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The effect of inoculation of Pasteurella haemolytica into the lactating mammary gland of mice, rats, rabbits, sows and cows
The Effect of Inoculation of Pasteurella haemolytica into the Lactating Mammary Gland of Mice, Rats, Rabbits, Sows and Cows G. H. Watkins, Ijepartment
q/‘:lnimal
Margaret
J. Scott
and J. E. T. Jones
Health, Ryal T’eterinarp CoIleLge. Ho1ton.r Park. Potterr Hnr. E.V6 I..C-B. @.A-.
Summary 41 isolate of’ Pasteurella
haemolytica (A9 I, which consistently produced st’vcrt
mastitis in ewes, was inoculated into the lactating mammaq’ glands of‘ ;I variet): of species.Mastitis did not develop after the inoculation of log-phase bacteria into the mammary gland of lactating mice, rats, rabbits or sow5bur did so in the mammary gland of two cows. Another A9 isolatefrom a ewe with mastitis and an Al isolate from a bovine pneumonic lung also induced mastiti5 in cows. Thus, in this study, P. haemolvti(.a produced mastitis only in ruminant ,mimals. Introduction haemo&ca biotype A causes pneumonia in sheep (Gilmour and Gilmour, 1989) and calves (Frank, 1989) and septicaemia in young lambs. Recently. it has been shown to be an important cause ofmastitis in ewes in the U.K. (Jones, 1985; Watkins, 1990) and has been reported to cause ovine and, to a lesser extent, caprine mastitis in many parts of the world. Like ovinr J)neumonic pasteurellosis, ovine mastitis in the U.K. is largely associated with serot ype A2 organisms. It has proved difficult to reproduce pneumonia consistently with P. haem(( tica in sheep without recourse to specific pathogen-free animals CGilmour. Thompson, Smith and Angus, 1975) and concurrent viral infection (Sharp. (;ilmour, Thompson and Rushton, 1978 I or in calves without use of‘vcry lar,q. numbers of bacteria (Gibbs, Alan, Wiseman and Selman, 1984; Jericho, 1987!. Septicaemic biotype A pasteurellosis has been produced by the intraperitoneai (i.p. 1 inoculation of young lambs with small numbers of bacteria (Smith, 1961 I. Likewise, mastitis, clinically and pathologically identical to that which occurs naturally, may be reproduced by- the injection of as few as 10 colon), forming units (cfu) of P. haemolytica into the lactating mammary gland of‘cw~s !El-Masannat, 1987; El-Masannat, Jones and Scott, 199 1j. Moreover, iso1atc.s from many sources possess the ability to produce severe mastitis experimentally, but some strains are of low patho,qenic-ity for the ovine mammar\ gland {Watkins and Jones, 1992). There have been many attempts to inducts disease with P. haemo@tiro in ;I Paskurelia
222
G. H. Watkins
et al.
variety of laboratory animals, most of which have been unsuccessful. Smith (1958) produced fatal infection in mice by intracerebral inoculation of small numbers of bacteria alone or by i.p. inoculation with hog gastric mucin Lhgm :. P. haemolytica was pathogenic for mice by the i.p. route if animals had first received haemoglobin (Chengappa, Carter and Chang, 1983) or an aqueous iron solution (Al-Sultan and Aitken, 1984). The potentiating effect of hgm may be due in part to the iron which it contains. However, dextran, a large molecular weight polysaccharide, also reduces the resistance of mice, guineapigs and hamsters (Wessman, 1967) to P. haemol_ytica, which suggests that the effect may be due to a physical property of the hgm.
El-Masannat
to reproduce mastitis in lactating mice by isolated from the milk of ewes with mastitis. He produced mastitis but only by means of the concurrent administration of aqueous iron solution and log-phase bacteria. In our study, P. haemolytica was injected into the mammary glands of different species in an effort to define more clearly the range of susceptible animals. intramammary
(1987) attempted
inoculation
with
P. haemo@ica
Materials
and
Methods
Animal5
Twenty primiparous, white, double caesarian derived mice, strain “CRH,
Glaxo
RBU” and 10 primiparous Wistar rats were caged individually, bedded on sawdust and fed on a proprietary pelleted feed. They were inoculated between 3 and 5 days post-partum. Five primiparous New Zealand White rabbits were penned individually on straw before and after parturition and fed on hay and a proprietary pelleted feed. They were inoculated 7 days post-partum. Four primiparous Large White X Landracc sows were housed in a farrowing pen in a controlled environment house on sawdust litter and fed a commercial sow diet. All had litter sizes within the normal range and were inoculated 2 weeks after farrowing. Two Friesian cows, which had yielded normal volumes of milk and had not had clinical mastitis during their current lactation, were used. Cow A was 7 years old and had calved 6 months previously. Cow B was 6 years old and had calved 4 months previously. The animals were housed on straw and fed hay and a small amount of dairy concentrate. They were milked twice daily by machine. Milk from the right fore-gland of both cows yielded coagulase-negative staphylococci on two separate occasrons before the experiment and, therefore, these glands were not inoculated. BacLe7-iai Inorula All animals were inoculated with an A9 isolate (Es26L) of P. haemoiytica which had consistently produced severe experimental mastitis in sheep / El-Masannat, 1987: El-Masannat et al., 1991; Watkins and Jones, 1992). Rats were also inoculated with another A9 (S328), an Al 1 (B32) and a TIS (B130) isolate of‘P. haemolvtica, all from ewes with mastitis. Additionally, the left fore-gland of cow A was inoculated with S328 and the right hind-gland of cow B with PG82/1, an ’ Al isolate of P. haemo(vticn from a bovine pneumonic lesion which has been used to induce bovine pneumonic pasteurellosis in calves (Gibbs, personal communication). Bacteria had been maintained in the lyophilized state after initial isolation. Onr day before inoculation, a vial of the required organism was reconstituted, streaked on Columbia blood agar (CBA) and incubated aerobically at 37°C. After 18 h, two colonies were suspended in 20 ml ofTodd-Hewitt broth and incubated at 37°C for 5 11.
P. haemolytica
in the
Mammary
9. ‘17 ..-.~
Gland
Inocula were prepared by making serial IO-fold dilutions of this broth phosphate-buffered saline (PBS) and adding between 0.02 and 0.10 appropriate dilution to PBS (for dose volumes see Table 1). The sizes of were determined by the plate count technique of Miles, Misra and Irwin
culture in ml of tht the inocula (1938j.
Inoculation Procedures ?vGce, rats and rabbits were anaesthetized with ether. In mice and rats, the inoculurn :0.05 ml) was injected at the teat base into the gland cistern by means of a 26-gaugt needle. In rabbits, the location of the openings of the numerous teat ducts wcr( ascertained by expressing a small amount of’milk. One opening was cannulatrd with :I i\’ A Howe and Co. Ltd, Ranhur). 25-gauge, round-ended, stainless steel cannula Ox&) and one lobe of the gland inoculated. A similar method was used in sows: one gland was inoculated by means 01‘ .t 2 1-gauge cannula inserted to a depth of 2.0 cm. The cows were inoculated in the teat sin& with similar cannulae. Details of the inoculation procedures are shown in ‘l‘abtt~ I. An appropriate volume of PBS was injected into control glands. Suckled young were removed from their mothers before maculation and rrturntd ‘ifter- 2 h. The cows were machine-milked 5 h after inoculation. .Monitoring
oj’E.uperimental
Animals
.1nimals were examined for signs of mastitis twice daily for 3 days after inoculation and then, except for the cows and sows, they were killed. The inoculated glands werc Ixxamined post-mortem and samples taken for bacteriological and histopatholo,+;II c.xamination. Milk samples were collected daily from the inoculated and control glancis of sows and from all four glands of the two cows at 5, 8, 12. 17 and 19 1~ Alice inoculation, for bacteriological and microscopical examination.
The
results
of the
inoculation
of mammary
Table glands
haemolytica
1 of various
animal
species
with
Pasteurella
224 Laboratorv
G. H. Watkins
et al.
Procedures
Milk samples collected from cows and sows were inoculated on to CBA and incubatvtl aerobically at 37°C for 48 h; films wcrc stainrd -by thv Gicmsa method. Samples 01’ inoculated and control mammary elands and of hver, spleen and heart blood front each of the mice, rats and rabbits wet-r collected post-mortem and t~saminrd bacteriologically in a similar manner. Samples of inoculatrd and control glands WV fixed and processed by standard methods for histopathological c.xamination.
Results Mice, Rats. Rabbits and Sows No clinical changes were observed in the inoculated or control mammary glands after inoculation. Post-mortem examination of mice, rats and rabbits 3 days after inoculation did not reveal any gross or histological abnormality 01 control or inoculated glands. No bacteria were cultured from the mammary glands, liver, spleen or heart blood of these animals. The inoculated and control glands of the sows remained clinically normal and milk was bacteriologically negative throughout the experiment.
cow A Mastitis developed in the three inoculated glands but not in the control gland. The changes seen in the left fore- and left hind-glands, which had been inoculated with 1.13 x 10” cfu of S328 (A9) and 0.98 x 10” cfu of Es26L ( A9 ), respectively, were similar and essentially the same as those described in ewes (El-Masannat, 1987). Inflammation of the gland and alterations in the milk (increased numbers of neutrophils initially, then development to a serous secretion) were first noted 12 h after inoculation and became progressively more severe by 19 h, when the animal was treated. The right hind-gland, which had been inoculated with approximatel) 200 cfu of Es26L, did not show any clinical changes, but the milk of this gland was muco-purulent after 15 h and sero-purulent after 19 h. The right foregland, which received PBS, remained unchanged. Bacteria
were
first
isolated
from
milk
samples
collected
after
12 h and
subsequently were present in large numbers. Pyrexia I 4 1°C 1, tachycardia, tachypnoca, anorexia, depression and lethargy were observed 12 to 17 h after inoculation
and
the
cow
became
recumbent
on
its
sternum
after
19 h.
Antibiotic therapy with oxytetracyclinc (Terramycin QlOO, Pfizer UK; 30 ml [3 g], intravenously [i.v.], twice daily) was instigated at this stage and continued for 5 days. During this time the systemic signs abated, but milk production in the control gland ceased. No bacteria were cultured from mammary secretions at the cessation of treatment and the mammary glands returned to clinical normality and underwent apparently normal involution.
P.
Cou,
haemolytica
in the
Mammary
Gland
wr, _...I
B
Both inoculated glands (approximately 150 cfu Es26L [A91 into the left forcgland and 0.53 x 10” cfu PG82/1 [Al] into the right hind-gland) developed severe acute mastitis. The right fore-gland, which received PBS, remained unchanged. The cow became very depressed 12 h after inoculation and antibiotictherapy (as for cow A) was instigated 5 h later. Initially, there was :i favourable response and no bacteria were cultured from mammary secretion after 2 days of treatment. Subsequently, the cow deteriorated. Laboratory rxamination of venous blood revealed uraemia, hypoalbuminaemia. hypochloraemia and a marked neutrophilia with many degenerative and immature neutrophils. The animal did not respond to oral and i.v. fluid therapy and was killed 10 days after inoculation. Abnormalities detected at necropsy were confined to the mammary gland and associated lymph nodes. In the left fore-gland, there were red. spherical hardened areas, 2 to 4 cm in diameter and the major ducts were filled with pus. Histologically, there was some necrosis of epithelial cells and a breakdown in the alveolar architecture. Intra-alveolar neutrophil accumulation was seen iii all parts of the gland and the inter-alveolar areas were massively distended b\, infiltration of inflammatory cells and proliferating fibrous tissue. The right hind-gland was uniformly swollen and firm but not hard and thcrc were no reddened areas in the parenchyma. Histopathological examination sholvcd that the alveolar structure was intact, but the inter-alveolar connective tissue was infiltrated with neutrophils and fibroblasts. Most alveoli were fillrd with neutrophils but some contained normal-staining secretion. ‘The gland cistern and major and minor ducts were full of thick, cream-coloured pus. Both left and right supra-mammary lymph nodes were approximately twic.t. their usual size, the cortices were reddened and the rortico-rncdullary junction \vas not discernible. iKo bacteria were isolated from the mammary ,glands, supra-mammary lymph nodes, heart blood, liver, spleen or kidneys, Discussion Inoculation of P. haemol_ytica into the lactating mammary gland of mice, rats. rabbits and sows did not result in the establishment of infection, but the inoculation of‘two lactating cows with three isolates i’l’able I ) of P. haemolvtirci rrsulted in acute, severe mastitis. The only report of an attempt to produce 1’. haemo&ictl mastitis expcrimentally in laboratory animals is that of El-Masannat (1987) who failed to establish infection in mice with the isolates and procedures used in our presrnt study. However, in his experiments, in which mice had previously been gi~~~~ i.s. injections of an aqueous iron solution, 13 (68 per centj of 19 inoculated mammae developed mastitis. Other workers have demonstrated that I’. haemo/ylica is of low pathogenicity for mice, rats and rabbits by the i.p., i.v. or subcutaneous routes unless an enhancer, such as h,gm or ferric ammonium
226
G. H. Watkins
et al.
citrate, is used (Smith, 19S8; Al-Sultan and Aitken, 1984). ‘Ihe present report shows that mice, rats, rabbits and sows are also resistant to intramammary infection. This finding has two main implications: first, that laboratory animal models of P. haemo(vtica mastitis are unlikely to be of use in future inv,estigations of this disease; second, that knowledge of the susceptibility of various species to infection may help in understanding the mechanisms of pathogenicity. The experimental production of mastitis in sheep with a bovine pneumonic. isolate of P. haemol_vtica has been described by Watkins and *Jones ( 1992 j In the present experiments, this isolate and two originating from ovine mastitic milk were used to inoculate two dairy cows. ‘411 five infected glands, two of w-hich received small doses of P. hnemo(_ytica, devreloped mastitis; both cows becamt severely ill, one failing to recover despite an apparent bacteriological cure. Unlike laboratory animals, cattle are susceptible to natural 1’. kaemo&tica infections, but mastitis caused by this organism is rarely reported (hlaplesden and Carter, 1955; Anderson and Jensen, 1977; Roberts, 1990). P. hnemolvtircz is carried in the upper respiratory tract of calves, as in sheep, but :A1 rather than A2 is the predominant serotype (Wray and Thompson, 197 1I. If cows arc susceptible to P. haemo!vtica mastitis and if the sucking calf is a potential sourc‘e of infection for the cow, in the way the lamb may’ be for the ewe (Watkins and eJo~~es, 1992), it is perhaps surprising that the disease does not occur more oficn in suckler cows. It is possible that the Al isolate used in this experiment was atypical and that the majority of strains carried by the calf are not capable 01 producing mastitis. Alternatively, differences in sucking behaviour between calves and lambs or in the anatomy and antibacterial properties of the teat duct may account for the low incidence of the disease in cows and its importance in ewes. Mastitis has been induced by the intramammary inoculation of lactating ewes with ovine pneumonic and bovine pneumonic isolates of P. hnemolvticm (Watkins and Jones, 1992). In our present study, mastitis in cows was produced by ovine mastitic and bovine pneumonic isolates of P. /memo[vtictl. The results suggest that these isolates arc capable of infecting the mammary gland of ruminant, but not non-ruminant animals. Properties of P. hamrt$rlic~ which constitute probable virulence factors in the development of’ mastitis might reasonably be expected to show similar activity in a particular range 01‘ host species. One such virulence factor is the cytotoxin \leucotoxin) which animals in vitro destroys leucocytes of ruminant, but not non-ruminant (Kaehler, Markham, Muscoplat and ,Johnson, 1980; Chang, Renshaw , Martens and Livingstone, 1986). The range of host species susceptible to the possible virulence factors of P. hnemo~vticarequires further investigation.
Acknowledgments
L niversity of Glasgow Dr Alison Gibbs of the Department of Veterinary Medicine, kindly provided the bovine P. haemo&cn (Al) isolate. This work was supported by a grant from the Agricultural and Food Research Council.
P. haemolytica
in the
Mammary
Gland
L’L’7
References
of Pasteurella haemolvtir-a infecti )II Al-Sultan, I. I. and Aitken, I. D. (1984). P romotion in mice by iron. Research in Veterinary Science, 36, 385-386. Anderson, 0. and Jensen, J. (1977). [Pasteurella haemo@ica as a cause of acute bn\~~nc~ mastitis. 1 Dansk Veterinaertidsskri, 60, 145- 146. Chang. Y.-F., Renshaw, H. W., Martens? R..J. and Livingstone, C. \V. 1)l!Ulti Pasteurella haemolytira leukotoxin: Chemilumtnrscent responses of periphrral ltlootl leukocytes from several different mammalian species to leukotosin atrtl opsonin-treated living and killed P. haemo/vtica and ,Etaphv/ococcu.r aureui. :Inre~~~a~/ ~-Journal of‘ T’eterinary Research? 47, 67-86. C:hengappa. ,2I. lf., Carter, G. R. and (:hany, ‘I‘. S. 1983 1. Hacmoglcrl)itt tmhancrmrnt of experimental infection of mice M ith Pn,steurella haemo/1,//i.a. :lmerican journal oJ‘ lTeterinarv Research, 44, 154.ip154C. El-~~fasannat, E. ‘1‘. S. ( 1987). PhD Thesis. Ovinc~ mastitis, with special rr~lercvtc.c~ 10 mastitis caused bv Pasteure/la haemolgtictr. I~.niversit) of‘ London, IT.K. El-.\lasannat. E. T. i.,
228
G. H. Watkins
et al.
q/‘:lnimal
Margaret
J. Scott
and J. E. T. Jones
Health, Ryal T’eterinarp CoIleLge. Ho1ton.r Park. Potterr Hnr. E.V6 I..C-B. @.A-.
Summary 41 isolate of’ Pasteurella
haemolytica (A9 I, which consistently produced st’vcrt
mastitis in ewes, was inoculated into the lactating mammaq’ glands of‘ ;I variet): of species.Mastitis did not develop after the inoculation of log-phase bacteria into the mammary gland of lactating mice, rats, rabbits or sow5bur did so in the mammary gland of two cows. Another A9 isolatefrom a ewe with mastitis and an Al isolate from a bovine pneumonic lung also induced mastiti5 in cows. Thus, in this study, P. haemolvti(.a produced mastitis only in ruminant ,mimals. Introduction haemo&ca biotype A causes pneumonia in sheep (Gilmour and Gilmour, 1989) and calves (Frank, 1989) and septicaemia in young lambs. Recently. it has been shown to be an important cause ofmastitis in ewes in the U.K. (Jones, 1985; Watkins, 1990) and has been reported to cause ovine and, to a lesser extent, caprine mastitis in many parts of the world. Like ovinr J)neumonic pasteurellosis, ovine mastitis in the U.K. is largely associated with serot ype A2 organisms. It has proved difficult to reproduce pneumonia consistently with P. haem(( tica in sheep without recourse to specific pathogen-free animals CGilmour. Thompson, Smith and Angus, 1975) and concurrent viral infection (Sharp. (;ilmour, Thompson and Rushton, 1978 I or in calves without use of‘vcry lar,q. numbers of bacteria (Gibbs, Alan, Wiseman and Selman, 1984; Jericho, 1987!. Septicaemic biotype A pasteurellosis has been produced by the intraperitoneai (i.p. 1 inoculation of young lambs with small numbers of bacteria (Smith, 1961 I. Likewise, mastitis, clinically and pathologically identical to that which occurs naturally, may be reproduced by- the injection of as few as 10 colon), forming units (cfu) of P. haemolytica into the lactating mammary gland of‘cw~s !El-Masannat, 1987; El-Masannat, Jones and Scott, 199 1j. Moreover, iso1atc.s from many sources possess the ability to produce severe mastitis experimentally, but some strains are of low patho,qenic-ity for the ovine mammar\ gland {Watkins and Jones, 1992). There have been many attempts to inducts disease with P. haemo@tiro in ;I Paskurelia
222
G. H. Watkins
et al.
variety of laboratory animals, most of which have been unsuccessful. Smith (1958) produced fatal infection in mice by intracerebral inoculation of small numbers of bacteria alone or by i.p. inoculation with hog gastric mucin Lhgm :. P. haemolytica was pathogenic for mice by the i.p. route if animals had first received haemoglobin (Chengappa, Carter and Chang, 1983) or an aqueous iron solution (Al-Sultan and Aitken, 1984). The potentiating effect of hgm may be due in part to the iron which it contains. However, dextran, a large molecular weight polysaccharide, also reduces the resistance of mice, guineapigs and hamsters (Wessman, 1967) to P. haemol_ytica, which suggests that the effect may be due to a physical property of the hgm.
El-Masannat
to reproduce mastitis in lactating mice by isolated from the milk of ewes with mastitis. He produced mastitis but only by means of the concurrent administration of aqueous iron solution and log-phase bacteria. In our study, P. haemolytica was injected into the mammary glands of different species in an effort to define more clearly the range of susceptible animals. intramammary
(1987) attempted
inoculation
with
P. haemo@ica
Materials
and
Methods
Animal5
Twenty primiparous, white, double caesarian derived mice, strain “CRH,
Glaxo
RBU” and 10 primiparous Wistar rats were caged individually, bedded on sawdust and fed on a proprietary pelleted feed. They were inoculated between 3 and 5 days post-partum. Five primiparous New Zealand White rabbits were penned individually on straw before and after parturition and fed on hay and a proprietary pelleted feed. They were inoculated 7 days post-partum. Four primiparous Large White X Landracc sows were housed in a farrowing pen in a controlled environment house on sawdust litter and fed a commercial sow diet. All had litter sizes within the normal range and were inoculated 2 weeks after farrowing. Two Friesian cows, which had yielded normal volumes of milk and had not had clinical mastitis during their current lactation, were used. Cow A was 7 years old and had calved 6 months previously. Cow B was 6 years old and had calved 4 months previously. The animals were housed on straw and fed hay and a small amount of dairy concentrate. They were milked twice daily by machine. Milk from the right fore-gland of both cows yielded coagulase-negative staphylococci on two separate occasrons before the experiment and, therefore, these glands were not inoculated. BacLe7-iai Inorula All animals were inoculated with an A9 isolate (Es26L) of P. haemoiytica which had consistently produced severe experimental mastitis in sheep / El-Masannat, 1987: El-Masannat et al., 1991; Watkins and Jones, 1992). Rats were also inoculated with another A9 (S328), an Al 1 (B32) and a TIS (B130) isolate of‘P. haemolvtica, all from ewes with mastitis. Additionally, the left fore-gland of cow A was inoculated with S328 and the right hind-gland of cow B with PG82/1, an ’ Al isolate of P. haemo(vticn from a bovine pneumonic lesion which has been used to induce bovine pneumonic pasteurellosis in calves (Gibbs, personal communication). Bacteria had been maintained in the lyophilized state after initial isolation. Onr day before inoculation, a vial of the required organism was reconstituted, streaked on Columbia blood agar (CBA) and incubated aerobically at 37°C. After 18 h, two colonies were suspended in 20 ml ofTodd-Hewitt broth and incubated at 37°C for 5 11.
P. haemolytica
in the
Mammary
9. ‘17 ..-.~
Gland
Inocula were prepared by making serial IO-fold dilutions of this broth phosphate-buffered saline (PBS) and adding between 0.02 and 0.10 appropriate dilution to PBS (for dose volumes see Table 1). The sizes of were determined by the plate count technique of Miles, Misra and Irwin
culture in ml of tht the inocula (1938j.
Inoculation Procedures ?vGce, rats and rabbits were anaesthetized with ether. In mice and rats, the inoculurn :0.05 ml) was injected at the teat base into the gland cistern by means of a 26-gaugt needle. In rabbits, the location of the openings of the numerous teat ducts wcr( ascertained by expressing a small amount of’milk. One opening was cannulatrd with :I i\’ A Howe and Co. Ltd, Ranhur). 25-gauge, round-ended, stainless steel cannula Ox&) and one lobe of the gland inoculated. A similar method was used in sows: one gland was inoculated by means 01‘ .t 2 1-gauge cannula inserted to a depth of 2.0 cm. The cows were inoculated in the teat sin& with similar cannulae. Details of the inoculation procedures are shown in ‘l‘abtt~ I. An appropriate volume of PBS was injected into control glands. Suckled young were removed from their mothers before maculation and rrturntd ‘ifter- 2 h. The cows were machine-milked 5 h after inoculation. .Monitoring
oj’E.uperimental
Animals
.1nimals were examined for signs of mastitis twice daily for 3 days after inoculation and then, except for the cows and sows, they were killed. The inoculated glands werc Ixxamined post-mortem and samples taken for bacteriological and histopatholo,+;II c.xamination. Milk samples were collected daily from the inoculated and control glancis of sows and from all four glands of the two cows at 5, 8, 12. 17 and 19 1~ Alice inoculation, for bacteriological and microscopical examination.
The
results
of the
inoculation
of mammary
Table glands
haemolytica
1 of various
animal
species
with
Pasteurella
224 Laboratorv
G. H. Watkins
et al.
Procedures
Milk samples collected from cows and sows were inoculated on to CBA and incubatvtl aerobically at 37°C for 48 h; films wcrc stainrd -by thv Gicmsa method. Samples 01’ inoculated and control mammary elands and of hver, spleen and heart blood front each of the mice, rats and rabbits wet-r collected post-mortem and t~saminrd bacteriologically in a similar manner. Samples of inoculatrd and control glands WV fixed and processed by standard methods for histopathological c.xamination.
Results Mice, Rats. Rabbits and Sows No clinical changes were observed in the inoculated or control mammary glands after inoculation. Post-mortem examination of mice, rats and rabbits 3 days after inoculation did not reveal any gross or histological abnormality 01 control or inoculated glands. No bacteria were cultured from the mammary glands, liver, spleen or heart blood of these animals. The inoculated and control glands of the sows remained clinically normal and milk was bacteriologically negative throughout the experiment.
cow A Mastitis developed in the three inoculated glands but not in the control gland. The changes seen in the left fore- and left hind-glands, which had been inoculated with 1.13 x 10” cfu of S328 (A9) and 0.98 x 10” cfu of Es26L ( A9 ), respectively, were similar and essentially the same as those described in ewes (El-Masannat, 1987). Inflammation of the gland and alterations in the milk (increased numbers of neutrophils initially, then development to a serous secretion) were first noted 12 h after inoculation and became progressively more severe by 19 h, when the animal was treated. The right hind-gland, which had been inoculated with approximatel) 200 cfu of Es26L, did not show any clinical changes, but the milk of this gland was muco-purulent after 15 h and sero-purulent after 19 h. The right foregland, which received PBS, remained unchanged. Bacteria
were
first
isolated
from
milk
samples
collected
after
12 h and
subsequently were present in large numbers. Pyrexia I 4 1°C 1, tachycardia, tachypnoca, anorexia, depression and lethargy were observed 12 to 17 h after inoculation
and
the
cow
became
recumbent
on
its
sternum
after
19 h.
Antibiotic therapy with oxytetracyclinc (Terramycin QlOO, Pfizer UK; 30 ml [3 g], intravenously [i.v.], twice daily) was instigated at this stage and continued for 5 days. During this time the systemic signs abated, but milk production in the control gland ceased. No bacteria were cultured from mammary secretions at the cessation of treatment and the mammary glands returned to clinical normality and underwent apparently normal involution.
P.
Cou,
haemolytica
in the
Mammary
Gland
wr, _...I
B
Both inoculated glands (approximately 150 cfu Es26L [A91 into the left forcgland and 0.53 x 10” cfu PG82/1 [Al] into the right hind-gland) developed severe acute mastitis. The right fore-gland, which received PBS, remained unchanged. The cow became very depressed 12 h after inoculation and antibiotictherapy (as for cow A) was instigated 5 h later. Initially, there was :i favourable response and no bacteria were cultured from mammary secretion after 2 days of treatment. Subsequently, the cow deteriorated. Laboratory rxamination of venous blood revealed uraemia, hypoalbuminaemia. hypochloraemia and a marked neutrophilia with many degenerative and immature neutrophils. The animal did not respond to oral and i.v. fluid therapy and was killed 10 days after inoculation. Abnormalities detected at necropsy were confined to the mammary gland and associated lymph nodes. In the left fore-gland, there were red. spherical hardened areas, 2 to 4 cm in diameter and the major ducts were filled with pus. Histologically, there was some necrosis of epithelial cells and a breakdown in the alveolar architecture. Intra-alveolar neutrophil accumulation was seen iii all parts of the gland and the inter-alveolar areas were massively distended b\, infiltration of inflammatory cells and proliferating fibrous tissue. The right hind-gland was uniformly swollen and firm but not hard and thcrc were no reddened areas in the parenchyma. Histopathological examination sholvcd that the alveolar structure was intact, but the inter-alveolar connective tissue was infiltrated with neutrophils and fibroblasts. Most alveoli were fillrd with neutrophils but some contained normal-staining secretion. ‘The gland cistern and major and minor ducts were full of thick, cream-coloured pus. Both left and right supra-mammary lymph nodes were approximately twic.t. their usual size, the cortices were reddened and the rortico-rncdullary junction \vas not discernible. iKo bacteria were isolated from the mammary ,glands, supra-mammary lymph nodes, heart blood, liver, spleen or kidneys, Discussion Inoculation of P. haemol_ytica into the lactating mammary gland of mice, rats. rabbits and sows did not result in the establishment of infection, but the inoculation of‘two lactating cows with three isolates i’l’able I ) of P. haemolvtirci rrsulted in acute, severe mastitis. The only report of an attempt to produce 1’. haemo&ictl mastitis expcrimentally in laboratory animals is that of El-Masannat (1987) who failed to establish infection in mice with the isolates and procedures used in our presrnt study. However, in his experiments, in which mice had previously been gi~~~~ i.s. injections of an aqueous iron solution, 13 (68 per centj of 19 inoculated mammae developed mastitis. Other workers have demonstrated that I’. haemo/ylica is of low pathogenicity for mice, rats and rabbits by the i.p., i.v. or subcutaneous routes unless an enhancer, such as h,gm or ferric ammonium
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citrate, is used (Smith, 19S8; Al-Sultan and Aitken, 1984). ‘Ihe present report shows that mice, rats, rabbits and sows are also resistant to intramammary infection. This finding has two main implications: first, that laboratory animal models of P. haemo(vtica mastitis are unlikely to be of use in future inv,estigations of this disease; second, that knowledge of the susceptibility of various species to infection may help in understanding the mechanisms of pathogenicity. The experimental production of mastitis in sheep with a bovine pneumonic. isolate of P. haemol_vtica has been described by Watkins and *Jones ( 1992 j In the present experiments, this isolate and two originating from ovine mastitic milk were used to inoculate two dairy cows. ‘411 five infected glands, two of w-hich received small doses of P. hnemo(_ytica, devreloped mastitis; both cows becamt severely ill, one failing to recover despite an apparent bacteriological cure. Unlike laboratory animals, cattle are susceptible to natural 1’. kaemo&tica infections, but mastitis caused by this organism is rarely reported (hlaplesden and Carter, 1955; Anderson and Jensen, 1977; Roberts, 1990). P. hnemolvtircz is carried in the upper respiratory tract of calves, as in sheep, but :A1 rather than A2 is the predominant serotype (Wray and Thompson, 197 1I. If cows arc susceptible to P. haemo!vtica mastitis and if the sucking calf is a potential sourc‘e of infection for the cow, in the way the lamb may’ be for the ewe (Watkins and eJo~~es, 1992), it is perhaps surprising that the disease does not occur more oficn in suckler cows. It is possible that the Al isolate used in this experiment was atypical and that the majority of strains carried by the calf are not capable 01 producing mastitis. Alternatively, differences in sucking behaviour between calves and lambs or in the anatomy and antibacterial properties of the teat duct may account for the low incidence of the disease in cows and its importance in ewes. Mastitis has been induced by the intramammary inoculation of lactating ewes with ovine pneumonic and bovine pneumonic isolates of P. hnemolvticm (Watkins and Jones, 1992). In our present study, mastitis in cows was produced by ovine mastitic and bovine pneumonic isolates of P. /memo[vtictl. The results suggest that these isolates arc capable of infecting the mammary gland of ruminant, but not non-ruminant animals. Properties of P. hamrt$rlic~ which constitute probable virulence factors in the development of’ mastitis might reasonably be expected to show similar activity in a particular range 01‘ host species. One such virulence factor is the cytotoxin \leucotoxin) which animals in vitro destroys leucocytes of ruminant, but not non-ruminant (Kaehler, Markham, Muscoplat and ,Johnson, 1980; Chang, Renshaw , Martens and Livingstone, 1986). The range of host species susceptible to the possible virulence factors of P. hnemo~vticarequires further investigation.
Acknowledgments
L niversity of Glasgow Dr Alison Gibbs of the Department of Veterinary Medicine, kindly provided the bovine P. haemo&cn (Al) isolate. This work was supported by a grant from the Agricultural and Food Research Council.
P. haemolytica
in the
Mammary
Gland
L’L’7
References
of Pasteurella haemolvtir-a infecti )II Al-Sultan, I. I. and Aitken, I. D. (1984). P romotion in mice by iron. Research in Veterinary Science, 36, 385-386. Anderson, 0. and Jensen, J. (1977). [Pasteurella haemo@ica as a cause of acute bn\~~nc~ mastitis. 1 Dansk Veterinaertidsskri, 60, 145- 146. Chang. Y.-F., Renshaw, H. W., Martens? R..J. and Livingstone, C. \V. 1)l!Ulti Pasteurella haemolytira leukotoxin: Chemilumtnrscent responses of periphrral ltlootl leukocytes from several different mammalian species to leukotosin atrtl opsonin-treated living and killed P. haemo/vtica and ,Etaphv/ococcu.r aureui. :Inre~~~a~/ ~-Journal of‘ T’eterinary Research? 47, 67-86. C:hengappa. ,2I. lf., Carter, G. R. and (:hany, ‘I‘. S. 1983 1. Hacmoglcrl)itt tmhancrmrnt of experimental infection of mice M ith Pn,steurella haemo/1,//i.a. :lmerican journal oJ‘ lTeterinarv Research, 44, 154.ip154C. El-~~fasannat, E. ‘1‘. S. ( 1987). PhD Thesis. Ovinc~ mastitis, with special rr~lercvtc.c~ 10 mastitis caused bv Pasteure/la haemolgtictr. I~.niversit) of‘ London, IT.K. El-.\lasannat. E. T. i.,
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