sample was preserved by the addition of two drops of formalin and sent to the Milk Marketing Board laboratory at Worcester where cells were counted by an electronic particle counter (Coulter Counter, Model MCC). At 3, 6, 9, 12, 18, 24, 48, 72 and 96 h after inoculation the ewes were examined clinically and milk samples were collected for bacteriological examination and, if the physical nature of the milk was suitable, for cell counts. Lambs were removed from their dams for 1 h before milk collection in order to obtain a sufficient volume of mammary secretion.
Bacterial Inoculum A P. haemolytica isolate, ES26L~ serotype A9, originating from a fatal field case of acute ovine mastltis and maintained in 5-h glycerol blood-broth cultures at - 20°C was used in all experiments. On the day before inoculation, the organism was subcultured from the broth onto Columbia sheep blood agar and, after 18 h incubation at 37°C, colonies were inoculated into 20ml of pre-warmed Todd-Hewitt broth and incubated aerobically for 5 h at 37°C. Several dilutions of the 5-h broth cultures in phosphate buffered saline (PBS), pH 7.3, were prepared so that each inoculum contained approximately 1000 to 2000, 100
457
I n d u c t i o n o f M a s t i t i s i n Sheep
to 200 or 10 colony forming units (cfu) of P. haemolytica in 2"0 ml as required in each of three experiments. Bacterial counts were determined by the method of Miles, Misra and Irwin (1938). Three replicate counts were made and the mean taken as the estimate of the dose given. The inocula were used immediately after preparation. Control inocula consisted of 2 ml sterile PBS.
Inoculation Procedure Ewes were inoculated 2 to 4 days after lambing. Lambs were removed from their dams 1 h before inoculation. Both teats &each ewe were cleansed with cotton soaked with 70 per cent ethanol and left for a few seconds to dry. The first streams of milk expressed from the glands were discarded and then 8 to 10 ml of milk were collected from each gland into a sterile container for subsequent laboratory examination. The bacterial inoculum was deposited in the cistern of the left gland via the teat canal by means of a 21 gauge, 25 ram, stainless steel cannula attached to a 2'0 ml syringe. PBS was injected into the right, control, gland. Two hours later the lambs were returned to their dams.
Experimental Design Three experiments were conducted. In the first, each of 11 Welsh Mountain ewes was inoculated with 1000 to 2000 cfu, In the second, 16 Poll Dorset ewes were inoculated with 100 to 200 cfu and in the third experiment, eight Welsh Mountain and three Poll Dorset ewes were inoculated with approximately 10 cfu. O f the 38 ewes, thirty were killed at predetermined intervals after inoculation, four died and four were allowed to survive (Table 1).
Necropsy Procedures The ewes that died were necropsied within 3 h of death and those killed at predetermined times post-inoculation (3, 6, 9, 12, 18, 24, 48, 72 and 96h) were necropsied immediately. Ewes were killed by intravenous injection of pentobarbitone
Table 1 The experimental production of mastitis in Welsh Mountain and Poll Dorset ewes by i n t r a m a m m a r y inoculation o£ Pasteurella haernolytica (A9) at one of three infective doses
Number of ewes killed (or that died*) after receiving the stated dose ( c.fit) in experiment Interval between inoculation and
death ( h)
I (IV) (1000 to 2000)
2 (D) (100 to 200)
3 (WandD) (10)
~,
(u
cfu
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2
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3
18 24 48 72 96 120 144 Not killed
2 '2' 2 2 1 . . . . . .
1 1 2 1 . . .
. . .
. . .
. . .
W Welsh Mountain breed, D Poll Dorset breed, cfu colony forming units.
l'(D) 3 (1W*,2D)
2 (W*) 1 (W*)
4 (W)
4:58
E.T.S.
et al.
E1-Masannat
sodium. The mammary gland and internal organs of each animal were examined. The presence, severity and extent of lesions were assessed by visual examination. Tissue samples from the right and left mammary glands were fixed in 10 per cent buffered formalin and processed by standard methods for histopathological examination. Samples from right and left mammary glands, supramammary lymph nodes, heart blood, liver and spleen of each ewe were cultured on Columbia blood agar and incubated aerobically and anaerobically for 48 h at 37°C. In the second experiment, on the Dorset ewes, samples of mammary secretions were collected for bacterial counts at 3, 6, 9, 12 and 18 h post-inoculation and immediately before killing. Cubes (1 cm3) &mammary tissue from the distal third of the gland were taken from the same animals, at the time of killing. The number of bacteria in 1 ml of secretion and I g of tissue was estimated by the method of Miles, Misra and Irwin
(1938).
Results Acute mastiffs developed in all animals inoculated. Neither the number of bacteria inoculated nor the breed of the ewe influenced the time of onset or the severity of mastiffs.
Clinical Findings An increase in rectal temperature (Fig. 1) and slight enlargement and hardness of the inoculated glands, accompanied by erythema, were evident within 12 h of inoculation. In most ewes, the skin over the affected glands became hot and discoloured 24 to 48 h after inoculation (Fig. 2). Signs of acute mastitis were maximal on day 3 or 4 after inoculation (Figs 4 and 6) and most of the skin
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M e a n rectal temperatures & W e l s h M o u n t a i n ( . . . . . i n t r a m a m m a r y inoculation ofP. haemolytica,
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96
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Induction of Mastitis in Sheep
459
over the inoculated glands became blue or black with a clear line of demarcation, separating areas of necrotic tissue from the adjoining living tissue. The gland became soft, cold and insensitive and the overlying subcutaneous tissue was oedematous. The nature of the m a m m a r y secretions varied greatly depending on the stage of the disease. Among the early changes observed were increased viscosity and the presence of clots or flakes; in more advanced stages, secretions were watery, straw-coloured or brown with thick clots and, later, blood-stained. Eventually, only a few drops of fluid could be expressed from the teats of affected glands. In a few ewes, the uninoculated control glands were also swollen and the milk was reduced in quantity but was of normal appearance. However, in three severely affected ewes (one died at 5 days) there was clinical and pathological evidence of mastitis in the control gland. Systemic signs of illness appeared on day 1 after inoculation and included loss of appetite, listlessness, rapid breathing and fever. The mean rectal temperature of ewes during the acute phase ofmastitis is shown in Fig. 1. Some ewes became very ill 3 to 4 days after inoculation and had diarrhoea and more pronounced dyspnoea and pyrexia. They became depressed and did not allow the lambs to suck; they remained standing but were reluctant to walk. Four ewes died between days 4 and 6 post-inoculation. In two of four ewes which survived the acute phase, sloughing of necrotic tissue began about a week after inoculation and was complete within two weeks. Healing of the skin took approximately 8 weeks. During the period following sloughing, appetite was restored. In the other two ewes, suppuration of necrotic tissue developed about a week after inoculation. Sloughing took place over the next few weeks and necrotic tissue (Fig. 7) was gradually replaced by fibrous tissue and healing was complete in 8 to 9 weeks. The uninfected control glands in all four ewes involuted and were restored to normal function during the subsequent lactation.
Necropsy Findings (i) Mammary Glands. At 12 h post-inoculation, glands were slightly enlarged; petechiae and ecchymoses were evident on the cut surface. At and beyond 18 h, the glands were markedly swollen and the affected parenchyma was intensely reddened as the result ofhyperaemia and haemorrhage (Fig. 3). The red areas were either multifocal, often giving the gland a mottled appearance, or confluent and sharply demarcated from adjacent tissue. Such lesions were extensive 3 to 4 days after inoculation and often involved the whole gland (Fig. 5). In many affected glands, there was marked distension of veins and, in some, thrombosis was commonly recognized by the presence of firm, grey clots adhering to the intima. At 1 to 2 days post-inoculation, ducts in the affected glands were plugged by threads of fibrin and clots of milk (Fig. 3). A serous, sero-sanguinous, or purulent exudate filled and distended the acini and the interlobular septae were clearly demarcated. The milk ducts and cistern were filled with brown
460
E . T . S . E l - M a s a n n a t e t al .
Induction of Mastitis in Sheep
461
masses of" inflammatory exudate containing threads of fibrin and necrotic debris. In the three ewes in which the uninoculated gland was clinically affected, there were haemorrhagic lesions. In many ewes, there was much oedema in the subcutaneous tissue overlying the glands, in the inguinal region and, occasionally, in the ventral abdomen, flank, perineum and thighs. Supramammary lymph nodes were oedematous and swollen; some were intensely reddened and haemorrhagic. (ii) Other Organs and Tissues. There was no evidence of any lesions which might have signified generalized infection.
Hislopathological Findings The earliest change detected was infiltration of many neutrophils into the alveoli. Twelve hours after inoculation, this infiltration was widespread; small areas of haemorrhage and hyperaemia were evident at this stage. There was degeneration of alveolar epithelium and many desquamated epithelial cells, neutrophils and fluid were present in the alveoli. Within 24 h of inoculation, necrotic areas had developed within the parenchyma and there was considerable haemorrhage. Venous thrombosis was frequently seen. In the course of 3 to 4 days, the entire parenchyma was involved in inflammatory and degenerative reactions characterized by a dense infiltration of'neutrophils, hyperaemia, haemorrhage and necrosis.
Bacteriological Findings (i) Microscopic Examination of Mammary Secretion. Films of secretion from inoculated glands collected up to 9 h post-inoculation revealed bacteria only. At a n d beyond 12 h post-inoculation, P. haemolytica and leucocytes, chiefly
Fig. 2.
Udder o[' ewe, 1 clay after inoculation of left gland with 1000 cfu of P. tzaemolytica; the gland is enlarged and there is discolouration of the skin.
Fig, 3.
Cut surface of udder illustrated in Fig. 2. The left gland is enlarged, clots of milk are present and there is a hyperaemic reaction anteriorly.
Fig. 4.
Udder of ewe, 3 clays after inoculation of left gland with 100 cfu of P. haemogytica. There is enlargement of both glands and a pronounced cyanotic reaction of the skin.
Fig. 5.
Cut surface of the udder illustrated in Fig. 4. There is a confluent hyperaemic, haemorrhaglc and necrotic reaction involving the whole of the left gland. The parenchyma has separated from surrounding connective tissue and there is subcutaneous oedema ventrally.
Fig, 6,
Uclder o['ewe, 3 days al'tcr inoculation of left gland with 100 cth o['P. haemolytica; the gland atld teat are enlarged but there is no discolouradon of the skin.
Fig. 7.
Udder of ewe, 21 clays after inoculation of the right gland with l0 cfu ofP. haemo!ylica. Much of the gland has been slonghed. There is a purulent cxudate from the l'emainlng uecrotie tissue. At tile periphery of the lesion there is evidence ~[' healing.
462
E. T. S. E 1 - M a s a n n a t e t al.
neutrophils, were present in substantial numbers. Some leucocytes contained engulfed P. haemo~tica but, in most cases, large masses of P. haem@tica were free in the altered secretion. Leucocytes showed degenerative changes. (ii) Cultural Examination. At 3, 6, 9, 12, 18, 24, 48, 72 and 9 6 h postinoculation, P. haemolylica (A9) was isolated in pure culture from the secretion of all inoculated glands except, at 72 h, in two ewes in which it was isolated together with Streptococcus acidominimus in one and with Strep. boris in the other. At necropsy, P. haemolytica (A9) was isolated from the secretions and tissues of all inoculated glands. In three ewes, killed at 12, 24 and 48 h, respectively, after inoculation, P. haemolytica was isolated from the control glands. Macroscopic lesions were not seen in these glands at necropsy, but there was a marked increase in the number of cells in the milk ( > 14x l0 s cells per ml) of two of these ewes sampled immediately before killing at 24 and 48 h, respectively. In three ewes in which the uninoculated half developed mastitis, P. haemolytica was isolated in large numbers from the haemorrhagic lesions at the time of necropsy 3, 4 and 5 days, respectively, after inoculation. Whenever there was well established mastitis, P. haemolytica was usually isolated from supramammary lymph nodes. P. haemolytica was not isolated from the ipsilateral lymph node (ipln) of any of the eight ewes killed within 9 h of intramammary inoculation, but it was isolated from the contralateral lymph node (cln) of one of two ewes killed at 9 h. In 26 ewes that were killed or died between 12 h and 6 days after inoculation, in which the m a m m a r y lymph nodes were examined bacteriologically, P. haemolytica was isolated from the ipln of 20 ewes and from the cln of 16 of these; in two ewes it was isolated from the cln only and in four ewes from neither ipln nor cln. In three of the ewes from which P. haemolytica was isolated from the cln, the control glands were infected and had signs of mastitis. In one ewe from which P. haemolytica was isolated from the cln, the uninoculated gland was infected but there were no signs of mastitis. Y. haemolytica was not isolated from heart blood, liver or spleen of any ewe. In all experiments, every isolate of P. haemolytica grown from various tissues and secretions was examined serologically and found to belong to serotype A9. (iii) Bacterial Counts in Infected Mammary Secretions and Tissues. In each of five pairs of ewes in which the numbers of bacteria in m a m m a r y secretions and tissues were counted at intervals of 3, 6, 9, 12 and 18 h, respectively, after inoculation, maximal numbers were detected at 12 and 18 h (Fig. 8).
Cell Counts of Milk in Uninfected and Infected Glands A 10-fold increase in the cell count of milk from inoculated glands occurred at approximately 12 h post-inoculation. In the period when milk samples were suitable for examination, this increase in cell count was maintained at about 107 per ml. Generally, samples taken from inoculated glands later than 24 h after inoculation were physically unsuitable for counting cells.
463
I n d u c t i o n of M a s t i t i s i n Sheep l
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Mean colony counts ofP. haemolylica in milk ( ,) and mammary tissue ( . . . . . . . )o[each of five pairs of Poll Dorset ewes at wu'ious intervals following intramammary inoculation of 100 cfu,
Discussion Inoculation of the mammary gland with P. haernolytica, ES26L, invariably resulted in acute mastitis of varying severity. The production of acute mastitis with as few as 10 cfu, inoculated via the teat canal, is perhaps the most significant finding of this experiment. It allows the reasonable speculation that only a few organisms would be required to produce the disease under natural conditions once they were beyond the teat canal. It seems that adult sheep are much more susceptible to pure P. haemolytica biotype A infection by the intramammary route than by any other route tested in the past. For example, the experimental production of pneumonic pasteurellosis requires the intrabronchial administration of huge doses of the organism or the utilization of the synergic effects of viruses or mycoplasmas (Smith, 1990). The rapidity with which mammary lesions developed was evident 12 h after inoculation by the presence of hyperaemia and multifocal haemorrhages. The clinical signs and the nature of well developed lesions were similar to those of acute mastitis described by Leyshon (1929) and seen by us during the course of our field work. The development of infection of the mammary gland with isolate ES26L was manifested in three phases: (i) a lag phase of less than 3 h duration, (ii) a
464
E. T. S. E1-Musunnat e t al.
phase of bacterial multiplication, peaking at approximately 7 x 106 organisms per ml milk and 4 x 106 organisms per g tissue, 12 h post-inoculation (Fig. 8), (iii) a reactive phase ofleucoeytic infiltration, hyperaemia and haemorrhage at about 12 h post-inoculation when the number of bacteria was maximal. This phase was manifested macroscopically by intense red mottling of the mammary parenchyma. After 25 h, infected glands developed severe, irreversible pathological changes (Fig. 5). The onset of inflammation was marked by an increase in the number of somatic cells in milk, evident 12 h post-inoculation, at which time the mean cell counts of secretion from inoculated glands were 10-fold higher than those from uninoculated glands. A delay of approximately 12 h occurred between inoculation and the appearance of large numbers of leucocytes in milk, a time lapse allowing sufficient opportunity for the bacteria to become established. In three ewes, the control glands developed haemorrhagic necrotizing lesions from which P. haernolytica was isolated. In another three ewes, P. haemolytica was isolated from the control gland in which there were no lesions. It may be that, in some ewes, infection was transmitted to the control gland by cross sucking between inoculated and control glands. Since there was no evidence of generalized infection in any ewe, haematogenous spread of infection to uninoculated glands seems unlikely. Some control glands were swollen but the milk appeared normal and no bacteria were isolated from it. In the majority of ewes, P. haemolSica was isolated from both the supramammary ipln and cln but, in some ewes, P. haemolytica was isolated only from supramammary lymph nodes of the control uninoculated glands. This may be explained by a cross-over of the lymph vessels from each of the two glands of the udders, as described by Vollmerhaus
(1981). Cultural examination of heart blood, liver and spleen in all inoculated animals failed to demonstrate generalized infection. It seems probable that severe systemic illness and, in some ewes, death, was brought about by toxin(s) produced by P. haemolytica during its multiplication in the mammary gland; it is possible that endotoxin played a part in the genesis of clinical signs and pathological changes. In ewes that survived the acute disease, recovery was characterized by complete loss of mammary tissue of affected glands and therefore permanent loss of function. In the unaffected glands of ewes that recovered, lactation was normal in the following lambing season. This consistently reproducible experimental model will enable various aspects of the pathogenesis of pasteurella mastitis to be studied and will facilitate comparison of different strains of P. haemolytica especially in relation to virulence determinants. Acknowledgments
We are grateful to Mr R. Goldsmith for care of the sheep. This work was supported by the Overseas Development Administration of the Foreign Office.
Induction of Mastitis in Sheep
465
References
Jones, J. E. T. (1985). An investigation of mastitis in sheep: preliminary phase. Proceedings Sheep Veterinary Society, 10, 48-51. Krzyzanowski, J. and Lakomy, M. (1972). [Clinical course and experimental treatment of mastitis in sheep produced by the administration of Pasteurella haemolytica]. Polskie Archiwum Wele~ynaryjne 15, Fasc. 2, 287-305. Abstract in Veterinary Bulletin (1973), 43, 2458. Leyshon, W . J . (1929). An examination of a number &cases of ovine mastitis. The Velerinary Journal, 85, 286-300 and 331-344,. Miles, A. A., Misra, S. S. and Irwin, J. O. (1938). The estimation of the bactericidal power of the blood. Journal of IIygiene, Cambridge, 38, 732-739. Simmons,.G.C. and Ryley, J. W. (1954). Ovine mas~tis with special reference to mastitis caused by Pasleurella mastitidis. O.jleenslandJournal of Agricullural and Animal Sciences, 11, 29-35. Smith, G. R. (1990). Pasteurella infections. In Topley and Wilson's Principles of Baeleriology, Virology and Immunily Vol. 3. G.R. Smith and C.S. Easmon, Eds, Edward Arnold, London, pp. 382-388. Vollmerhaus, B. ( 1981). Lymphatic system. In The Anatomy of the Domestic Animals Vol. 3. A. Schummer, H. Wilkens, B. Volmerhaus and K. H. Habermehl, Eds, Paul Parey, Berlin, p. 4.17.
I Received, June 8lh, 1991 ~ Accepted, July 17th, 1991_]