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Experimental production of tick pyaemia
Veterinary Parasitology, 34 (1989) 129-133 Elsevier Science Publishers B.V., A m s t e r d a m - Printed in The Netherlands
129
Experimental Production of Tick Pyaemia K.A. WEBSTER* and G.B.B. MITCHELL
Scottish Agricultural Colleges Veterinary Investigation Centre, Auchincruive, A yr (Gt. Britain) (Accepted for publication 7 March 1989 )
ABSTRACT Webster, K.A. and Mitchell, G.B.B., 1989. Experimental production of tick pyaemia. Vet. Parasitol., 34: 129-133. Lambs aged 2 weeks were inoculated with a tick-borne fever (TBF) stabilate on Day 0 and
Staphylococcus aureus-contaminated ticks were applied on Day 5. Tick pyaemia was produced experimentally for the first time using Ixodes ricinus as a mechanical vector of S. aureus. Lambs aged 18 weeks were rechallenged with a homologous strain of TBF, and S. aureus-infected ticks applied 5 days later. No significant changes were noted at post-mortem examination.
INTRODUCTION
Tick pyaemia is a serious staphylococcal infection of young lambs, common in animals from tick-infested areas, but is not found in tick-free regions of the United Kingdom. Foggie (1959) suggested that the tick Ixodes ricinus is not a direct vector of staphylococci, but that the immunodepressive symptoms produced by tick-borne fever (TBF), especially the profound neutropaenia, allowed staphylococci normally present on the skin to invade. Recent work (Brodie et al., 1986) has shown that while only 20% (4/20) of lambs inoculated intravenously with Staphylococcus aureus alone developed pyaemia, 57% of those inoculated following TBF became pyaemic; moreover, 9/10 lambs inoculated with S. aureus during the febrile phase of a TBF infection (usually 5 days post-infection) developed pyaemia. These workers could only produce the syndrome consistently if S. aureus was injected intravenously. However, Brodie (1985) successfully produced pyaemic lesions by injecting S. aureus 9 days post TBF infection via the intradermal route, when massive numbers of organisms were used. *Present address: Department of Medicine 2, General Hospital, Southampton, Gt. Britain.
0304-4017/89/$03.50
© 1989 Elsevier Science Publishers B.V.
130
K.A. WEBSTER AND G.B.B.MITCHELL
M A T E R I A L S AND M E T H O D S
Four Scottish Blackface ewes with twin lambs, obtained from a tick-free farm, were treated as follows (Table 1 ). When 2 weeks old, one of each pair of lambs (Group A) was infected intravenously with 1 ml of a 10 -1 dilution (in phosphate-buffered saline, pH 7.2) of the Old Sourhope strain of TBF, while siblings remained as uninfected controls (Group B). TBF-free nymphal and larval I. ricinus from a laboratory-maintained culture were placed on a blood agar plate with a confluent growth ofS. a u r e u s for 30 min. Five days after TBF infection, 10 nymphs and 100 larvae were placed in ear and tail bags, respectively, on all eight lambs. The ticks were allowed to attach and feed normally. Two pairs of twins were killed on Days 20 and 27 post TBF infection for post-mortem examination. Lamb liveweight was recorded prior to post-mortern examination and spleens were removed entire and also weighed. At necroscopy, viscera were examined macroscopically and gross lesions noted. Swabs were taken from lesions, tick bites, skin and from stifle and carpal joints (which were cut open with a sterile blade) and placed in 5-ml volumes of brain-heart infusion broth (BHI). The broths were inoculated onto blood agar and mannitol salt agar (MSA) 24 h later. Samples of liver, lung, heart, spleen, brain and kidney were cultured on blood agar and MSA. All plates were incubated at 37 ° C overnight and bacterial growth examined the following morning. The densities of haemolytic S. a u r e u s were scored on a scale of 0-4 corresponding to gradations from nil to confluent growth, respectively. A second experiment (2) was designed to mimic the TBF challenge associated with the autumn peak of tick activity reported in some areas (MacLeod, 1939; Milne, 1945). Although the experiment grossly simplifies the period of tick challenge in that it supposes no contact between sheep and ticks in the interim, the approach enabled the immune status of the animals to be measured and interpreted with some accuracy. Four Scottish Blackface ewes with twin lambs were obtained from the same tick-free source and treated as follows (Table 2). At the age of 2 weeks, all lambs were infected intravenously with TBF as described for Experiment 1. This resulted in normal TBF reactions, i.e. pyrexia, parasitaemia, lymphocyTABLE1 Design of Experiment 1
Group A Group B
T B F infection (Day 0)
Application of S. aureus-infected ticks (Day 5)
+ -
+ +
Two pairs of twins were killed on Days 20 a n d 27 post T B F infection.
131
TICK PYAEMIA TABLE
2
Design of Experiment 2
Group C Group D
TBF at 2 weeks of age
TBF at 18 weeks of age (Day 0)
Application of S. aureus-infected ticks (Day 5)
+ +
+ -
+ +
Two pairs of twins were killed on Days 20 and 27 post secondary TBF infection.
topaenia, neutropaenia and production of specific antibody. Sixteen weeks after initial infection one of each pair of twins was rechallenged with T B F (Group C), while siblings remained as unchallenged controls (Group D ) . S. aureusinfected ticks were allowed to feed on all lambs 5 days after reinfection with TBF. The lambs were killed 20 days post T B F challenge for post-mortem examination. RESULTS
Post-mortem examination of the lambs in Experiment i revealed no significant changes in Group B lambs, while all Group A lambs had a number of small abscesses in the right diaphragmatic lobe of the lung. Liver abscesses were present in half of the lambs, while splenomegaly, associated with TBF infection (Gordon et al., 1932; Hudson, 1950) was noted in every case. S. aureus was recovered from abscesses, the area of skin surrounding bite wounds and from the heart blood of Group A lambs. All isolates had cultural and biochemical reactions identical to the original strain of S. aureus used for infection. Post-mortem examination of the lambs in the second experiment revealed only non-specific changes in all animals except for the presence of splenomegaly in some lambs. DISCUSSION
This is believed to be the first recorded experimental production of tick pyaemia by I. ricinus. Previously, Foggie (1962) failed to reproduce the disease in both normal and neutropaenic lambs by subcutaneous or intradermal inoculation of staphylococci with or without an extract of tick salivary gland, or by the feeding of ticks on areas of skin heavily contaminated with S. aureus. However, lambs in the neutropaenic phase of T B F were shown to be 100-fold more susceptible to the intravenous inoculation of staphylococci than healthy lambs (Foggie, 1962). In the present study the staphylococcal challenge was administered 5 days after T B F infection, at the peak of parasitaemia, prior to devel-
132
K.A. WEBSTER AND G.B.B. MITCHELL
opment of neutropaenia, whereas Foggie always administered staphylococci during the neutropaenic phase of T B F infection. In Experiment 1, 2-week-old lambs all developed pyaemic abscesses in the lungs and some also in the liver, while in older lambs undergoing a secondary TBF infection (Experiment 2), which produced less severe haematological changes than a primary infection and a reduced serological response (Webster and Mitchell, 1988), abscessation was absent. These results reflect the field situation where tick pyaemia primarily occurs in the late spring, when lambs born in upland areas are about 4 weeks old, but is not normally present in other seasons of the year, despite tick activity. The occurrence of tick pyaemia at this time may well be related to the immunodepression associated with T B F infection (Webster, 1987) coinciding with the waning of colostral-derived staphylococcal antitoxin, at around the 20th day of life, allowing invasion and multiplication of pathogenic staphylococci. This may explain why only a percentage of lambs at risk develop tick pyaemia. In Experiment 2, S. aureus-infected ticks were allowed to attach and feed on lambs 5 days after the initiation of a secondary TBF infection. Reactions to secondary TBF infections are known to be less severe than to primary infections (Woldehiwet and Scott, 1982; Webster and Mitchell, 1988) in terms of pyrexia, parasitaemia, neutropaenia and lymphocytopaenia. It is therefore likely that S. aureus inoculated by ticks in Experiment 2 encountered a more effective immune attack. In field outbreaks the pathogenesis of staphylococcal infection remains obscure. Foggie (1947) showed that haemolytic staphylococci commonly occurring on the skin of lambs have the same characteristics as staphylococci isolated from cases of tick pyaemia, while McDiarmid (1948) noted that the sites of tick bites are usually inflamed and abscesses may be present from which S. aureus can be isolated. This was substantiated by postmortem examination of experimental and field cases of tick pyaemia at the Auchincruive Veterinary Investigation Centre. It has been suggested that staphylococci may be released from a latent state by the immunodepression associated with TBF, or that staphylococci invading through minor cuts and abrasions can multiply when the host is stressed by TBF infection (Anderson, 1983). However, many lambs have been experimentally infected with TBF and there have been no reports of the development of spontaneous pyaemia, which might be expected if the above explanations were valid. Further experimental studies are required to determine whether or not I. ricinus is directly responsible for the disease by inoculation of pathogenic staphylococci or only indirectly responsible by virtue of TBF and penetration of the dermis. ACKNOWLEDGEMENT The authors wish to thank Prof. G.M. Urquhart for helpful discussion.
TICK PYAEMIA
133
REFERENCES Anderson, J.C., 1983. Staphylococci and Staphylococcal Infections. Academic Press, London, p. 193. Brodie, T.A., 1985. Some aspects of tick-borne fever in sheep. Ph.D. Thesis, University of Glasgow, 240 pp. Brodie, T.A., Holmes, P.H. and Urquhart, G.M., 1986. Some aspects of tick-borne diseases of British sheep. Vet. Rec., 118: 415-418. Foggie, A., 1947. Studies on the source of staphylococcal infection found in tick pyaemia of lambs. J. Comp. Pathol., 57: 245-260. Foggie, A., 1959. Studies on the relationship of tick-bite to tick pyaemia of lambs. Ann. Trop. Med. Parasitol., 53: 27-34. Foggie, A., 1962. Studies on tick pyaemia and tick-borne fever. Symp. Zool. Sot. London, 6: 5158. Gordon, W.S., Brownlee, A., Wilson, D.R. and MacLeod, J., 1932. Tick-borne fever (a hitherto undescribed disease of sheep). J. Comp. Pathol., 45: 301-312. Hudson, J.R., 1950. The recognition of tick-borne fever as a disease of cattle. Br. Vet. J., 106: 317. McDiarmid, A., 1948. Leader. Proc. R. Sot. Med., 41: 429-430. MacLeod, J., 1939. The seasonal and annual incidence of the sheep tick, Zxodes ricinus, in Britain. Bull. Entomol. Res., 30: 103-118. Mime, A., 1945. The ecology of sheep tick Zxodes ricinus L. The seasonal activity in Britain with particular reference to Northern England. Parasitology, 36: 142-152. Webster, K.A., 1987. Zxodes ricinus, the sheep tick: ecology and disease. Ph.D. Thesis, University of Glasgow, 342 pp. Webster, K.A. and Mitchell, G.B.B., 1988. Use of counter immunoelectrophoresis in detection of antibodies to tickborne fever. Res. Vet. Sci., 45: 28-30. Woldehiwet, Z. and Scott, G.R., 1982. Immunological studies of tick-borne fever in sheep. J. Comp. Pathol., 92: 457-467.
129
Experimental Production of Tick Pyaemia K.A. WEBSTER* and G.B.B. MITCHELL
Scottish Agricultural Colleges Veterinary Investigation Centre, Auchincruive, A yr (Gt. Britain) (Accepted for publication 7 March 1989 )
ABSTRACT Webster, K.A. and Mitchell, G.B.B., 1989. Experimental production of tick pyaemia. Vet. Parasitol., 34: 129-133. Lambs aged 2 weeks were inoculated with a tick-borne fever (TBF) stabilate on Day 0 and
Staphylococcus aureus-contaminated ticks were applied on Day 5. Tick pyaemia was produced experimentally for the first time using Ixodes ricinus as a mechanical vector of S. aureus. Lambs aged 18 weeks were rechallenged with a homologous strain of TBF, and S. aureus-infected ticks applied 5 days later. No significant changes were noted at post-mortem examination.
INTRODUCTION
Tick pyaemia is a serious staphylococcal infection of young lambs, common in animals from tick-infested areas, but is not found in tick-free regions of the United Kingdom. Foggie (1959) suggested that the tick Ixodes ricinus is not a direct vector of staphylococci, but that the immunodepressive symptoms produced by tick-borne fever (TBF), especially the profound neutropaenia, allowed staphylococci normally present on the skin to invade. Recent work (Brodie et al., 1986) has shown that while only 20% (4/20) of lambs inoculated intravenously with Staphylococcus aureus alone developed pyaemia, 57% of those inoculated following TBF became pyaemic; moreover, 9/10 lambs inoculated with S. aureus during the febrile phase of a TBF infection (usually 5 days post-infection) developed pyaemia. These workers could only produce the syndrome consistently if S. aureus was injected intravenously. However, Brodie (1985) successfully produced pyaemic lesions by injecting S. aureus 9 days post TBF infection via the intradermal route, when massive numbers of organisms were used. *Present address: Department of Medicine 2, General Hospital, Southampton, Gt. Britain.
0304-4017/89/$03.50
© 1989 Elsevier Science Publishers B.V.
130
K.A. WEBSTER AND G.B.B.MITCHELL
M A T E R I A L S AND M E T H O D S
Four Scottish Blackface ewes with twin lambs, obtained from a tick-free farm, were treated as follows (Table 1 ). When 2 weeks old, one of each pair of lambs (Group A) was infected intravenously with 1 ml of a 10 -1 dilution (in phosphate-buffered saline, pH 7.2) of the Old Sourhope strain of TBF, while siblings remained as uninfected controls (Group B). TBF-free nymphal and larval I. ricinus from a laboratory-maintained culture were placed on a blood agar plate with a confluent growth ofS. a u r e u s for 30 min. Five days after TBF infection, 10 nymphs and 100 larvae were placed in ear and tail bags, respectively, on all eight lambs. The ticks were allowed to attach and feed normally. Two pairs of twins were killed on Days 20 and 27 post TBF infection for post-mortem examination. Lamb liveweight was recorded prior to post-mortern examination and spleens were removed entire and also weighed. At necroscopy, viscera were examined macroscopically and gross lesions noted. Swabs were taken from lesions, tick bites, skin and from stifle and carpal joints (which were cut open with a sterile blade) and placed in 5-ml volumes of brain-heart infusion broth (BHI). The broths were inoculated onto blood agar and mannitol salt agar (MSA) 24 h later. Samples of liver, lung, heart, spleen, brain and kidney were cultured on blood agar and MSA. All plates were incubated at 37 ° C overnight and bacterial growth examined the following morning. The densities of haemolytic S. a u r e u s were scored on a scale of 0-4 corresponding to gradations from nil to confluent growth, respectively. A second experiment (2) was designed to mimic the TBF challenge associated with the autumn peak of tick activity reported in some areas (MacLeod, 1939; Milne, 1945). Although the experiment grossly simplifies the period of tick challenge in that it supposes no contact between sheep and ticks in the interim, the approach enabled the immune status of the animals to be measured and interpreted with some accuracy. Four Scottish Blackface ewes with twin lambs were obtained from the same tick-free source and treated as follows (Table 2). At the age of 2 weeks, all lambs were infected intravenously with TBF as described for Experiment 1. This resulted in normal TBF reactions, i.e. pyrexia, parasitaemia, lymphocyTABLE1 Design of Experiment 1
Group A Group B
T B F infection (Day 0)
Application of S. aureus-infected ticks (Day 5)
+ -
+ +
Two pairs of twins were killed on Days 20 a n d 27 post T B F infection.
131
TICK PYAEMIA TABLE
2
Design of Experiment 2
Group C Group D
TBF at 2 weeks of age
TBF at 18 weeks of age (Day 0)
Application of S. aureus-infected ticks (Day 5)
+ +
+ -
+ +
Two pairs of twins were killed on Days 20 and 27 post secondary TBF infection.
topaenia, neutropaenia and production of specific antibody. Sixteen weeks after initial infection one of each pair of twins was rechallenged with T B F (Group C), while siblings remained as unchallenged controls (Group D ) . S. aureusinfected ticks were allowed to feed on all lambs 5 days after reinfection with TBF. The lambs were killed 20 days post T B F challenge for post-mortem examination. RESULTS
Post-mortem examination of the lambs in Experiment i revealed no significant changes in Group B lambs, while all Group A lambs had a number of small abscesses in the right diaphragmatic lobe of the lung. Liver abscesses were present in half of the lambs, while splenomegaly, associated with TBF infection (Gordon et al., 1932; Hudson, 1950) was noted in every case. S. aureus was recovered from abscesses, the area of skin surrounding bite wounds and from the heart blood of Group A lambs. All isolates had cultural and biochemical reactions identical to the original strain of S. aureus used for infection. Post-mortem examination of the lambs in the second experiment revealed only non-specific changes in all animals except for the presence of splenomegaly in some lambs. DISCUSSION
This is believed to be the first recorded experimental production of tick pyaemia by I. ricinus. Previously, Foggie (1962) failed to reproduce the disease in both normal and neutropaenic lambs by subcutaneous or intradermal inoculation of staphylococci with or without an extract of tick salivary gland, or by the feeding of ticks on areas of skin heavily contaminated with S. aureus. However, lambs in the neutropaenic phase of T B F were shown to be 100-fold more susceptible to the intravenous inoculation of staphylococci than healthy lambs (Foggie, 1962). In the present study the staphylococcal challenge was administered 5 days after T B F infection, at the peak of parasitaemia, prior to devel-
132
K.A. WEBSTER AND G.B.B. MITCHELL
opment of neutropaenia, whereas Foggie always administered staphylococci during the neutropaenic phase of T B F infection. In Experiment 1, 2-week-old lambs all developed pyaemic abscesses in the lungs and some also in the liver, while in older lambs undergoing a secondary TBF infection (Experiment 2), which produced less severe haematological changes than a primary infection and a reduced serological response (Webster and Mitchell, 1988), abscessation was absent. These results reflect the field situation where tick pyaemia primarily occurs in the late spring, when lambs born in upland areas are about 4 weeks old, but is not normally present in other seasons of the year, despite tick activity. The occurrence of tick pyaemia at this time may well be related to the immunodepression associated with T B F infection (Webster, 1987) coinciding with the waning of colostral-derived staphylococcal antitoxin, at around the 20th day of life, allowing invasion and multiplication of pathogenic staphylococci. This may explain why only a percentage of lambs at risk develop tick pyaemia. In Experiment 2, S. aureus-infected ticks were allowed to attach and feed on lambs 5 days after the initiation of a secondary TBF infection. Reactions to secondary TBF infections are known to be less severe than to primary infections (Woldehiwet and Scott, 1982; Webster and Mitchell, 1988) in terms of pyrexia, parasitaemia, neutropaenia and lymphocytopaenia. It is therefore likely that S. aureus inoculated by ticks in Experiment 2 encountered a more effective immune attack. In field outbreaks the pathogenesis of staphylococcal infection remains obscure. Foggie (1947) showed that haemolytic staphylococci commonly occurring on the skin of lambs have the same characteristics as staphylococci isolated from cases of tick pyaemia, while McDiarmid (1948) noted that the sites of tick bites are usually inflamed and abscesses may be present from which S. aureus can be isolated. This was substantiated by postmortem examination of experimental and field cases of tick pyaemia at the Auchincruive Veterinary Investigation Centre. It has been suggested that staphylococci may be released from a latent state by the immunodepression associated with TBF, or that staphylococci invading through minor cuts and abrasions can multiply when the host is stressed by TBF infection (Anderson, 1983). However, many lambs have been experimentally infected with TBF and there have been no reports of the development of spontaneous pyaemia, which might be expected if the above explanations were valid. Further experimental studies are required to determine whether or not I. ricinus is directly responsible for the disease by inoculation of pathogenic staphylococci or only indirectly responsible by virtue of TBF and penetration of the dermis. ACKNOWLEDGEMENT The authors wish to thank Prof. G.M. Urquhart for helpful discussion.
TICK PYAEMIA
133
REFERENCES Anderson, J.C., 1983. Staphylococci and Staphylococcal Infections. Academic Press, London, p. 193. Brodie, T.A., 1985. Some aspects of tick-borne fever in sheep. Ph.D. Thesis, University of Glasgow, 240 pp. Brodie, T.A., Holmes, P.H. and Urquhart, G.M., 1986. Some aspects of tick-borne diseases of British sheep. Vet. Rec., 118: 415-418. Foggie, A., 1947. Studies on the source of staphylococcal infection found in tick pyaemia of lambs. J. Comp. Pathol., 57: 245-260. Foggie, A., 1959. Studies on the relationship of tick-bite to tick pyaemia of lambs. Ann. Trop. Med. Parasitol., 53: 27-34. Foggie, A., 1962. Studies on tick pyaemia and tick-borne fever. Symp. Zool. Sot. London, 6: 5158. Gordon, W.S., Brownlee, A., Wilson, D.R. and MacLeod, J., 1932. Tick-borne fever (a hitherto undescribed disease of sheep). J. Comp. Pathol., 45: 301-312. Hudson, J.R., 1950. The recognition of tick-borne fever as a disease of cattle. Br. Vet. J., 106: 317. McDiarmid, A., 1948. Leader. Proc. R. Sot. Med., 41: 429-430. MacLeod, J., 1939. The seasonal and annual incidence of the sheep tick, Zxodes ricinus, in Britain. Bull. Entomol. Res., 30: 103-118. Mime, A., 1945. The ecology of sheep tick Zxodes ricinus L. The seasonal activity in Britain with particular reference to Northern England. Parasitology, 36: 142-152. Webster, K.A., 1987. Zxodes ricinus, the sheep tick: ecology and disease. Ph.D. Thesis, University of Glasgow, 342 pp. Webster, K.A. and Mitchell, G.B.B., 1988. Use of counter immunoelectrophoresis in detection of antibodies to tickborne fever. Res. Vet. Sci., 45: 28-30. Woldehiwet, Z. and Scott, G.R., 1982. Immunological studies of tick-borne fever in sheep. J. Comp. Pathol., 92: 457-467.