Veterinary Research Communications, 14 (1990) 175-180 Copyright
8 Kluwer
Academic
Publishers
bv - Printed
in the Netherlands
EFFECTS OF PASTEURELLA HAEMOLYTICA LEUKOTOXIN ON NEUTROPHILS FROM WHITE-TAILED DEER AND SEVERAL EXOTIC RUMINANT SPECIES A.W. CONFER’, K.R. SIMONS’, M.T. BARRIE2 AND K.D. CLINKENBEARD’ ‘Department of Veterinary Pathology and Department of Veterinary Parasitology, Microbiology and Public Health, College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma, OK 74078, USA 20kllahoma City Zoo, Oklahoma City, OK 73111, USA
ABSTRACT Confer, A.W., Simons, K.R, Barrie, M.T. and Clinkenbeard, K.D., 1990. Effects of Pasteurella huemolyrica leukotoxin on neutrophils from white-tailed deer and several exotic ruminant species. Veterinary Research Communications, 14 (J), 175-180
Pasteurella haemolytica leukotoxin
is a pore-forming cytolrjin which acts as a virulence factor in pasteurellosis of domestic ruminants. Leukocytes from cattle, sheep and goats are susceptible to leukotoxin-induced lysis; however, leukocytes from non-ruminant species so far tested are resistant to leukotoxin-induced lysis. Neutrophils obtained from three white-tailed deer, four Saiga antelope, an Addra gazelle, a Grant’s gazelle and a Sable antelope were tested for susceptibility to the lytic effects of P. huemoiydca leukotoxin using lactate dehydrogenase release. Results were compared to those obtained using neutrophils from a steer and cultured bovine lymphoma cells. Neutrophils obtained from all these ruminants, except the Addra gazelle, were susceptible to P. haemolytca leukotoxin. Individual variation among the Saiga and the deer did not appear to be due to the percentages of neutrophils or the percentage of contaminating erythrocytes in the cell preparations.
Keywvrak antelope,
deer, gazelle,
leukotoxin,
neutrophils,
Pasteurella huemolybca
INTRODUCTION haemolytica, a gram-negative bacterium, is a major cause of fibrinous pleuropneumonia in cattle, sheep and goats and of septicaemia in sheep (Carter, 1967). Pneumonic pasteurellosis has been described in big-horn sheep which were in association with domestic sheep (Foreyt, 1989). Reports of P. haemolyfica infections in other ruminants have been infrequent (Mwangota et al., 1978; Aikimbaev et al., 1985; Hayashidani, 1988). P. haemolytica is part of the nasal flora of,cattle and sheep, and during stress, such as viral infection, shipping, and/or inclement weather, the bacteria replicate and are inhaled into the pulmonary alveoli (Yates, 1982) where they are associated with severe inflammation and vascular damage (Allan et al., 1985). One of the virulence factors produced by P. haemolytica is a pore-forming cytolysin, leukotoxin (LKT), which causes lysis of leukocytes from ruminants, but leukocytes from non-ruminant species appear resistant to LKT (Shewen and Wilkie, 1982; O’Brien and Duffus, 1987; Clinkenbeard et al., 1989a,b,c). Lysis of leukocytes in the alveoli may cause impaired clearance of the bacteria and leakage of leukocytic lysosomal contents may damage the pulmonary parenchyma (Shewen and Wilkie, 1982).
Pasteurella
176
Pneumonic pasteurellosis due to P. haemolytica is rare in non-domestic ruminants (Griner, 1983; Heuschele, 1986). This may be because P. haemolytica is not carried in their nasal flora or because those ruminants are not susceptible to the virulence factors from P. huemolyficu, such as LKT. However, big-horn sheep are more susceptible than domestic sheep to P. huemolyticu infection (Onderka et al., 1988; Foreyt, 1989). The purpose of these studies was to investigate the susceptibility of neutrophils from various ruminants from the families Cervidae and Bovidae to the cytolytic effects of P. huemolyticu leukotoxin.
MATERIALS
AND METHODS
Bacterial culture
Lyophilized cultures of P. huemolyticu serotype 1 were suspended in distilled water, plated onto brain-heart infusion agar containing 5% bovine blood and grown at 37°C under 5% CO for 18 h. Isolated colonies were transferred to 50 ml of brain-heart infusion brothfn a 250 ml Erlenmeyer flask and grown at 37°C with rotatory shaking at 80 oscillations/min for 4 h. These cultures were used to inoculate media for the production of LKT (Clinkenbeard et al., 1989a).
Leukotoxin production
Cultures of P. huemolyficu were collected by centrifugation at 12 000 g for 30 min, suspended in 10 ml of 0.5% bovine serum albumin (BSA) in RPMI-1640 medium (RPMI-BSA), pH 7.2 and used to inoculate 100-400 ml of RPMI-BSA in Erlenmeyer flasks, which were incubated at 37°C for 1 h (Clinkenbeard et al., 1989a,b). Cultures were then centrifuged at 12 000 g for 30 min; the supernatant (leukotoxin solution) was filtered through a 0.22 pm filter and stored at -70°C. Heat-inactivated LKT (HI-LKT) was prerpared by heating LKT to 60°C for 10 min.
Ruminant species txumined
Blood was obtained several times from a yearling cross-bred beef steer (Bos taurus) and once from three white-tailed deer (Udocoileus viqiniunus) housed at the College of Veterinary Medicine research facilities, Oklahoma State University. Blood was obtained from four Saiga antelope (Suigu tututica tufuricu, three adults and one neonate), a Sable antelope (Hippotrugus niger), an Addra gazelle (Gazella damu ruficollis) and a Grant’s gazelle (Gaze/la granti roosevelti) housed at the Oklahoma City Zoo.
177
Cytotoxicity ussuy Bovine lymphoma cells (BL3 cells, originally obtained from Dr Gordon Theilen, University of California-Davis) were grown at 37°C with 5% COZ in suspension culture in 50% Leibovitz L-15-50% Eagle’s Minimal Essential Medium with 20% fetal bovine serum to which rglutamine, gentamicin and NaHCO, were added (Clinkenbeard et aI., 1989b). Cells for LKT assays were grown for 1 day prior to use. Neutrophils were isolated from 30 ml of titrated blood by differential sedimentation and hypotonic lysis (Henson and Oades, 1975; Clinkenbeard et al., 1989c). In each case, processing of blood began within 1.5 h of its withdrawal. BL3 cells in 100 ~1 of RPM1 1640 medium or neutrophils (106) in Ca2’ and Mg2+-free Hank’s solution were added to 150 ~1 of RPM1 1640 medium in 1.5 ml conical tubes. Each sample was assayed for lactate dehydrogenase (LDH) release in triplicate by the method described by Clinkenbeard et al. (1989a). The percentage specific release (%SR - the release specifically resulting from the action of LKT) of LDH was calculated using the means of the triplicate samples, such that: %SR = [LDH activity for LKT treated cells - LDH activity for HI-LKT treated cells] / [total releasable LDH activity in 1% triton - LDH activity for HI-LKT treated cells]
RESULTS The lysis and differential centrifugation technique provided adequate numbers of reasonably pure populations of neutrophils. The percentage of neutrophils ranged from 82% (Sable Antelope) to 98% (Saiga 1) although the cell preparation from Saiga 4 (the neonate) contained only 66% neutrophils (29% lymphocytes, 3% monocytes, 2% eosinophils). Cell preparations from the steer ranged from 86% to 92% neutrophils. In experiments 1 and 2, the effects of P. haemolyticu LKT on neutrophils obtained from the steer and deer were examined (Table I). The bovine neutrophils appeared slightly more susceptible to the effects of P. huemoZyticu LKT than were the cervine neutrophils, especially compared to deer 2 in experiment 2. In experiments 3 and 4 (Table I), neutrophils obtained from the steer seemed to be more susceptible to the lytic effects of P. huemolyticu LKT than those obtained from the Sable antelope or Addra gazelle. This was indicated by a higher %SR at lower dilutions of LKT and lysis of bovine neutrophils at higher dilutions of LKT than for the Sable antelope’s neutrophils. There was an absence of lysis for the Addra gazelle’s neutrophils. Neutrophils obtained from the Grant’s gazelle and the steer seemed to be similar in susceptibility to the lytic effects of LKT. Neutrophils obtained from Saiga were of variable susceptibility to LKT. Neutrophils obtained from Saigas 1 and 4 were of similar susceptibility to the neutrophils from the steer, whereas those from Saigas 2 and 3 seemed less susceptible.
178
TABLE I Percentage specific lactate dehydrogenase (LDH) release from cultured bovine lymphoma (BL3) cells and from neutrophils collected from various ruminants and exposed to P. haemolyfica leukotoxin (LKT)
Cells
Species of origin
Percentage specific LDH release at each LKT dilutiona
1:2.5
Experiment BL3
Cattle
57.1
45.4
41.9
ND
Cattle Deer
73.2 62.2
68.2 54.6
65.1 46.7
ND ND
Cattle
57.4
Cattle Deer 1 Deer 2
62.3 49.9
Cattle
63.6
Cattle Sable antelope Addra azelle Saiga 1fT Saiga 2b Saiga 3b Saiga 4’
38.8 17.5 0.0 51.2 24.4 26.8 42.7
Cattle
69.8
Cattle Grant’s gazelle
52.2 66.0
68.5
ND 59.7 51.0 19.2
ND 55.8 37.1 4.3
ND 34.9 12.8 3.4
3
Neutrophils
Experiment BL3
155
2
Neutrophils
Experiment BL3
1:lO
1
Neutrophils
Experiment BL3
15
ND 25.9 3.2 0.0 36.7 0.0 15.0 22.1
ND
24.1 0.2 0.0 23.5 0.0 0.0 2.8
ND 0.0 0.0 0.0 0.0 0.0 0.0 0.0
4
Neutrophils
aValues represent results of triplicate assays bAdult Saiga ‘Nursing baby Saiga ND = not done
ND 50.0 45.8
ND 7.2 13.8
ND 6.8 11.8
179
DISCUSSION Although neutrophils from each of the ruminants examined, with the exception of the Addra gazelle, were susceptible to the lytic effects of P. huemol’ca LKT, there was individual animal variation. In previous studies, bovine neutrophils were more susceptible to the lytic effects of P. haemolytica LKT than were bovine mammary or pulmonary alveolar macrophages (O’Brien and Duffus, 1987). It is possible that the variations in neutrophil susceptibilities within species in the present study could have been due to variation in the percentage of neutrophils in the cell preparations. However, this did not appear to be the case because cell preparations obtained from Saigas 1 and 4 had similar responses to LKT despite a 32% variation in percentage of neutrophils. Variations in susceptibility of cell preparations to LKT could also have reflected variations in erythrocyte contamination. Erythrocyte receptors for LKT might compete with receptors on neutrophils, or large numbers of erythrocytes might non-specifically inhibit LKT binding to neutrophils. However, when total erythrocyte counts were determined in experiments 3 and 4, the %SR was not inversely proportional to the number of erythrocytes present as the ratios of leukocytes to erythrocytes were almost identical among the Saigas. The lack of susceptibility of neutrophils obtained from the Addra gazelle was of special interest because neutrophils obtained from the Grant’s gazelle and from the steer, which are closely phylogenetically related, were of similar susceptibility. It is unfortunate that blood from another Addra gazelle or from the same one was not available for follow-up examination. It may be that neutrophils from Addra gazelle are not susceptible to the effect of LKT, or that there are individual animals within a species which are resistant to the toxin.
ACKNOWLEDGEMENTS This manuscript is published as journal article number 5477 from the College of Veterinary Medicine and the Oklahoma Agricultural Experiment Station. Supported in part by Special Grants numbers 85CRSR-2-2618 and S6-CRSR-2-2880 from the USDA. The authors thank Dr Alan Kocan for assistance in obtaining deer blood,
REFERENCES Aikimbaev, MA., Martinevskii, LL., Altukhov, AA., Ivanov, S.I. and Surov, V.F., 198s. Isolation of Pusreurella huemolytica from Saiga antelopes (Suigu turfutica) between February and March 1984 in the Uralsk region of Kazakhstan (in Russian). hvest& Akademii Nauk Kuzak-hskoi SS& Seriyu
Biologicheskgyu, 4,39-41 Allan,
E.M., Gibbs, HA., Wiseman, A. and Selman, I.E., 1985. Sequential lesions of bovine pneumonic pasteurellosis. Veterinary Record, 117,438~442 Carter, G.R, 1967. Pasteurellosis: Pusteurelkz nudtocida and Pasteurella huemolytia. Advunces
in
Veferitfa?yScience and Comparative Medicine, l&321-378 Clinkenbeard, swelling
420-42.5
K.D., Mosier, DA. and Confer, A.W., 1989a. Transmembrane in cytotoxicity caused by Pusteurella huemolyica leukotozin.
pore
size and role of cell
Infection und immunity, 57,
180 Clinkenbeard, K.D., Mosier, DA., Timko, A.L. and Confer, A.W., 1989b. Effects of Pasteurellu haemo&ica on cultured bovine lymphoma cells. American Journal of Veterinary Research, SO,271-275 Clinkenbeard, KD., Mosier, DA. and Confer, A.W., 1989c. Effects of Pasteurella haemolytica leukotoxin on isolated bovine neutrophils. Toticon, 27,797-804 Foreyt, W.J., 1989. Fatal Pasteurella haemolytica pneumonia in bighorn sheep after direct contact with clinically normal domestic sheep. American Journal of Veterinary Research, 50,341-344 Griner, LA:, 1983. Pathology of Z& Animals, (Zoological Society of San Diego, San Diego), 495-574 Havashidani. H.. Honda. E.. Nakamura. T.. Mori. Y.. Sawada. T. and Oeawa. M.. 1988. Outbreak of - pneumonia caused by Pkteurella huemo&ica infection in Shiba goats & Japan. japanese Journal of Veterinary Science, 50,960-962 Henson, P.M. and Oades, Z.G., 1975. Stimulation of human neutrophils by soluble and insoluble immunoglobulin aggregates. Journal of Clinical Investigation, 56,1053-1061 Heuschele, W.P., 1986. Infectious diseases. In: M.E. Fowler (ed.), Zoo and Wild Animal Medicine, 2nd edn., (Saunders, Philadelphia), 57-62 Mwangota, A.U., Muhammed, S.I. and Thomson, RG., 1978. Serological type of Pasteurella haemorytica in Kenya. Cornell Veterinarian, 68,84-93 O’Brien, J.K. and Duffus, W.P.H., 1987. Pasteurella haemolytica cytotoxin: relative susceptibility of bovine leucocytes. Veterinary Microbiology, 13,321-324 Onderka, D.K., Rawluk, S.A. and Wishart, W.D., 1988. Susceptibility of Rocky Mountain bighorn sheep and domestic sheep to pneumonia induced by bighorn and domestic livestock strains of PosteureUa huemoIytica. Canadian Journal of Veterinary Research, 52,439-444 Shewen, P.E. and Wilkie, B.N., 1982. Cytotoxin of Pasteurellu haemolytica acting on bovine leukocytes. Infection and Immunity, 35,91-94 Yates, W.D.G., 1982. A review of infectious bovine rhinotracheitis, shipping fever pneumonia and viral-bacterial synergism in respiratory disease of cattle. Canadian Journal of Comparative Medicine, 46,225263 (Accepted: 19 January 1990)
8 Kluwer
Academic
Publishers
bv - Printed
in the Netherlands
EFFECTS OF PASTEURELLA HAEMOLYTICA LEUKOTOXIN ON NEUTROPHILS FROM WHITE-TAILED DEER AND SEVERAL EXOTIC RUMINANT SPECIES A.W. CONFER’, K.R. SIMONS’, M.T. BARRIE2 AND K.D. CLINKENBEARD’ ‘Department of Veterinary Pathology and Department of Veterinary Parasitology, Microbiology and Public Health, College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma, OK 74078, USA 20kllahoma City Zoo, Oklahoma City, OK 73111, USA
ABSTRACT Confer, A.W., Simons, K.R, Barrie, M.T. and Clinkenbeard, K.D., 1990. Effects of Pasteurella huemolyrica leukotoxin on neutrophils from white-tailed deer and several exotic ruminant species. Veterinary Research Communications, 14 (J), 175-180
Pasteurella haemolytica leukotoxin
is a pore-forming cytolrjin which acts as a virulence factor in pasteurellosis of domestic ruminants. Leukocytes from cattle, sheep and goats are susceptible to leukotoxin-induced lysis; however, leukocytes from non-ruminant species so far tested are resistant to leukotoxin-induced lysis. Neutrophils obtained from three white-tailed deer, four Saiga antelope, an Addra gazelle, a Grant’s gazelle and a Sable antelope were tested for susceptibility to the lytic effects of P. huemoiydca leukotoxin using lactate dehydrogenase release. Results were compared to those obtained using neutrophils from a steer and cultured bovine lymphoma cells. Neutrophils obtained from all these ruminants, except the Addra gazelle, were susceptible to P. haemolytca leukotoxin. Individual variation among the Saiga and the deer did not appear to be due to the percentages of neutrophils or the percentage of contaminating erythrocytes in the cell preparations.
Keywvrak antelope,
deer, gazelle,
leukotoxin,
neutrophils,
Pasteurella huemolybca
INTRODUCTION haemolytica, a gram-negative bacterium, is a major cause of fibrinous pleuropneumonia in cattle, sheep and goats and of septicaemia in sheep (Carter, 1967). Pneumonic pasteurellosis has been described in big-horn sheep which were in association with domestic sheep (Foreyt, 1989). Reports of P. haemolyfica infections in other ruminants have been infrequent (Mwangota et al., 1978; Aikimbaev et al., 1985; Hayashidani, 1988). P. haemolytica is part of the nasal flora of,cattle and sheep, and during stress, such as viral infection, shipping, and/or inclement weather, the bacteria replicate and are inhaled into the pulmonary alveoli (Yates, 1982) where they are associated with severe inflammation and vascular damage (Allan et al., 1985). One of the virulence factors produced by P. haemolytica is a pore-forming cytolysin, leukotoxin (LKT), which causes lysis of leukocytes from ruminants, but leukocytes from non-ruminant species appear resistant to LKT (Shewen and Wilkie, 1982; O’Brien and Duffus, 1987; Clinkenbeard et al., 1989a,b,c). Lysis of leukocytes in the alveoli may cause impaired clearance of the bacteria and leakage of leukocytic lysosomal contents may damage the pulmonary parenchyma (Shewen and Wilkie, 1982).
Pasteurella
176
Pneumonic pasteurellosis due to P. haemolytica is rare in non-domestic ruminants (Griner, 1983; Heuschele, 1986). This may be because P. haemolytica is not carried in their nasal flora or because those ruminants are not susceptible to the virulence factors from P. huemolyficu, such as LKT. However, big-horn sheep are more susceptible than domestic sheep to P. huemolyticu infection (Onderka et al., 1988; Foreyt, 1989). The purpose of these studies was to investigate the susceptibility of neutrophils from various ruminants from the families Cervidae and Bovidae to the cytolytic effects of P. huemolyticu leukotoxin.
MATERIALS
AND METHODS
Bacterial culture
Lyophilized cultures of P. huemolyticu serotype 1 were suspended in distilled water, plated onto brain-heart infusion agar containing 5% bovine blood and grown at 37°C under 5% CO for 18 h. Isolated colonies were transferred to 50 ml of brain-heart infusion brothfn a 250 ml Erlenmeyer flask and grown at 37°C with rotatory shaking at 80 oscillations/min for 4 h. These cultures were used to inoculate media for the production of LKT (Clinkenbeard et al., 1989a).
Leukotoxin production
Cultures of P. huemolyficu were collected by centrifugation at 12 000 g for 30 min, suspended in 10 ml of 0.5% bovine serum albumin (BSA) in RPMI-1640 medium (RPMI-BSA), pH 7.2 and used to inoculate 100-400 ml of RPMI-BSA in Erlenmeyer flasks, which were incubated at 37°C for 1 h (Clinkenbeard et al., 1989a,b). Cultures were then centrifuged at 12 000 g for 30 min; the supernatant (leukotoxin solution) was filtered through a 0.22 pm filter and stored at -70°C. Heat-inactivated LKT (HI-LKT) was prerpared by heating LKT to 60°C for 10 min.
Ruminant species txumined
Blood was obtained several times from a yearling cross-bred beef steer (Bos taurus) and once from three white-tailed deer (Udocoileus viqiniunus) housed at the College of Veterinary Medicine research facilities, Oklahoma State University. Blood was obtained from four Saiga antelope (Suigu tututica tufuricu, three adults and one neonate), a Sable antelope (Hippotrugus niger), an Addra gazelle (Gazella damu ruficollis) and a Grant’s gazelle (Gaze/la granti roosevelti) housed at the Oklahoma City Zoo.
177
Cytotoxicity ussuy Bovine lymphoma cells (BL3 cells, originally obtained from Dr Gordon Theilen, University of California-Davis) were grown at 37°C with 5% COZ in suspension culture in 50% Leibovitz L-15-50% Eagle’s Minimal Essential Medium with 20% fetal bovine serum to which rglutamine, gentamicin and NaHCO, were added (Clinkenbeard et aI., 1989b). Cells for LKT assays were grown for 1 day prior to use. Neutrophils were isolated from 30 ml of titrated blood by differential sedimentation and hypotonic lysis (Henson and Oades, 1975; Clinkenbeard et al., 1989c). In each case, processing of blood began within 1.5 h of its withdrawal. BL3 cells in 100 ~1 of RPM1 1640 medium or neutrophils (106) in Ca2’ and Mg2+-free Hank’s solution were added to 150 ~1 of RPM1 1640 medium in 1.5 ml conical tubes. Each sample was assayed for lactate dehydrogenase (LDH) release in triplicate by the method described by Clinkenbeard et al. (1989a). The percentage specific release (%SR - the release specifically resulting from the action of LKT) of LDH was calculated using the means of the triplicate samples, such that: %SR = [LDH activity for LKT treated cells - LDH activity for HI-LKT treated cells] / [total releasable LDH activity in 1% triton - LDH activity for HI-LKT treated cells]
RESULTS The lysis and differential centrifugation technique provided adequate numbers of reasonably pure populations of neutrophils. The percentage of neutrophils ranged from 82% (Sable Antelope) to 98% (Saiga 1) although the cell preparation from Saiga 4 (the neonate) contained only 66% neutrophils (29% lymphocytes, 3% monocytes, 2% eosinophils). Cell preparations from the steer ranged from 86% to 92% neutrophils. In experiments 1 and 2, the effects of P. haemolyticu LKT on neutrophils obtained from the steer and deer were examined (Table I). The bovine neutrophils appeared slightly more susceptible to the effects of P. huemoZyticu LKT than were the cervine neutrophils, especially compared to deer 2 in experiment 2. In experiments 3 and 4 (Table I), neutrophils obtained from the steer seemed to be more susceptible to the lytic effects of P. huemolyticu LKT than those obtained from the Sable antelope or Addra gazelle. This was indicated by a higher %SR at lower dilutions of LKT and lysis of bovine neutrophils at higher dilutions of LKT than for the Sable antelope’s neutrophils. There was an absence of lysis for the Addra gazelle’s neutrophils. Neutrophils obtained from the Grant’s gazelle and the steer seemed to be similar in susceptibility to the lytic effects of LKT. Neutrophils obtained from Saiga were of variable susceptibility to LKT. Neutrophils obtained from Saigas 1 and 4 were of similar susceptibility to the neutrophils from the steer, whereas those from Saigas 2 and 3 seemed less susceptible.
178
TABLE I Percentage specific lactate dehydrogenase (LDH) release from cultured bovine lymphoma (BL3) cells and from neutrophils collected from various ruminants and exposed to P. haemolyfica leukotoxin (LKT)
Cells
Species of origin
Percentage specific LDH release at each LKT dilutiona
1:2.5
Experiment BL3
Cattle
57.1
45.4
41.9
ND
Cattle Deer
73.2 62.2
68.2 54.6
65.1 46.7
ND ND
Cattle
57.4
Cattle Deer 1 Deer 2
62.3 49.9
Cattle
63.6
Cattle Sable antelope Addra azelle Saiga 1fT Saiga 2b Saiga 3b Saiga 4’
38.8 17.5 0.0 51.2 24.4 26.8 42.7
Cattle
69.8
Cattle Grant’s gazelle
52.2 66.0
68.5
ND 59.7 51.0 19.2
ND 55.8 37.1 4.3
ND 34.9 12.8 3.4
3
Neutrophils
Experiment BL3
155
2
Neutrophils
Experiment BL3
1:lO
1
Neutrophils
Experiment BL3
15
ND 25.9 3.2 0.0 36.7 0.0 15.0 22.1
ND
24.1 0.2 0.0 23.5 0.0 0.0 2.8
ND 0.0 0.0 0.0 0.0 0.0 0.0 0.0
4
Neutrophils
aValues represent results of triplicate assays bAdult Saiga ‘Nursing baby Saiga ND = not done
ND 50.0 45.8
ND 7.2 13.8
ND 6.8 11.8
179
DISCUSSION Although neutrophils from each of the ruminants examined, with the exception of the Addra gazelle, were susceptible to the lytic effects of P. huemol’ca LKT, there was individual animal variation. In previous studies, bovine neutrophils were more susceptible to the lytic effects of P. haemolytica LKT than were bovine mammary or pulmonary alveolar macrophages (O’Brien and Duffus, 1987). It is possible that the variations in neutrophil susceptibilities within species in the present study could have been due to variation in the percentage of neutrophils in the cell preparations. However, this did not appear to be the case because cell preparations obtained from Saigas 1 and 4 had similar responses to LKT despite a 32% variation in percentage of neutrophils. Variations in susceptibility of cell preparations to LKT could also have reflected variations in erythrocyte contamination. Erythrocyte receptors for LKT might compete with receptors on neutrophils, or large numbers of erythrocytes might non-specifically inhibit LKT binding to neutrophils. However, when total erythrocyte counts were determined in experiments 3 and 4, the %SR was not inversely proportional to the number of erythrocytes present as the ratios of leukocytes to erythrocytes were almost identical among the Saigas. The lack of susceptibility of neutrophils obtained from the Addra gazelle was of special interest because neutrophils obtained from the Grant’s gazelle and from the steer, which are closely phylogenetically related, were of similar susceptibility. It is unfortunate that blood from another Addra gazelle or from the same one was not available for follow-up examination. It may be that neutrophils from Addra gazelle are not susceptible to the effect of LKT, or that there are individual animals within a species which are resistant to the toxin.
ACKNOWLEDGEMENTS This manuscript is published as journal article number 5477 from the College of Veterinary Medicine and the Oklahoma Agricultural Experiment Station. Supported in part by Special Grants numbers 85CRSR-2-2618 and S6-CRSR-2-2880 from the USDA. The authors thank Dr Alan Kocan for assistance in obtaining deer blood,
REFERENCES Aikimbaev, MA., Martinevskii, LL., Altukhov, AA., Ivanov, S.I. and Surov, V.F., 198s. Isolation of Pusreurella huemolytica from Saiga antelopes (Suigu turfutica) between February and March 1984 in the Uralsk region of Kazakhstan (in Russian). hvest& Akademii Nauk Kuzak-hskoi SS& Seriyu
Biologicheskgyu, 4,39-41 Allan,
E.M., Gibbs, HA., Wiseman, A. and Selman, I.E., 1985. Sequential lesions of bovine pneumonic pasteurellosis. Veterinary Record, 117,438~442 Carter, G.R, 1967. Pasteurellosis: Pusteurelkz nudtocida and Pasteurella huemolytia. Advunces
in
Veferitfa?yScience and Comparative Medicine, l&321-378 Clinkenbeard, swelling
420-42.5
K.D., Mosier, DA. and Confer, A.W., 1989a. Transmembrane in cytotoxicity caused by Pusteurella huemolyica leukotozin.
pore
size and role of cell
Infection und immunity, 57,
180 Clinkenbeard, K.D., Mosier, DA., Timko, A.L. and Confer, A.W., 1989b. Effects of Pasteurellu haemo&ica on cultured bovine lymphoma cells. American Journal of Veterinary Research, SO,271-275 Clinkenbeard, KD., Mosier, DA. and Confer, A.W., 1989c. Effects of Pasteurella haemolytica leukotoxin on isolated bovine neutrophils. Toticon, 27,797-804 Foreyt, W.J., 1989. Fatal Pasteurella haemolytica pneumonia in bighorn sheep after direct contact with clinically normal domestic sheep. American Journal of Veterinary Research, 50,341-344 Griner, LA:, 1983. Pathology of Z& Animals, (Zoological Society of San Diego, San Diego), 495-574 Havashidani. H.. Honda. E.. Nakamura. T.. Mori. Y.. Sawada. T. and Oeawa. M.. 1988. Outbreak of - pneumonia caused by Pkteurella huemo&ica infection in Shiba goats & Japan. japanese Journal of Veterinary Science, 50,960-962 Henson, P.M. and Oades, Z.G., 1975. Stimulation of human neutrophils by soluble and insoluble immunoglobulin aggregates. Journal of Clinical Investigation, 56,1053-1061 Heuschele, W.P., 1986. Infectious diseases. In: M.E. Fowler (ed.), Zoo and Wild Animal Medicine, 2nd edn., (Saunders, Philadelphia), 57-62 Mwangota, A.U., Muhammed, S.I. and Thomson, RG., 1978. Serological type of Pasteurella haemorytica in Kenya. Cornell Veterinarian, 68,84-93 O’Brien, J.K. and Duffus, W.P.H., 1987. Pasteurella haemolytica cytotoxin: relative susceptibility of bovine leucocytes. Veterinary Microbiology, 13,321-324 Onderka, D.K., Rawluk, S.A. and Wishart, W.D., 1988. Susceptibility of Rocky Mountain bighorn sheep and domestic sheep to pneumonia induced by bighorn and domestic livestock strains of PosteureUa huemoIytica. Canadian Journal of Veterinary Research, 52,439-444 Shewen, P.E. and Wilkie, B.N., 1982. Cytotoxin of Pasteurellu haemolytica acting on bovine leukocytes. Infection and Immunity, 35,91-94 Yates, W.D.G., 1982. A review of infectious bovine rhinotracheitis, shipping fever pneumonia and viral-bacterial synergism in respiratory disease of cattle. Canadian Journal of Comparative Medicine, 46,225263 (Accepted: 19 January 1990)
