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A Multivalent Mannheimia / Bibersteinia Vaccine Protects Bighorn Sheep Against 1
Mannheimia haemolytica Challenge 2
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Renuka Subramaniam1, Sudarvili Shanthalingam1 , Jegarubee Bavananthasivam1, Abirami 4
Kugadas1, Kathleen A. Potter1,2 , William J. Foreyt1, Douglas C. Hodgins3, Patricia E. Shewen3, 5
George M. Barrington4, Donald P. Knowles1,5 Subramaniam Srikumaran.1* 6
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Department of Veterinary Microbiology and Pathology1, Washington Animal Disease Diagnostic 8
Laboratory2, College of Veterinary Medicine, Washington State University, Pullman, WA 9
99164-7040, USA; Department of Pathobiology3, Ontario Veterinary College, University of 10
Guelph, Guelph, Ontario, N1G 2W1,Canada. Department of Veterinary Clinical Medicine and 11
Surgery4, College of Veterinary Medicine, Washington State University, Pullman, WA 99164-12
7040, USA; Animal Disease Research Unit, USDA Agricultural Research Service5, Pullman, 13
WA 99164-6630, USA. 14
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Running title: Vaccination of bighorn sheep 16
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*Corresponding Author: 18
Dr. S. Srikumaran 19
Department of Veterinary Microbiology and Pathology, 20
Washington State University, 21
Pullman,WA 99164 -7040, USA. 22
Phone: 509-335-4572; Fax: 509-335-8529 23
E-mail: [email protected] 24
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Clin. Vaccine Immunol. doi:10.1128/CVI.05276-11 CVI Accepts, published online ahead of print on 10 August 2011
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Abstract 25
Bighorn sheep (BHS) are more susceptible than domestic sheep (DS) to Mannheimia 26
haemolytica pneumonia. Although both species carry M. haemolytica as a commensal bacterium 27
in the nasopharynx, DS carry mostly leukotoxin (Lkt)-positive strains while BHS carry Lkt-28
negative strains. Consequently, antibodies to surface antigens and Lkt are present at much higher 29
titers in DS, but lower in BHS. The objective of this study was to determine whether repeated 30
immunization of BHS with multivalent Mannheimia / Bibersteinia vaccine will protect them 31
upon M.haemolytica challenge. Four BHS were vaccinated with a culture supernatant vaccine 32
prepared from M. haemolytica A1 and A2, and Biberstienia trehalosi T10, on days 0, 21, 35, 49 33
and 77. Four other BHS were used as nonvaccinated controls. On the day of challenge, 12 days 34
after last immunization, the mean serum titers of Lkt-neutralizing antibodies and antibodies to 35
surface antigens against M.haemolytica were 1:160 and 1:4000, respectively. Following 36
intranasal challenge with M.haemolytica A2 (1x105 cfu), all four control BHS died within 48 37
hours. Necropsy revealed acute fibrinonecrotic pneumonia characterisitic of M.haemolytica 38
infection. None of the vaccinated BHS died during the eight weeks post-challenge observation 39
period. Radiography at three weeks post-challenge revealed no lung lesions in two vaccinated 40
BHS and mild lesions in the other two, which resolved by eight weeks post-challenge. These 41
results indicate that if BHS can be induced to develop high titers of Lkt-neutralizing antibodies 42
and antibodies to surface antigens, they are likely to survive M.haemolytica challenge which is 43
likely to reduce BHS population decline due to pneumonia. 44
Key words: Bighorn sheep, Mannheimia haemolytica , leukotoxin, vaccine 45
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Introduction 48
The Bighorn sheep (BHS, Ovis canadensis) population in North America has declined drastically 49
during the last century due to a combination of factors including loss of habitat, competition for 50
forage with domestic livestock, predation, and disease. Pneumonia is the primary disease that 51
causes significant mortality in BHS (2, 26). Although Mannheimia haemolytica, Bibersteinia 52
trehalosi and Pasteurella multocida have been isolated from several pneumonia outbreaks, only 53
M. haemolytica has been shown to consistently cause fatal pneumonia in BHS under 54
experimental conditions (4, 7, 9, 22). Virulence factors of M. haemolytica include capsule, outer 55
membrane proteins, neuraminidase, lipopolysaccharide and a potent exotoxin called leukotoxin 56
(Lkt), which is cytolytic to all subsets of ruminant leukocytes (3,14). Based on the fact that Lkt- 57
deletion mutants cause no mortality in BHS (4), and lower mortality and mild lung lesions in 58
cattle (12, 19, 24), Lkt has been accepted as the major virulence factor of this organism. 59
Although, M. haemolytica causes pneumonia in all ruminants, BHS are highly susceptible to this 60
disease (4, 7, 9. 22).There are a number of potential factors contributing to the apparent 61
difference in susceptibility to pneumonia. Enhanced susceptibility of PMNs from BHS to Lkt-62
induced cytolysis is one of the factors responsible for the higher susceptibility of BHS (20, 22), 63
which underscores the importance of Lkt-neutralizing antibodies (Lkt-nAb) for protection of 64
BHS against M. haemolytica pneumonia. 65
Vaccination studies in cattle have shown that antibodies against Lkt and surface antigens 66
of M. haemolytica confer protection against experimental challenge (13, 21), and that such 67
antibodies can be induced by vaccination of cattle or BHS with log-phase Culture supernatant 68
fluid (1, 13, 16, 27). However, to-date systematic analysis of the response of BHS to vaccination 69
and subsequent challenge with M. haemolytica has not been performed. Thus, unambiguous data 70
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on the role of antibodies in protection of BHS against pneumonia caused by M. haemolytica are 71
not available. Several pneumonia outbreaks have occurred in the recent past resulting in the 72
death of large numbers of BHS (17). Thus, there is a critical need for development of efficacious 73
vaccines to protect BHS against pneumonia. This study represents an initial step in addressing 74
this need. 75
BHS are more susceptible to M. haemolytica pneumonia than the related species 76
domestic sheep (Ovis aries, DS, 4, 9, 18, 22). Although both species carry M. haemolytica and B. 77
trehalosi as a commensal bacteria in their nasopharynx, DS carry mostly leukotoxin (Lkt)-78
positive strains while BHS carry Lkt-negative strains (23, Shanthalingam et.al., 2011, 79
Unpublished data). It is very likely because of the difference in the Lkt expression by the M. 80
haemolytica strains inhabiting the nasopharynx of DS and BHS, and the consequent difference in 81
exposure to the immune system, Lkt-nAb are present at much higher titers in DS, but at 82
negligibly low titers in BHS (11). We hypothesized that if BHS can be induced to develop high 83
titers of antibodies to surface antigens and Lkt, they will withstand subsequent challenge with M. 84
haemolytica. B. trehalosi also was used in this vaccine because Lkt positive B. trehalosi has 85
been shown to cause fatal pneumonia in BHS (Dassanayake et.al., 2011, Unpublished data). 86
Therefore the objective of this study was to determine whether repeated immunization with a 87
multivalent Mannheimia /Bibersteinia culture supernatant vaccine would protect BHS against 88
experimental M. haemolytica challenge. 89
Materials and Methods 90
Experimental animals 91
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All experimental protocols were reviewed and approved by the Institutional Animal Care and 92
Use Committee (IACUC) at Washington State University (WSU) before the onset of the study. 93
Eight (two-year-old) BHS from the hand-raised captive herd at WSU were divided into two 94
groups of four each. At the onset of the experiment, all the animals were healthy and did not 95
have any history of respiratory disease. Nasal and pharyngeal swabs were collected to determine 96
the presence of M. haemolytica and B. trehalosi in the nasopharynx before the onset of the 97
study. Blood samples were collected to determine serum antibody titers against Lkt and surface 98
antigens of M. haemolytica and B. trehalosi. 99
Detection of M. haemolytica and B. trehalosi in nasopharynx 100
Nasal and pharyngeal swabs were submitted to Washington Animal Disease Diagnostic 101
laboratory (WADDL) at WSU, for detection of M. haemolytica and B. trehalosi using cultural 102
and biochemical tests. Culture results were further confirmed by genus-specific PCR as 103
previously described (5). All M. haemolytica and B. trehalosi cultures were tested for the 104
presence of the lktA gene by lktA gene-specific PCR (6). 105
Determination of antibody titers 106
Antibodies against surface antigens of M. haemolytica A1, A2 and B. trehalosi T10, and Lkt 107
containing culture supernatant fluids were measured by ELISA (11). Lkt neutralizing antibodies 108
were determined using MTT dye-reduction cytotoxicity assay (10). 109
Antibodies against bacterial surface antigens 110
Serum antibodies specific for surface antigens of M. haemolytica and B. trehalosi were measured 111
by indirect ELISA. Briefly, bacteria were immobilized onto the wells of an ELISA plate (Becton 112
Dickinson, Franklin Lakes, NJ) at 2 x 106 cfu /50 μl/ well in coating buffer [15mM Na2CO3 and 113
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35mM NaHCO3, pH 9.6] overnight at 4 oC. The plate was rinsed three times with 1:20 NAP 114
blocker (G-biosciences, St Louis, MO) in 1x PBS. Serially two fold diluted serum samples (50 115
μl) were added to the wells and incubated for 45 min at 37 oC. After washing the plate three 116
times, rabbit anti-sheep IgG (H+L) conjugated to horseradish peroxidase (50 μl of 1:5000 117
dilution, Pierce, Rockford, IL) was added. After 45 min of incubation, the substrate (100 μl of 118
ABTS, KPL, Gaithersburg, MD) was added and the plate was incubated at room temperature for 119
30 min. The optical density (OD405nm) of the final product was measured using an ELISA plate 120
reader (Becton Dickinson, Franklin Lakes, NJ). The highest dilution of serum yielding an OD 121
405nm value equal to or greater than twice the OD 405nm value given by a comparable dilution of 122
negative control serum was considered the antibody titer of the serum. 123
Antibodies against Lkt and other soluble antigens in the culture supernatant fluid 124
Lkt in the form of culture supernatant fluid from M. haemolytica A1 (50µl per well) was 125
immobilized onto a 96 well ELISA plate overnight at 4oC. The plate was blocked with 1:4 NAP 126
blocker (G-biosciences) in 1x PBS for 1 h at 37 oC. The remaining steps were performed as 127
described above for determination of surface antigen titers. 128
Neutralizing antibodies against Lkt 129
Lkt-nAb in serum were determined using the MTT (3-[4,5-dimethylthiazoyl-2-yl]-2-5-diphenyl 130
tetrazolium bromide [Sigma Chemical Co., St. Louis, MO]) dye-reduction cytotoxicity assay, as 131
previously described (10). Fifty μl of each serially diluted serum sample was incubated with 132
50μl of log phase culture supernatant fluid containing Lkt at a dilution which caused 50% 133
cytotoxicity to bovine lymphoma cells (BL-3 cells) in the wells of a U-bottom microtiter plate 134
for 1h at 4oC. BL-3 cells (50 μl of 2.5 x106/ml) were then added to the serum-Lkt mixture, and 135
incubated at 37oC for another 1h. The plate was centrifuged at 600×g for 5 min and supernatant 136
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fluid was removed. The cells were resuspended in 100 μl of colorless RPMI 1640 (Invitrogen, 137
Carlsbad CA) and 20 μl of MTT dye (5mg/ml) and incubated at 37oC for 1h. The plate was 138
centrifuged again at 600×g for 5 min, the fluid was decanted, and 100 μl of acid isopropanol was 139
added to dissolve the precipitate. The OD540nm of the final product was measured using an 140
ELISA plate reader (Becton Dickinson). Percent inhibition was calculated as described 141
previously (11) and outlined below. 142
% inhibition = (cytotoxicity Lkt + medium) – (cytotoxicity Lkt + serum) x 100 143
(cytotoxicity Lkt + medium) 144
The titer was the highest dilution of the serum which yielded minimum of 50% inhibition of Lkt-145
induced cytotoxicity. 146
Vaccines 147
The commercial vaccine Presponse SQTM which consists of logarithmic phase culture 148
supernatant from M. haemolytica serotype A1 (ATCC 43270) as the immunogen was purchased 149
from Boehringer Ingelheim, Ridgefield, CT (lot # 084331A). An experimental vaccine against 150
M. haemolytica serotype A2 and B. trehalosi serotype T10 was prepared as described below. 151
Preparation of experimental vaccine 152
The experimental vaccine was prepared using the logarithmic phase culture supernatant fluid 153
from M. haemolytica serotype A2 (WSU 1) and B. trehalosi serotype T10 (ATCC 33374) as the 154
immunogen. Formulation of the vaccine was similar to that described previously (13, 16). 155
Briefly, the culture supernatant fluid was prepared using separate logarithmic cultures of each 156
strain in RPMI 1640, and filtered using 0.22µm filter (Millipore, Billerica, Massachusetts), then 157
dialyzed across a 3000MWC membrane (Spectrum, Rancho Dominiguez, CA) against distilled 158
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water. Culture supernatant fluids were frozen (-80 0C) overnight and lyophilized. Lyophilized 159
powder was reconstituted in RPMI 1640 to obtain 20X concentration. The vaccine consisted of 160
equal amounts of culture supernatant fluid and adjuvant complex. The adjuvant complex 161
consisted of Al (OH) 3 (23% rehydra gel HPA, General Chemical, Parsipanny, NJ) and Quil A 162
(0.005%, Brenntag Biosector, Germany). 163
Immunization protocol 164
Four BHS were inoculated subcutaneously with 2ml each of PresponseSQ TM and experimental 165
vaccine at two different sites, on day 0. Vaccination was repeated on days 21, 35, 49 and 77. 166
Serum samples were collected from all vaccinated and control animals immediately before each 167
administration of vaccine and before the challenge on day 89. 168
Challenge of BHS 169
All animals were challenged intranasally with M. haemolytica A2 (WSU 1), cultured overnight 170
at 37 oC on brain heart infusion (BHI) agar supplemented with 5% sheep blood (Remel, Lenexa, 171
KS). The BHI agar plate was washed with colorless RPMI 1640 and the bacteria were scraped 172
from the plate and resuspended in RPMI 1640 to obtain an OD 600nm of 0.4. The suspension was 173
cultured for 1.5-2 h until the OD 600nm reached 0.6. Following centrifugation at 4000 rpm for 20 174
mins, the bacterial pellet was resuspended in colorless RPMI 1640 to obtain a concentration of 175
approximately equivalent to 1×105 colony forming units (cfu)/ 5ml. The bacterial count was 176
extrapolated based on the standard curve plotted with colony forming units (cfu) against OD. 177
One BHS in the control group was challenged with 1×106 cfu on day 57. The remaining three 178
BHS in the control group and all four BHS in the vaccinated group were challenged intra-nasally 179
with 1×105 cfu on day 89. Animals in each group were monitored daily for clinical signs of 180
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pneumonia including anorexia, lethargy, cough, nasal discharge and dyspnea. Necropsy was 181
performed on all dead animals. Lung tissue was collected aseptically from the lesional areas for 182
histological examination and bacterial isolation. The degree of involvement of the lung lobes was 183
scored as percent pneumonic tissue (percentage of lung that was consolidated on gross 184
examination). Animals that survived the challenge were not euthanized, but were radiographed to 185
assess the lung consolidation at 3 and 8 weeks post-challenge. 186
Statistical analysis 187
Antibody titers against surface antigens and Lkt were logarithmically transformed to satisfy the 188
assumption of normal distribution, and geometric mean titers (GMT) were calculated. Repeated 189
measures ANOVA were used to analyse the titers and the Bonferroni test was used to make 190
pairwise comparison between days of vaccination. Antibody titers against surface antigens of M. 191
haemolytica A1 were analyzed using Student's t test. GMT was transformed back to the 192
corresponding reciprocal values to present the titers in the results. Results were analysed using 193
GraphPad prism version 5.0 software. P ≤ 0.05 was considered significant . 194
195
Results 196
Pre-vaccination microbial profile of the nasopharynx of BHS 197
No Pasteurellaceae of interest (M. haemolytica and B. trehalosi) were detected by standard 198
aerobic culture or biochemical analyses in samples obtained prior to vaccination from the nasal 199
cavities of the control or vaccinated BHS, but three controls and all four vaccinates carried B. 200
trehalosi in the pharynx. The other BHS in the control group had M. haemolytica in the pharynx. 201
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However, neither the B. trehalosi nor the M. haemolytica isolates possessed the lktA gene, as 202
determined by the lktA gene-specific PCR (Table 1). 203
Vaccinated BHS exhibited high serum titers of antibodies against surface antigens of M. 204
haemolytica A1 and A2 and B. trehalosi T10. 205
An indirect ELISA revealed that, pre-vaccination, all the BHS had low titers (<1:150) of serum 206
antibodies against surface antigens of M. haemolytica A1 and A2. Following vaccination, serum 207
antibody titer against surface antigens of M. haemolytica A2 increased significantly in all four 208
BHS, in comparison to the control group (P ≤0.0001, Fig.1A). A significant increase in 209
antibodies was also observed between priming and the subsequent two injections (P <0.05). On 210
the day of challenge, the mean serum antibody titer of the vaccinated BHS against surface 211
antigens on M. haemolytica A2 was 1:4000 whereas that of the control group was 1:125. 212
Similarly, the vaccinated group had significantly higher serum antibodies against surface 213
antigens of M. haemolytica A1 (1:2000), in comparison to the control group (1:150, P ≤0.001, 214
Fig.1B) on the day of challenge. 215
Pre-vaccination, BHS in the control and the vaccinated groups had serum titers of 1:375 216
and 1:700, respectively, against the surface antigens of B. trehalosi. BHS in the vaccinated group 217
responded to vaccination, which resulted in significantly higher antibody titers against surface 218
antigens of B. trehalosi, in comparison to the control group (Fig. 2, P≤0.0001). The mean 219
serum antibody titer of the vaccinated group was 1:3200, whereas that of the control group 220
remained at 1:375, on the day of challenge. 221
Vaccination of BHS resulted in high titers of serum antibodies against Lkt 222
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The ELISA revealed that BHS in the vaccinated group developed significantly higher titers of 223
anti-Lkt antibodies, in comparison to the control group, at all time points following vaccination 224
(Fig. 3A, P≤0.0001). The mean titer of these antibodies in the vaccinated group was 1:6400 225
whereas that of the control group was 1:100, on the day of challenge. Obviously, this ELISA 226
measures antibodies to the spectrum of soluble antigens in culture supernatant fluids, including 227
both neutralizing and non-neutralizing antibodies to Lkt. Determination of the Lkt-nAb titers 228
using the MTT-dye-reduction cytotoxicity assay revealed significantly higher Lkt-nAb titers in 229
the vaccinated group following vaccination, compared to the controls (P≤ 0.007 Fig.3B). The 230
mean serum Lkt-nAb titer in the vaccinated group on the day of challenge was 1:160 whereas 231
that of the control group was <1:10. 232
Vaccination protects BHS against M. haemolytica A2 challenge 233
Following challenge with M. haemolytica A2 (1×105 cfu), the BHS in the control group rapidly 234
developed clinical signs of pneumonia. Nasal discharge, anorexia and lethargy were apparent at 235
20 hours post- challenge (hpc). All controls were recumbent at 30 hpc and died at 40-45 hpc. At 236
necropsy, gross pathological examination revealed lesions characteristic of acute pneumonia 237
including severe lung consolidation, fibrin deposition and haemorrhage. Lesions were present in 238
the entire cranial and middle lobes of the right lung (30-60%) and the cranial lobe of the left lung 239
(0-25%, Fig. 4A). Large numbers of bacteria were recovered from lung tissue (1×108 cfu/g of 240
lung tissue), which were confirmed as M. haemolytica by culture and biochemical method, and 241
by PCR. Histological sections of lesional lung tissue revealed extremely congested pulmonary 242
vasculature, expanded and edematous interlobular septa and alveoli, and clusters of necrotic 243
neutrophils, intermixed with bacterial colonies (Fig. 4B). Two BHS in the vaccinated group were 244
slightly lethargic during the first two days post-challenge, but recovered quickly. All of them 245
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were alert, active, and exhibited good appetite throughout the study period. Radiography at 3 246
weeks post-challenge revealed no lesions in two BHS (Fig. 5A). The other two BHS had 247
moderately increased interstitial density within the cranial and /or caudal lung field and increased 248
soft tissue opacity within the cranial lung field, suggestive of mild pneumonia (Fig.5B). 249
However, lungs of these two BHS did not exhibit any abnormalities when they were 250
radiographed again at eight weeks post-challenge (Fig.5C). All four vaccinated BHS remain 251
healthy up to the date of preparation of this manuscript (16 weeks post challenge). 252
Discussion 253
Two previous attempts to immunize BHS against M. haemolytica have not succeeded. In 254
the first study, 100% of BHS (n=3) vaccinated with an experimental bacterin-toxoid vaccine 255
comprised of Pasteurella haemolytica A2, T3 and T10 (currently classified as M. haemolytica 256
A2, B. trehalosi T3 and T10, respectively) died of pneumonia following commingling with 257
healthy DS known to carry P. haemolytica A1 and A2 in their nasal cavities (7). In the second 258
study, 100% of BHS in two groups (n=2 in each group) vaccinated by intratracheal inoculation 259
of live cultures of cytotoxic or noncytotoxic strains, of M. haemolytica A11 (currently classified 260
as M. glucosida), succumbed after challenge with M. haemolytica A2 (8). In these two studies 261
induction of antibodies in response to the vaccination was not evaluated: hence, the reason(s) for 262
vaccine failure could not be determined. In the only study in which the development of 263
antibodies in response to the vaccine (A1, A2 and T10 culture supernatant fluid) was measured, 264
the BHS were not challenged with M. haemolytica. Instead, P. haemolytica T10 (now B. 265
trehalosi T10) was used for challenge. Even with B. trehalosi challenge, the percent protection 266
was only 60-80 in the vaccinated BHS. Twenty percent of the controls also survived. 267
Furthermore, cumulative post-mortem scores based on gross lesions and bacteriology did not 268
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differ between vaccinated and control animals (15). Thus, unambiguous data on the role of 269
vaccination in protection of BHS against pneumonia caused by M. haemolytica are not currently 270
available. Therefore we designed this study to test the hypothesis that vaccination to induce high 271
titers of antibodies to M. haemolytica surface and soluble antigens, and Lkt-neutralizing 272
antibodies will protect BHS against subsequent challenge with M. haemolytica. As a ‘proof of 273
concept’ experiment, we determined whether immunization of BHS with a multivalent (A1,A2, 274
T10) vaccine would protect against M. haemolytica A2 challenge. Our hypothesis was generated 275
by the observation that DS, which are relatively resistant to M. haemolytica pneumonia, carry 276
mostly Lkt-positive M. haemolytica in the nasopharynx and consequently have higher serum 277
Lkt-nAb and antibodies against surface antigens of this organism (11, 22). 278
The pre-vaccination microbial profile of the BHS used in this study indicated that the B. 279
trehalosi and M. haemolytica isolates from these animals did not possess the lkt gene (Table 1), 280
which is in agreement with previous reports (18, 23) and supports our notion that the lack of Lkt-281
nAb in BHS is primarily due to the absence of Lkt-positive M. haemolytica and B. trehalosi as 282
commensal bacteria in the nasopharynx. The single study (25) that suggested that there was no 283
difference between the M. haemolytica strains isolated from BHS and DS, utilized β-hemolysis 284
assay, which, unlike the cytotoxicity assay, is not absolutely confirmative of leukotoxicity 285
(Shanthalingam et.al., 2011, Unpublished data). M. haemolytica has been shown to consistently 286
cause pneumonia in BHS under experimental conditions (4, 9, 22). This organism has also been 287
identified in pneumonic BHS lungs (4, 9, 22, Shanthalingam et.al, 2011, Unpublished data). 288
Although B. trehalosi also has been isolated from pneumonic lungs, the great majority of these 289
isolates is Lkt-negative, and hence cannot be the pathogens that cause fatal pneumonia in BHS 290
(Shanthalingam et.al., 2011, Unpublished data). However, since the small number of Lkt-291
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positive strains of B. trehalosi can cause fatal pneumonia in BHS (Dassanayake et al., 2011, 292
Unpublished data), we included the culture supernatant from both species in the experimental 293
vaccine. Although M. haemolytica A2 is the serotype that is commonly found in DS and BHS, 294
serotype A1 also can cause fatal pneumonia in BHS (4). Hence we also included the commercial 295
vaccine PresponseSQ which contains A1 culture supernatant as the immunogen. Culture 296
supernatant fluid that we used as the immunogen in our experimental vaccine has Lkt and other 297
soluble antigens, including outer membrane proteins which can contribute to efficacy, for 298
example by inducing opsonising antibodies. Earlier studies in BHS used challenge doses ranging 299
from 1x107 to 6x109 cfu per animal intratracheally or oropharyngeally. However, in this study 300
we administered 1x10 6 cfu intranasally to one control animal, and 1x105 cfu by the same route to 301
the other three control animals and all the four vaccinated animals. All control animals died of 302
pneumonia within 48 hours post-challenge, suggesting that intranasal administration of 1x105 cfu 303
of the M. haemolytica A2 strain that we used (WSU-1) is adequate for causing fatal pneumonia. 304
Presence of significantly higher titers of Lkt-nAb and anti-surface antigen antibodies in 305
all four vaccinated BHS, but not in nonvaccinated control BHS (Fig. 1-3), is consistent with the 306
hypothesis that the vaccinated BHS survived the challenge because of the protection conferred 307
by these antibodies. Even the two BHS that had mild lesions at three weeks post-challenge, 308
recovered and their lungs were normal in the radiographs taken at 8 weeks post-challenge (Fig. 5 309
C). More importantly, all four vaccinated BHS remain healthy up to the last observation, 16 310
weeks post-challenge. 311
Although we included M. haemolytica serotypes A1, A2, and B. trehalosi T10 in the 312
vaccine, we challenged the vaccinated group only with M. haemolytica A2 in this phase of the 313
study for two reasons. First, it is A2 that predominantly infects BHS, and secondly, inclusion of 314
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all three organisms in the challenge inoculum would preclude us from knowing which one of 315
them killed the BHS, if they didn’t survive the challenge. In the experiment reported here, the 316
vaccine was administered five times. However, anti-surface antigen antibodies and Lkt-nAb did 317
not increase further after the third injection suggesting that three injections might have given the 318
same outcome as five, if BHS were challenged after 12 days of last immunization. Nevertheless, 319
one might question the practicality of giving even three injections to wild sheep. It should be 320
emphasized here that this was a ‘proof-of concept’ study, designed to answer the question 321
whether BHS would withstand challenge if they carried sufficiently high titers of Lkt-nAb and 322
anti-surface antigen antibodies. All controls died while 100% of vaccinated BHS survived the 323
challenge with M. haemolytica A2, which, to our knowledge, has not been reported previously. 324
With this new knowledge we will now focus on developing a vaccine that does not require 325
repeated injections. A live vector-based vaccine may satisfy this requirement. A vaccine 326
deliverable noninvasively, for example through the feed or water, would represent the ideal 327
vaccine for wild BHS. 328
329
330
Acknowledgements: 331
This research was supported by funds from the Foundation for North American Wild Sheep and 332
its Eastern, Idaho, Oregon, and Washington Chapters, and the US Forest Service. We thank 333
Jeremy Brown for his assistance with animal handling. 334
335
336
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426
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Figure legends 427
Table 1: Pre-vaccination microbial profile of the nasopharynx of experimental BHS. 428
Isolates were tested by cultural and biochemical tests, and confirmed by genus-specific PCR. 429
Presence of the lktA gene was tested by lktA gene-specific PCR. 430
Fig 1: Antibody titers against surface antigens of M. haemolytica in vaccinated and control 431
groups of BHS 432
Serum antibody (Ab) titers against M. haemolytica surface antigens were determined by indirect 433
ELISA. Three independent assays were performed using serum samples from all the animals. 434
Each data point represents the geometric mean titer of one animal in the respective group. 435
Horizontal bar represents the geometric mean titer of the group at each time point. 436
(A).Vaccinated group had significantly higher mean titers against surface antigens of M. 437
haemolytica A2 than the control group after vaccination (P <0.0001). (B). Vaccinated group had 438
significantly higher mean titers against surface antigens of M. haemolytica A1 than the control 439
group (P <0.001) on the day of challenge (Day 89). Titers are shown as the natural logarithm 440
(LN) of the reciprocal dilution. Open circles represent control animals, closed triangles indicate 441
vaccinated animals. DOC and gp means group and day of challenge, respectively. Titers <100 442
was considered as 100 for the purpose of analysis. 443
Fig 2: Antibody titers against surface antigens of B.trehalosi in vaccinated and control 444
groups of BHS. 445
Serum antibody (Ab) titers against B.trehalosi T10 surface antigens were determined by indirect 446
ELISA. Three independent assays were performed using serum samples from all the animals. 447
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Each data point represents the geometric mean titer of one animal in the respective group. 448
Horizontal bar represents the geometric mean titer of the group at each time point. Vaccinated 449
group had significantly higher mean titers than the control group (P ≤0.0001) following 450
vaccination. Titers are shown as the natural logarithm (LN) of the reciprocal dilution. Open 451
circles represent control animals, closed triangles indicate vaccinated animals. DOC and gp 452
means group and day of challenge, respectively. Titers <100 was considered as 100 for the 453
purpose of analysis. 454
Fig 3: Lkt antibody titers of vaccinated and control groups of BHS 455
(A) Antibodies (Ab) against soluble antigens (Ag) including Lkt in the culture supernatant (CS) 456
fluid were measured by an indirect ELISA. The vaccinated group had significantly higher 457
geometric mean titers than the control group (P ≤0.0001) following vaccination. Titers <100 was 458
considered as 100 for the purpose of analysis. (B) Lkt neutralizing antibodies were measured by 459
the MTT dye-reduction cytotoxicity assay. Vaccinated BHS had significantly higher Lkt-nAb 460
than controls (P≤0.007) following vaccination. Titers <10 was considered as 10 for the puropose 461
of analysis. Each data point represents the geometric mean titer of one animal in the respective 462
group. Horizontal bar represents the geometric mean titer of the group at each time point. Three 463
independent assays were performed using serum samples from all the animals. Titers are shown 464
as the natural logarithm (LN) of the reciprocal dilution. Open circles represent control animals, 465
closed triangles indicate vaccinated animals. DOC and gp means group and day of challenge, 466
respectively. 467
Fig 4: Representative gross lesions and histopathological appearance of the lungs of 468
unvaccinated control BHS 469
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(A) Gross pathology showing severe consolidation in the right cranial, middle and left cranial 470
lobes of the lung. Arrow indicates the consolidation of the lungs. (B) Histopathology showing 471
extremely congested pulmonary vasculature, expanded and edematous interlobular septa and 472
alveoli, and clusters of necrotic neutrophils, intermixed with bacterial colonies. Sections were 473
stained with haematoxylin and eosin. (Magnification 100x). 474
475
Fig 5: Radiographs of vaccinated BHS at three weeks (A&B) and eight weeks (C) post- 476
challenge. 477
(A) Radiograph of the thorax of one of the two healthy BHS at three weeks post-challenge. (B) 478
Radiograph of the thorax of one of the two BHS, suggestive of mild pneumonic lesions at three 479
weeks post-challenge. Arrow indicates interstitial density and increased soft tissue opacity in the 480
cranial field of the lungs (C) Radiograph of the thorax of one of the two BHS (that had evidence 481
of mild pneumonic lesions at three weeks post-challenge) at eight weeks post–challenge. 482
483
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Table 1
Groups M. haemolytica B. trehalosi Lkt gene
by PCR
Animal ID
Nasal swabs
Pharyngeal swabs
Nasal swabs
Pharyngeal swabs
Control 16 - + - - -
24 - - - + -
39 - - - + -
45 - - - + -
Vaccination 20 - - - + -
22 - - - + -
23 - - - + -
50 - - - + -
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