Laboratory Animals (1975) 9, 197-200.
197
AN EYE INFECTION
IN LABORATORY
MICE
ASSOCIATED WITH PASTEURELLA
PNEUMOTROPICA by
J. R. NEEDHAM
and J. E. COOPER
Animal Division, Medical Research Council, Clinical Research Centre, Watford Road, Harro\\!, HAl 3UJ SUMMARY
Pasteurella pneumotropica was isolated from cases of clinical conjunctivitis in mice. No isolates were obtained from normal mice. At this Centre there is a yearly turnover of about 72 000 conventional mice, which are held in polypropylene boxes with wood chips for bedding. Over a period of 25 months, eye infections were observed in 45 stock mice. A literature search revealed a scarcity of reports on such conditions. Saunders (1967) reported Staphylococcus aureus from eyes of rats and a reference was made to the isolation of a Pasteurella sp. from inflamed rat eyes. Young & Hill (1974) isolated 'J?.asteurella pneumotropica from rats with conjunctivitis. The 45 mice e'Xamined presented with closed eyes, without purulent discharge but with increased lachrymal secretion; only 1 showed a grossly disTable 1. Distribution
Strain AKR C57BIOD2 BALB/C CBA CBA BALB/C BALB/C
CBA
Number of mice
Age (where known)
1
1 1 1
6 weeks 6 weeks
5 6 .1
3 weeks
10
NUDE
7
A
1 1
NZB
of mice with infected eyes.
A A AKR
2 1
AKR
3
CBA
1
8 months
3 11 weeks
Downloaded from lan.sagepub.com at NORTHERN ARIZONA UNIVERSITY on May 26, 2015
Sex
198
J. R. NEEDHAM
AND
J. E. COOPER
tended eye, the conjunctival surface of which was covered with a hard scablike plaque. They were distributed throughout the colony and ranged from 3 to 32 weeks of age. Table 1 shows the strains and numbers of animals involved. All animals were killed by the intraperitoneal injection of sodium pentobarbitone. With sterile forceps the lower eyelid of each infected eye was everted and material was collected using a sterile wire loop (Jarvis, 1973). The sample was cultured on 2 blood-agar plates and on a Loeffler's serumagar slope. The media were incubated at 37°C overnight; one blood agar plate was incubated anaerobically, the other media aerobically. Any plate showing no growth after overnight incubation was incubated for a further 24 hours. After incubation, identification tests were performed and the organisms classified according to Cowan & Steele (1966). No attempt was made to examine specimens for the presence of Mycoplasma spp. The internal organs of each animal were examined macroscopically but were not subjected to any cultural techniques. The eyes, heart, kidneys, spleen, liver and lungs of 5 affected mice were examined histologically as was the grossly distended eye. Specimens were placed in 10 % formol-saline prior to subsequent staining with Cole's haematoxylin and eosin. The eyes of 10 healthy mice from a barrier-maintained (SPF) breeding unit were examined microbiologically and the eyes of 2 of them histologically in the manner described. Of the 45 mice examined 39 yielded a pure growth of Pasteurella pneumotropica, 2 yielded a pure growth of Staphylococcus aureus, and no isolates were obtained from 4 mice. No bacterial isolates were obtained from the 10 control mice. Histopathological examination of the eyes of the 5 mice showed a keratoconjunctivitis in each case. The cornea was grossly thickened and oedematous and the substantia propria was infiltrated by large numbers of inflammatory cells which were mainly neutrophils but also included occasional plasma cells. There was a purulent exudate on the surface of the conjunctiva and within the fornices (the latter showed hyperkeratinisation). In 3 cases there was a more generalised inflammation with a polymorphonuclear infiltration of the limbus and, to a lesser extent, sclera; the blood vessels in these areas were dilated. There was an inflammatory cell infiltration around the lachrymal glands, but no apparent glandular involvement (Fig. 1). In no case was there abnormality of the intraocular structures. The eyelids showed no abnormalities other than possible hyperkeratinisation. The grossly distended eye showed histological evidence of a long-standing panophthalmitis. The conjunctival plaque consisted of fibrin which contained many neutrophils, some of them pyknotic. Intraocular structures showed considerable disruption and a severe inflammatory reaction. There was an extensive fibrosis both within the eye and in the eyelid adjacent to the lachrymal glands. Heart, kidney and spleen contained no histopathological lesions. All 5
Downloaded from lan.sagepub.com at NORTHERN ARIZONA UNIVERSITY on May 26, 2015
PASTEURELLA
INFECTION OF MOUSE EYES
199
livers showed varying degrees of vacuolation of hepatocytes and small numbers of scattered cellular aggregates-mainly composed of neutrophils. 4 of the lungs appeared normal but 1 showed a considerable degree of peri bronchiolar lymphocytic cuffing.
Fig. 1. Portion of cye and eyelid of affected mouse. Showing extensive polymorphonuclear infiltration but no involvement of fornix or lachrymal gland. Cole's haematoxylin and eosin. Line represents 500 j.Im.
The histopathological diagnosis of keratoconjunctivitis would agree with the condition described by Saunders (1967), from which Staphylococcus aureus was isolated. In the present study it is probable that the origin of the Pasteurella pneumotropica was the upper respiratory tract as all mice in the conventional rooms (where the infections occurred) were known to be colonised by this organism. However, other factors may be involved in the pathogenesis of eye infections. In this survey only 1 species of bacterium was isolated from the eyes in contrast to the findings of Young & Hill (1974). Hill (1974) demonstrated multiplication of a mixture of organisms, including P. pneumotropica, in rat eyes following experimental inoculation, but there was no evidence of clinical conjunctivitis. Similarly at this Centre conjunctivitis was not observed macroscopically when over 400 mice were inoculated intranasally with P. pneumotropica and spillage into the eye of the bacterial suspension occurred (work to be reported). This evidence, together with that of Young & Hill (1974), suggests that predisposing factors, for example eye damage, may be necessary before ocular infection with P. pneumotropica can occur.
Downloaded from lan.sagepub.com at NORTHERN ARIZONA UNIVERSITY on May 26, 2015
200
J. R. NEEDHAM
AND
J. E. COOPER
REFERENCES Cowan, S. T. & Steel, K. J. (1966). Manualfor the identification of medical bacteria. Cambridge: Cambridge University Press. Hill, A. (1974). Experimental and natural infection of the conjunctiva of rats. Laboratory Animals 8,305-310. Jarvis, J. :D. (1973). Collection of specimens for culture. In Basic clinical bacteriology. London: Butterworth. Saunders, L. Z. (1967). Ophthalmic pathology in rats and mice. In Pathology of laboratory rats and mice (ed. E. Cotchin & F. J. C. Roe), pp. 349-371. Oxford & Edinburgh: Blackwell. Young, C. & Hill, A. (1974). Conjunctivitis in a colony of rats. Laboratory Animals 8, 301-304.
Downloaded from lan.sagepub.com at NORTHERN ARIZONA UNIVERSITY on May 26, 2015
197
AN EYE INFECTION
IN LABORATORY
MICE
ASSOCIATED WITH PASTEURELLA
PNEUMOTROPICA by
J. R. NEEDHAM
and J. E. COOPER
Animal Division, Medical Research Council, Clinical Research Centre, Watford Road, Harro\\!, HAl 3UJ SUMMARY
Pasteurella pneumotropica was isolated from cases of clinical conjunctivitis in mice. No isolates were obtained from normal mice. At this Centre there is a yearly turnover of about 72 000 conventional mice, which are held in polypropylene boxes with wood chips for bedding. Over a period of 25 months, eye infections were observed in 45 stock mice. A literature search revealed a scarcity of reports on such conditions. Saunders (1967) reported Staphylococcus aureus from eyes of rats and a reference was made to the isolation of a Pasteurella sp. from inflamed rat eyes. Young & Hill (1974) isolated 'J?.asteurella pneumotropica from rats with conjunctivitis. The 45 mice e'Xamined presented with closed eyes, without purulent discharge but with increased lachrymal secretion; only 1 showed a grossly disTable 1. Distribution
Strain AKR C57BIOD2 BALB/C CBA CBA BALB/C BALB/C
CBA
Number of mice
Age (where known)
1
1 1 1
6 weeks 6 weeks
5 6 .1
3 weeks
10
NUDE
7
A
1 1
NZB
of mice with infected eyes.
A A AKR
2 1
AKR
3
CBA
1
8 months
3 11 weeks
Downloaded from lan.sagepub.com at NORTHERN ARIZONA UNIVERSITY on May 26, 2015
Sex
198
J. R. NEEDHAM
AND
J. E. COOPER
tended eye, the conjunctival surface of which was covered with a hard scablike plaque. They were distributed throughout the colony and ranged from 3 to 32 weeks of age. Table 1 shows the strains and numbers of animals involved. All animals were killed by the intraperitoneal injection of sodium pentobarbitone. With sterile forceps the lower eyelid of each infected eye was everted and material was collected using a sterile wire loop (Jarvis, 1973). The sample was cultured on 2 blood-agar plates and on a Loeffler's serumagar slope. The media were incubated at 37°C overnight; one blood agar plate was incubated anaerobically, the other media aerobically. Any plate showing no growth after overnight incubation was incubated for a further 24 hours. After incubation, identification tests were performed and the organisms classified according to Cowan & Steele (1966). No attempt was made to examine specimens for the presence of Mycoplasma spp. The internal organs of each animal were examined macroscopically but were not subjected to any cultural techniques. The eyes, heart, kidneys, spleen, liver and lungs of 5 affected mice were examined histologically as was the grossly distended eye. Specimens were placed in 10 % formol-saline prior to subsequent staining with Cole's haematoxylin and eosin. The eyes of 10 healthy mice from a barrier-maintained (SPF) breeding unit were examined microbiologically and the eyes of 2 of them histologically in the manner described. Of the 45 mice examined 39 yielded a pure growth of Pasteurella pneumotropica, 2 yielded a pure growth of Staphylococcus aureus, and no isolates were obtained from 4 mice. No bacterial isolates were obtained from the 10 control mice. Histopathological examination of the eyes of the 5 mice showed a keratoconjunctivitis in each case. The cornea was grossly thickened and oedematous and the substantia propria was infiltrated by large numbers of inflammatory cells which were mainly neutrophils but also included occasional plasma cells. There was a purulent exudate on the surface of the conjunctiva and within the fornices (the latter showed hyperkeratinisation). In 3 cases there was a more generalised inflammation with a polymorphonuclear infiltration of the limbus and, to a lesser extent, sclera; the blood vessels in these areas were dilated. There was an inflammatory cell infiltration around the lachrymal glands, but no apparent glandular involvement (Fig. 1). In no case was there abnormality of the intraocular structures. The eyelids showed no abnormalities other than possible hyperkeratinisation. The grossly distended eye showed histological evidence of a long-standing panophthalmitis. The conjunctival plaque consisted of fibrin which contained many neutrophils, some of them pyknotic. Intraocular structures showed considerable disruption and a severe inflammatory reaction. There was an extensive fibrosis both within the eye and in the eyelid adjacent to the lachrymal glands. Heart, kidney and spleen contained no histopathological lesions. All 5
Downloaded from lan.sagepub.com at NORTHERN ARIZONA UNIVERSITY on May 26, 2015
PASTEURELLA
INFECTION OF MOUSE EYES
199
livers showed varying degrees of vacuolation of hepatocytes and small numbers of scattered cellular aggregates-mainly composed of neutrophils. 4 of the lungs appeared normal but 1 showed a considerable degree of peri bronchiolar lymphocytic cuffing.
Fig. 1. Portion of cye and eyelid of affected mouse. Showing extensive polymorphonuclear infiltration but no involvement of fornix or lachrymal gland. Cole's haematoxylin and eosin. Line represents 500 j.Im.
The histopathological diagnosis of keratoconjunctivitis would agree with the condition described by Saunders (1967), from which Staphylococcus aureus was isolated. In the present study it is probable that the origin of the Pasteurella pneumotropica was the upper respiratory tract as all mice in the conventional rooms (where the infections occurred) were known to be colonised by this organism. However, other factors may be involved in the pathogenesis of eye infections. In this survey only 1 species of bacterium was isolated from the eyes in contrast to the findings of Young & Hill (1974). Hill (1974) demonstrated multiplication of a mixture of organisms, including P. pneumotropica, in rat eyes following experimental inoculation, but there was no evidence of clinical conjunctivitis. Similarly at this Centre conjunctivitis was not observed macroscopically when over 400 mice were inoculated intranasally with P. pneumotropica and spillage into the eye of the bacterial suspension occurred (work to be reported). This evidence, together with that of Young & Hill (1974), suggests that predisposing factors, for example eye damage, may be necessary before ocular infection with P. pneumotropica can occur.
Downloaded from lan.sagepub.com at NORTHERN ARIZONA UNIVERSITY on May 26, 2015
200
J. R. NEEDHAM
AND
J. E. COOPER
REFERENCES Cowan, S. T. & Steel, K. J. (1966). Manualfor the identification of medical bacteria. Cambridge: Cambridge University Press. Hill, A. (1974). Experimental and natural infection of the conjunctiva of rats. Laboratory Animals 8,305-310. Jarvis, J. :D. (1973). Collection of specimens for culture. In Basic clinical bacteriology. London: Butterworth. Saunders, L. Z. (1967). Ophthalmic pathology in rats and mice. In Pathology of laboratory rats and mice (ed. E. Cotchin & F. J. C. Roe), pp. 349-371. Oxford & Edinburgh: Blackwell. Young, C. & Hill, A. (1974). Conjunctivitis in a colony of rats. Laboratory Animals 8, 301-304.
Downloaded from lan.sagepub.com at NORTHERN ARIZONA UNIVERSITY on May 26, 2015
