Evaluation of a vaccine against Mannheimia haemolytica and

Pasteurella multocida in sheep

Honors Thesis

Presented to the College of Agriculture and Life Sciences,

Department of Animal Science

of Cornell University

in Partial Fulfillment of the Requirements for the

Research Honors Program

by

Tao Sun

May 2009

Supervised by Dr. Jerrie Gavalchin

1
 Evaluation of a vaccine against Mannheimia haemolytica and

Pasteurella multocida in sheep

Of the 20 currently identified species in the genus Pasteurella, Mannheimia

haemolytica (formerly called Pasteurella haemolytica) and Pasteurella multocida
are the most important respiratory pathogens affecting domestic ruminants,
especially in sheep and cattle, together causing fibrinous, necrotic pneumonia.
This disease, commonly called shipping fever, is a leading cause of economic
loss in the sheep industry. Factors such as transportation, viral infection, and
overcrowded housing, may predispose the opportunistic infection by the pathogen.
At present, several commercial vaccines have been developed to control
pasteurellosis in sheep, including types such as bacterins, live attenuated,
leukotoxin, capsule, lipopolysaccaride, subunit vaccines, sodium salicilate extract
and potassium thiocyanate. Previous studies have shown that effective specific
antibody response against M. haemolytica whole-cell antigen could be achieved
via vaccination. However, for P. multocida, vaccines did not induce strong specific
antibody response and some reports even suggested an increase in severity of
infection by P. multocida after vaccination.
In this study, the efficacy of an autogenous vaccine made from antigens from
both M. haemolytica and P. multocida, was evaluated by measuring specific
serum antibody titers produced against both bacteria in immunized sheep. Lambs
were vaccinated on Nov. 23 and sera were obtained from all control (unvaccinated)
and vaccinated animals on Nov. 16, Nov. 30, Dec. 7 and Dec. 14. The results
showed that the vaccine induced significant antibody response against M.
haemolytica in both ewes and rams after 7 days post vaccination. For the
response to P. multocida, specific antibodies were induced in ewes; however, the
vaccine failed to stimulate specific antibody production against P. multocida in
rams.

2
Acknowledgements
This work was completed under the supervision of Dr. Jerrie Gavalchin at Cornell

University. Without her patient guidance and consistent support, the completion of

this work could not be possible.

We would like to thank Dr. Michael L. Thonney for providing the lamb serum

samples and Dr. Yung-Fu Chang for providing the vaccine.

Finally, I would like to thank all my family members and friends who, directly or

indirectly, gave me confidence and courage to accomplish this work.

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Table of Contents
1. Introduction
2. Review of Literature
3. Materials and Methods
3.1 Animals
3.2 M. haemolytica and P. multocida vaccine
3.3 M. haemolytica and P. multocida antigen preparations for ELISA
3.4 ELISA for specific antibody
3.5 Statistical analysis
4. Results and Discussions
4.1 Determination of specific antibody titers
4.2 Data analysis
4.2.1 Vaccine efficacy against M. haemolytica
4.2.1.1 Factors affecting specific antibody titers against M. haemolytica
4.2.1.2 Discussion on vaccine efficacy against M. haemolytica
(1) Interaction between treatment and dates of sampling in ewe lambs
(2) Interaction between treatment and dates of sampling in ram lambs
(3) Vaccine efficacy against M. haemolytica
4.2.2 Vaccine efficacy against P. multocida
4.2.2.1 Factors affecting specific antibody titers against P. multocida
4.2.2.2 Discussion on vaccine efficacy against P. multocida
(1) Interactions between treatment and dates of sampling in ewe lambs
(2) Interactions between treatment and dates of sampling in ram lambs
(3) Vaccine efficacy against P. multocida
5. Reference
6. Appendix
6.1 Raw data from ELISA tests
6.2 Specific antibody titers

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1. Introduction
Mannheimia haemolytica (formerly called Pasteurella haemolytica) and

Pasteurella multocida are known to be the most prominent pathogens in the family

Pasteurellaceae causing great economic losses in the domestic animal industry

[1] [2]. Of the various M. haemolytica strains, M. haemolytica serotype A2 is the

major pathogen responsible for diseases in sheep, causing a fibrinous, necrotic

pneumonia, also called shipping fever. But this strain was not as

well-characterized as strain A1, which mainly causes pneumonia in cattle [2].

Factors such as transportation, viral infection, and overcrowded housing, may

also facilitate the opportunistic infection by the pathogen [3]. P. multocida was

found present in lung lesions of sheep affected by pneumonia as well [4] [5], but

due to its high antigenic variability, its molecular pathogenic mechanisms still

remain to be investigated [6].

Our experiment was part of a long-term project to develop and tested the

effectiveness of an autogenous vaccine to prevent pneumonia in young lambs.

The vaccine was developed from antigens of both M. haemolytica and P.

multocida. The goal of this experiment was to test this vaccines efficacy in

inducing specific antibody responses to the two bacteria in both ewe lambs and

ram lambs.

Currently there are two types of serological tests for evaluating vaccine

efficacy against M. haemolytica and P. multocida. One is the leukotoxin

5
neutralization test (LNT) and the other is an ELISA test. Mosier et al. 1986 [14]

showed that ELISA was a more convenient and faster tool compared with LNT. In

this experiment, we measured the level of serum antibodies against both M.

haemolytica and P. multocida using a whole-cell ELISA assay [15].

2. Review of Literature
At present, several commercial vaccines have been developed to control

pasteurellosis in sheep. For example, Once PMH, One Shot, Presponse, use

antigens including bacterins, leukotoxin, capsule, different surface antigens and

lipopolysaccaride [7] [8] [15]. However, researchers found that each vaccine

induced variable levels of protection in response to different bacteria strains.

Diker et al. 2000 [15] reported monovalent combined immunogens induced high

antibody titers against homologous serotypes, but low titers against heterologous

serotypes; and antigens from M. haemolytica Serotype A1 and A7 were found to

be more antigenic than A2 and T4. There were also species differences in vaccine

efficacy with variability in the induction of protective response in sheep and cattle

seen [12].

Previous studies have shown that effective specific antibody response against

M. haemolytica whole-cell antigen could also be achieved via vaccination.

Mehmet Akan et al. 2006 [9] reported One Shot Ultra 8 vaccine induced specific

antibody production against M. haemolytica using ELISA tests, with higher

6
specific antibody titers in vaccinated groups than in control groups. In addition,

LNT (leukotoxin neutralization test) also revealed a higher specific antibody titer in

vaccinated sheep compared to unvaccinated sheep. The study also evaluated

lung lesion scores of both vaccinated and control animals and statistically

significant differences (P<0.001) were observed between the vaccinated and

control groups, with fewer lesion in the vaccinated groups. S. Srinand et al 1996

[10] examined the efficacy of four vaccines: One Shot (SmithKline Beecham,

West Chester, PA, a bacterin-toxoid), Presponse (Langford Laboratories, Guelph,

Ontario, an Lkt-rich culture supernatant), Once PMH (BioCor Inc., Omaha, NE, a

modified live vaccine), and Septimune (Fort Dodge laboratories, Fort Dodge, IA,

an outer membrane extract). The study showed that One Shot, and Once PMH

vaccinated animals showed a significant (P < 0.05) increase in antibody levels

against leukotoxin at 28 days post vaccination. Once PMH vaccinated animals

also showed significant (P < 0.05) increase in antibody levels against IROMPs

(iron regulated outer membrane proteins) at 28 days after vaccination compared

to the other groups. Presponse, Once PMH, and One Shot vaccinated animals

showed a significant (P < 0.05) increase in antibody levels against CP (capsular

polysaccharide) over time. These groups also had significantly higher antibody

levels against CP, compared to controls and Septimune vaccinated at 14 and 28

days (P < 0.05).

However, other studies suggested that vaccination did not effectively protect

7
sheep from infection. Chandrasekaran S et al. 1991 [11] tested an oil adjuvant

vaccine incorporating locally isolated strains of M. haemolytica type 7 and P.

multocida types A and D, and found that the vaccine significantly reduced the lung

lesions at P<0.05 level in vaccinated sheep compared with the control groups

when all animals were challenged with M. haemolytica alone. However, when

animals were challenged with P. multocida or the combination of M. haemolytica

and P. multocida, the vaccine failed to induce significant levels of protection in

vaccinated animals. Interestingly, the study also compared the efficacy of a

commercial vaccine named Carovax (Wellcome Laboratories, UK, antigen

information unavailable, protecting sheep and pigs from M. haemolytica) with this

oil adjuvant vaccine containing locally isolated bacteria strains and found the

Carovax vaccine did not produce any significant reduction in lung lesions caused

by M. haemolytica and/or P. multocida. This observation indicated that geological

differences in terms of isolation of bacteria strains could also influence the efficacy

of vaccine, which would further complicate the development of a vaccine which

could protect sheep from diverse strains of the bacteria. E. F. Cassirer et al. 2001

[12] tested whether a combination of an experimental P. trehalosi and M.

haemolytica vaccine and a commercially-available bovine P. multocida and M.

haemolytica vaccine would increase lamb survival following a pneumonia

epidemic. They found that lamb survival differed among flocks (range 22% to

100%), and, unexpectedly, survival of lambs born to vaccinated ewe lambs was

8
lower (P = 0.08) than survival of lambs born to unvaccinated ones. Antibody titers

were high in ewes prior to vaccination, and vaccines failed to enhance antibody

titers in treated ewes. None of the lambs born to vaccinated ewe lambs survived.

These data suggested that, using existing technology, vaccinating ewe lambs

following pneumonia epidemics would have little chance of increasing neonatal

survival and population recovery, for example, in bighorn sheep.

Currently there is no effective vaccine that protects sheep from strains of both

M. haemolytica and P. multocida or strains derived from different geological

origins. However, given the significant economic losses due to sheep pneumonia

that is caused by these two bacteria, it is necessary to develop such a vaccine for

the benefits of sheep industry. It is likely that this can be realized. Jerry K.

McVicker et al. 2002 [13] indicated the development of an effective vaccine could

be possible based on the fact that animals that were naturally infected did develop

resistance to subsequent infection.

3. Materials and Methods

3.1 Animals

Sheep were from flocks at the Cornell Sheep Farm, composed of Dorsets and

Finnsheep. Twenty ewe lambs and twenty ram lambs born in August 2005 were

randomly selected and weaned on October, 2005. Blood samples were obtained

from each animal on November 16 before vaccination. On November 23, 10

9
randomly selected ewes and 10 randomly selected rams were vaccinated, while

an additional 10 animals of each sex comprised control groups. Blood samples

were obtained from both control and vaccinated animals on November 30,

December 7 and December 14.

3.2 M. haemolytica and P. multocida vaccine preparation

The vaccine used in this study was prepared by Dr. Yung-Fu Chang at the

Animal Health Diagnostic Center of Cornell University. Isolates of Mannheimia

haemolytica and Pasteurella multocida were obtained from Cornell lambs dying of

pneumonia. They were grown in culture and then killed and enriched for

leukotoxin (the toxin produced by the bacteria that causes lung damage) to create

the bacterin. The vaccine was expected to produce antibodies and prime

cell-mediated immune responses against both bacterial antigens and leukotoxin.

3.3 M. haemolytica and P. multocida antigen preparation for

ELISA tests

We prepared whole-cell antigens of both bacteria for ELISA testing. Both

bacteria were streaked onto BHI (Brain heart infusion broth) agar and then

collected separately into formaldehyde (4%) containing peptone water. After being

washed three times in phosphate buffered saline (PBS), the cell collection was

diluted to108 bacteria/ml for use in the assay [9] [15].

3.4 ELISA tests for specific antibody

Bacteria prepared in 3.3 were diluted to 106 cells per ml into carbonate coating

10
buffer (0.05M Na2CO3, 0.05M NaHCO3, pH 9.6). Each was then dispensed into

the wells of an Immunolon 1B plate (Thermo Fisher Scientific Inc, Waltham, MA)

in a volume of 50l per well. After overnight incubation at 4C, the plates were

washed twice with PBS containing 1% Tween20 and twice with PBS. Then serum

samples that had been serially diluted from 1/40 to 1/1280 into PBS containing

0.05% Tween 20, were added in duplicate to the wells, at 50l per well. After

overnight incubation at 4C, the plates were washed as described above. Then

50l anti-sheep IgG conjugated to alkaline phosphatase (Sigma Chemical Co., St.

Louis, MO) diluted 1:1000, was added to each well [9] [15]. The plates were

incubated overnight again and then washed as before. Finally, p-nitrophenyl

phosphate substrate (Sigma Chemical Co., St Louis, MO), with 1mM of MgCl2 was

added to each well in a volume of 50l per well. Absorbance values were read at

OD 405 nm after 20 min incubation by an ELISA reader.

3.5 Statistical analysis

Specific antibody titers in control and vaccinated sera were evaluated. We

used JMP data-analysis software to analyze our data; Fit Model tests examined

the regression relationship between two variables. The null hypothesis of the Fit

Model is that two variables are independent of each other. When P<0.05, the null

hypothesis is rejected.

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4 Results and Discussions
4.1 Determination of specific antibody titers

We did not have access to serum samples from known affected lambs;

therefore we had to determine ELISA titers using another approach. We reasoned

that if the vaccine was effective in inducing specific antibody, we would expect

that vaccinated animals would have higher specific antibody titers than control

animals would. Similar to the methods described by Mehmet et al. [9], we

determined the specific antibody titer as the reciprocal of the last dilution of each

serum sample that gave an OD that was greater than three standard deviations

above the mean value of the OD of sera from control sheep at the 1: 1280 dilution

(Table 1-8).

Table 1 Development of specific antibody titers against M. haemolytica over the four sampling dates in control ewes

Dates/Control
1 2 5 9 10 12 15 19 24 35
ewes ID
Nov. 16 160 640 640 320 320 640 160 640 640 160
Nov. 30 160 80 320 160 160 320 160 80 640 80
Dec. 7 40 40 640 160 80 640 40 80 80 40
Dec. 14 40 160 80 80 80 160 80 160 640 80

Table 2 Development of specific antibody titers against M. haemolytica over the four sampling dates in vaccinated ewes

Dates/Vaccinated
17 20 21 23 25 26 27 30 34 37
ewes ID
Nov. 16 320 1280 1280 640 320 320 320 320 160 320
Nov. 30 640 640 1280 1280 1280 1280 1280 1280 1280 1280
Dec. 7 160 320 1280 1280 1280 1280 640 160 40 320
Dec. 14 1280 640 640 640 640 640 640 320 640 320

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Table 3 Development of specific antibody titers against M. haemolytica over the four sampling dates in control rams

Dates/Control
3 7 13 14 28 29 31 33 38 40
rams ID
Nov. 16 160 80 320 40 320 320 320 40 80 40
Nov. 30 40 160 640 160 320 320 320 80 40 40
Dec. 7 160 160 160 160 160 160 160 160 320 160
Dec. 14 320 40 640 160 320 160 320 320 320 80

Table 4 Development of specific antibody titers against M. haemolytica over the four sampling dates in vaccinated rams

Dates/Vaccinated
4 6 8 11 16 18 22 32 36 39
rams ID
Nov. 16 1280 1280 640 640 640 320 320 640 320 320
Nov. 30 1280 1280 1280 1280 1280 1280 1280 1280 1280 1280
Dec. 7 640 40 40 40 40 40 40 40 40 40
Dec. 14 1280 1280 1280 1280 1280 320 1280 1280 1280 1280

Table 5 Development of specific antibody titers against P. multocida over the four sampling dates in control ewes

Dates/Control
1 2 5 9 10 12 15 19 24 35
ewes ID
Nov. 16 640 320 640 320 640 320 160 640 320 320
Nov. 30 160 160 320 160 160 320 320 160 640 320
Dec. 7 40 80 640 160 80 160 40 80 320 160
Dec. 14 80 320 320 160 160 320 320 640 320 320

Table 6 Development of specific antibody titers against P. multocida over the four sampling dates in vaccinated ewes

Dates/Vaccinated
17 20 21 23 25 26 27 30 34 37
ewes ID
Nov. 16 640 640 320 1280 320 1280 1280 640 640 640
Nov. 30 1280 1280 640 1280 640 1280 640 640 1280 1280
Dec. 7 40 160 160 320 160 320 80 160 80 160
Dec. 14 1280 1280 1280 1280 1280 640 1280 1280 1280 1280

Table 7 Development of specific antibody titers against P. multocida over the four sampling dates in control rams

Dates/Control
3 7 13 14 28 29 31 33 38 40
rams ID
Nov. 16 40 80 80 80 40 80 80 40 320 40
Nov. 30 160 320 640 320 320 640 160 160 160 80
Dec. 7 160 160 640 320 320 640 160 40 80 160
Dec. 14 640 160 640 160 640 320 320 320 320 320

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Table 8 Development of specific antibody titers against P. multocida over the four sampling dates in vaccinated rams

Dates/Vaccinated
4 6 8 11 16 18 22 32 36 39
rams ID
Nov. 16 320 320 320 320 160 320 320 320 160 80
Nov. 30 320 320 320 320 640 160 160 320 320 320
Dec. 7 1280 80 320 80 160 80 40 160 80 40
Dec. 14 1280 160 320 160 160 160 320 80 320 160

4.2 Data analysis and discussion

ELISA titer values were analyzed by the Fit Model tests of JMP. We examined

regression relationship of specific antibody titer with each of the three variables:

treatment (control or vaccination), sex (ewes or rams) and dates of sampling (Nov.

16, Nov. 30, Dec. 7 and Dec. 24).

4.2.1 Vaccine efficacy against M. haemolytica

4.2.1.1 Factors affecting specific antibody titers against M. haemolytica

Models showed that the vaccine did induce significantly (P<.0001<.05)

increased levels of specific antibodies against M. haemolytica in vaccinated

lambs over unvaccinated animals. We also found that there was a significant

regression relationship (P<0.0001<0.05) between the dates of sampling and

specific antibody titers. However, there was no difference in specific antibody

titers (P=0.9067>0.05) between ewe lambs and ram lambs.

4.2.1.2 Discussion of vaccine efficacy against M. haemolytica

(1) Interaction between treatment and dates of sampling in Ewes

According to Figure 1, after vaccination on Nov. 23, specific antibody titers

14
peaked on Nov. 30, and were much higher than the titers on Nov. 16. However,

the titer decreased sharply from Nov. 30 to Dec. 7, and then remained constant to

Dec. 14. The specific antibody titer levels on Dec. 7 and Dec. 14 were still about

100 titer units higher than that of ewes on Nov. 16 before vaccination. For control

ewes, there was a slight decrease in specific antibody titer from Nov. 16 to Nov. 30,

and then the titer remained constant during the remaining three sampling periods.

Development of specific antibody titers

against M. haemolytica in Ewes

1400
1200
1000
800 Control Ewes
Titers

600 Vaccinated Ewes

400
200
0
Nov. 16 Nov. 30 Dec. 7 Dec. 14
Treatment X Date

Figure 1 Development of specific antibody titers against M. haemolytica in ewes on Nov. 16, Nov. 30, Dec. 7 and Dec. 14.

To understand the trends in more detail, we also analyzed the specific

antibody titer development for each ewe over the entire sampling period (Figure 2

and 3). In control ewes, specific antibody titers decreased in 7 out of the 10

animals from Nov. 16 to Nov. 30. Since antibodies from passive transfer of

colostrum usually existed in lambs for only one to two weeks, it was not likely that

the decrease was due to colostrum antibody decline. Instead, we proposed that

15
the decline happened as a result of natural decrease of antibodies, which were

probably induced by previous exposure or infection of M. haemolytica. Then from

Nov. 30 to Dec. 7, 6 out of 10 control ewes titers were again slightly decreased

and remained constant on Dec. 14. In vaccinated ewes, specific antibody titers

increased sharply in 8 out of 10 vaccinated ewes from Nov. 16 to Nov. 30. On Nov.

30, 8 out 10 animals titers were 1280 while the remaining two were 640. Titers

then declined sharply in 6 ewes and remained the same in the remaining 4

animals on Nov. 30 as on Dec. 7. Finally, from Dec. 7 to Dec. 14, titers decreased

in 4 ewes, increased in 4 ewes and remained constant in the remaining 2 ewes.

Development of specific antibody titers

against M. haemolytica in Control Ewes

1400
1200
1000 Nov. 16
Titers

800
600 Nov. 30
400
200 Dec. 7
0
1 2 5 9 10 12 15 19 24 35 Dec. 14

Animal ID

Figure 2 Specific antibody titers development against M. haemolytica over the four sampling times in control ewes. The
X-axis denotes the ID of control ewes and Y-axis shows specific antibody titers.

16
 Development of specific antibody titers
against M. haemolytica in Vaccinated Ewes
1400
1200 Nov. 16
1000
Nov. 30
Titers

800
600 Dec. 7
400
200 Dec. 14
0
17 20 21 23 25 26 27 30 34 37
Animal ID

Figure 3 Specific antibody titers development against M. haemolytica over the four sampling dates in vaccinated ewes. The
X-axis denotes the ID of vaccinated ewes and Y-axis shows specific antibody titers.

The results indicate that the autogenous vaccine did induce significant

specific antibody production against M. haemolytica in the majority of the ewes

immediately following vaccination.

(2) Interaction between treatment and dates of sampling in Rams

According to Figure 4, specific antibody titers in vaccinated rams were higher

than that of control rams on all sampling dates except Dec. 7. For control rams,

titers generally remained low and constant.

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 Development of specific antibody titers
agaisnt M. haemolytica in Rams

1600
1400
1200
1000
Control Rams
Titers

800
Vaccinated Rams
600
400
200
0
Nov. 16 Nov. 30 Dec. 7 Dec. 14
Treatment X Date

Figure 4 Development of specific antibody titers against M. haemolytica in rams on Nov. 16, Nov. 30, Dec. 7 and Dec. 14.

To understand the trends in more detail, we also analyzed specific antibody

titer development in each ram over time (Figure 5 and 6). In the control group,

specific antibody titer values in most of the rams were below 320 and there were

no significant changes in titers over the four sampling times. In the vaccinated

group, specific antibody titers in 8 out of 10 rams increased from Nov. 16 to Nov.

30; on Nov. 30, the titers of all vaccinated rams were 1280. But the titers of all the

rams declined sharply from Nov. 30 to Dec. 7. On Dec. 7, 9 out of the 10

vaccinated rams titers dropped to 40 but rose again to 1280 in 9 vaccinated rams.

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 Development of specific antibody titers
against M. haemolytica in Control Rams

1400
1200
1000 Nov. 16
Titers

800
600 Nov. 30
400
200
0 Dec. 7
3 7 13 14 28 29 31 33 38 40 Dec. 14
Animal ID

Figure 5 Specific antibody titers development against M. haemolytica over the four sampling dates in control rams. The
X-axis denotes the ID of control rams and Y-axis shows specific antibody titers.

Development of specific antibody titers

against M. haemolytica in Vaccinated Rams

1400
1200 Nov. 16
1000
Nov. 30
Titers

800
600 Dec. 7
400
200 Dec. 14
0
4 6 8 11 16 18 22 32 36 39
Animal ID

Figure 6 Specific antibody titers development against M. haemolytica over the four sampling dates in vaccinated rams. The
X-axis denotes the ID of vaccinated rams and Y-axis shows specific antibody titers.

The results showed that the vaccine also induced significant antibody

production against M. haemolytica in rams, which declined quickly, within 1-2

weeks.

(3) Vaccine efficacy against M. haemolytica

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 There are several previous studies examining vaccine efficacy against whole

cell antigens of M. haemolytica. Srinand et al. [10] investigated antibody

responses against M. haemolytica whole cell antigens in animals treated with four

commercial vaccines and reported significant antibody level increases on day 28

post vaccination at P<0.05 level in animals vaccinated with Once PMH, One Shot,

Presponse. Mehmet et al. [9] examined one vaccines efficacy in inducing

specific antibodies against whole cell antigens of M. haemolytica in lambs

(including both ewes and rams) three weeks post vaccination using ELISA tests,

and found ELISA titers differed from 80 and 320, and between 160 and 320 in

lambs vaccinated once and twice, respectively, while titers in unvaccinated

controls were negative. Lung lesion scores were also compared and significant

decreases were observed in the trials compared with control groups at P<0.001.

In our study, we found that the overall production of specific antibody against

M. haemolytica in both vaccinated ewe lambs and ram lambs was significantly

higher than that of control lambs. This indicates that vaccination did induce

specific antibody response in lambs, and agrees with the previous studies. In

addition, in our study, vaccinated ewes had antibody titers at three weeks post

vaccination ranging from 320 to 1280 (320 in 2 ewes, 640 in 7 ewes and 1280 in 1

ewe). Compared with titers of vaccinated groups in Mehmet et al. study, the titer

level in our study was higher. In control ewes in our study, titers were from 40 to

640 (40 in 1 ewe, 80 in 5 ewes, 160 in 3 ewes and 640 in 1 ewe). Titers in 9

20
vaccinated rams were 1280; in control rams, titers ranged from 40 to 640 (40 in 1

ram, 80 in 1 ram, 160 in 2 rams, 320 in 5 rams, and 640 in 1 ram). The fact that we

were able to detect some antibody in control animals may have been due to

natural infection or exposure.

Interestingly, from Nov. 30 to Dec. 7, antibody titers in both vaccinated ewes

and rams decreased dramatically. On Dec. 7, for vaccinated ewes, titers in all the

other 6 ewes decreased except that 4 animals titers remained at the titer level of

1280 as they were on Nov. 30. For vaccinated rams from Nov. 30 to Dec. 7, titers

in 9 out of all 10 rams dropped from 1280 to 40. We may rule out the possibility of

assay failure based on the fact that high antibody titers in sera taken on Dec. 7

were detected in 4 ewes. Since all tests for sera on Dec. 7 were performed at the

same time, if the assay had failed, there would have been no antibody detected in

every sera sample. On the other hand, A.W. Confer et al. 2009 [16] also reported

decrease of specific antibody against whole cell M. haemolytica antigens in

vaccinated animals between Day 14 and Day 21 after vaccination, which

remained constant after Day 21, but the average response still remained higher

than that of control animals; we did not see this in the present study. For the

vaccinated ewes and rams in our experiment, we proposed that the titer decline

from Nov. 30 to Dec. 7 may have also been due to natural antibody decrease,

which usually declined to basal level within 14 days. It is possible that the titer

increase from Dec. 7 to Dec. 14 in vaccinated rams may have been induced by

21
exposure to M. haemolytica in the environment that would act as a booster, and

induce a secondary immune response. This hypothesis is supported by the fact

that the antibody titers against M. haemolytica in control rams, although slightly

lower, also increased from Dec. 7 to Dec. 14, and again may have been due to

exposure to the organism(s) in the environment.

Finally, the results suggest there were no gender differences in antibody

response against M. haemolytica induced by vaccination.

4.2.2 Vaccine efficacy against P. multocida

4.2.2.1 Factors affecting specific antibody titers against P. multocida

The JMP model indicated that the vaccine did induce a significant increase

(P<0.0001<0.05) in the production of specific antibody against P. multocida in

vaccinated lambs over unvaccinated lambs. It also showed there was significant

regression relationship (P<0.0001<0.05) between sex and specific antibody. In

addition, titers of specific antibodies varied significantly (P<0.0001<0.05) across

the four sampling dates.

4.2.2.2 Discussion of vaccine efficacy against P. multocida

(1) Interactions between treatment and dates of sampling in Ewes

In Figure 7, the specific antibody titers in the vaccinated group were higher

than the corresponding titers in the control group over the experimental period

except on Dec. 7. After vaccination on Nov. 23, there was an increase in specific

22
antibody titer on Nov. 30, compared to Nov. 16, and also that of titers in the control

group. However, there was a sudden titer decrease on Dec. 7, and a sharp

increase followed on Dec. 14 for vaccinated sera. In the control group, the titer

decreased from Nov. 16 to Dec. 7.

Development of specific antibody titers

against P. multocida in Ewes

1400
1200
1000
800 Control Ewes
Titers

600 Vaccinated Ewes

400
200
0
Nov. 16 Nov. 30 Dec. 7 Dec. 14
Dates of Sampling

Figure 7 Development of specific antibody titers against P. multocida in ewes on Nov. 16, Nov. 30, Dec. 7 and Dec. 14.

To understand the trend in more detail, we analyzed the specific antibody titer

in each ewe over time (Figures 8 and 9). In control ewes, from Nov. 16 to Nov. 30,

titers in 6 out of 10 animals decreased, while titers for two animals increased and

the remaining two were unchanged. Then from Nov. 30 to Dec. 7, titers decreased

again in 8 animals. Finally, titers increased from Dec. 7 to Dec. 14 in 7 out of 10

animals. The titer on Dec. 14 was close to that on Nov. 30. Since antibodies from

passive transfer of colostrum usually existed in lambs for only one to two weeks, it

was not likely that the titer level decline in the majority of control ewes from Nov.

16 to Nov. 30 was due to colostrum antibody decrease. Instead, we proposed that

23
the decline happened as a result of natural decrease of antibodies, which were

probably induced by previous exposure or infection of P. multocida. In vaccinated

ewes, titers increased from Nov. 16 to Nov. 30 in 6 out of 10 animals, while titers in

three ewes remained the same and one decreased. On Nov. 30, titers for 6 ewes

were 1280 and that of the remaining four were 640. Then titers decreased sharply

from Nov. 30 to Dec. 7 in all 10 animals and titers on Dec. 7 ranged from 40 to 320.

Finally, titers increased in all animals from Dec. 7 to Dec. 14, to 1280 in 9 animals

on Dec. 14.

Development of specific antibody titers

against P. multocida in Control Ewes

1400
1200 Nov. 16
1000
Nov. 30
Titers

800
600 Dec. 7
400
200 Dec. 14
0
1 2 5 9 10 12 15 19 24 35
Animal ID

Figure 8 Development of specific antibody titers against P. multocida in control ewes over the four sampling dates. The
X-axis denotes the ID of control ewes and the Y-axis shows the specific antibody titers.

24
 Development of specific antibody titers
against P. multocida in Vaccinated Ewes

1400
1200 Nov. 16
1000
Nov. 30
Titers

800
600 Dec. 7
400
200 Dec. 14
0
17 20 21 23 25 26 27 30 34 37
Animal ID

Figure 9 Development of specific antibody titers against P. multocida in vaccinated ewes over the four sampling dates. The
X-axis denotes the ID of vaccinated ewes and the Y-axis shows specific antibody titers.

The results indicated that the vaccine did induce strong specific antibody

production against P. multocida in ewes, which declined quickly in 1-2 weeks.

(2) Interactions between treatment and dates of sampling in Rams

In Figure 10, there was no significant difference in specific titers between

vaccinated ram lambs and control ram lambs over the four sampling dates.

Development of specific antibody titers

against P. multocida in Rams

1400
1200
1000
800 Control Rams
Titers

600 Vaccinated Rams

400
200
0
Nov. 16 Nov. 30 Dec. 7 Dec. 14
Dates of Sampling

Figure 10 Development of specific antibody titers against P. multocida in rams on Nov. 16, Nov. 30, Dec. 7 and Dec. 14.

25
 To understand the phenomenon in more details, we also analyzed the

development of specific antibody titers in each ram over the four sampling dates

(Figures 11 and 12). Specific antibody titers in almost all vaccinated animals

across the four sampling times were below 400. Unexpectedly, more

unvaccinated rams had titers greater than 400 compared to vaccinated rams. Also,

after immediate vaccination titers were increased only in three vaccinated rams,

from Nov. 16 to Nov. 30.

Development of specific antibody titers

against P. multocida in Control Rams

1400
1200 Nov. 16
1000
Nov. 30
Titers

800
600 Dec. 7
400
200 Dec. 14
0
3 7 13 14 28 29 31 33 38 40
Animal ID

Figure 11 Development of specific antibody titers against P. multocida in control rams over the four sampling dates. The
X-axis denotes the ID of control rams and the Y-axis shows specific antibody titers.

26
 Development of specific antibody titers
against P. multocida in Vaccinated Rams

1400
1200 Nov. 16
1000
Nov. 30
Titers

800
600 Dec. 7
400
200 Dec. 14
0
4 6 8 11 16 18 22 32 36 39
Animal ID

Figure 12 Development of specific antibody titers against P. multocida in vaccinated rams over the four sampling dates.
The X-axis denotes the ID of vaccinated rams and the Y-axis shows the specific antibody titers.

The result indicated that the vaccine failed to induce significant specific

antibody response in rams.

(3) Vaccine efficacy against P. multocida

Several previous studies investigated the efficacy of vaccination against P.

multocida and found that vaccination against P. multocida was not as effective as

against M. haemolytica. Chandrasekaran S et al. 1991 [11] tested an oil

adjuvant vaccine incorporating antigens from both M. haemolytica and P.

multocida, and reported significant pneumonic lesion reduction in vaccinated

lambs after challenge with M. haemolytica, but not after challenge with P.

multocida. Carmen et al. 1983 [17] argued that the limited protection of specific

antibody against P. multocida via vaccination was probably because P. multocida

strains did not share universal antigens, and they proposed that finding a new

27
strain with wide spectrum of antigens would help solve this problem.

According to the data presented in section 4.2.2.2, we observed significant

overall specific antibody response against P. multocida in vaccinated ewes

compared with control ewes, while there were no significant differences between

titers of control rams and vaccinated rams. The results indicated a gender

difference in specific antibody response against P. multocida vaccines. The exact

reason for gender difference in antibody response to vaccination is still uncertain,

but several studies have shown that some immune responses in humans are

higher in females compared to males, including numbers of CD4+ T cells [18] [19]

[20] [21]. In particular, Alberto Amadori et al. 1995 [19] examined CD4 and CD8

T cell numbers in healthy donors and reported the CD4/CD8 T cells ratio was

significantly higher in females than that in males, which, they proposed, was due

to genetic regulation in humans. Bret J. Rudy et al. 2002 [21] performed flow

cytometry analysis of lymphocyte subset markers on a group of sexually active,

human immunodeficiency virus (HIV)-negative adolescents, and found that

females had higher total CD4+ T cell and CD4+ memory T cell counts compared

to males. These observations suggest, at least in part, why females may have

responded better than males in terms of antibody production. Furthermore, the

function of these T cells (TH1 or TH2) rather than number, may also be important

[22]. In addition, considering the differences in the immune systems of humans

and sheep, other factors may play a role in the gender differences in antibody

28
response in lambs.

Interestingly, the antibody titer in vaccinated ewes declined sharply from Nov.

30 to Dec. 7. On Nov. 30, titers for 6 vaccinated ewes were 1280 and the other 4

ewes titers were 640. On Dec.7, titers were as low as 40 to 320. The possibility

that the ELISA assay failed was ruled out because other samples that were tested

in the same assay showed significant antibody titers. It is possible that sampling

or storage issues may have resulted in our inability to detect specific antibody in

the sera of vaccinated ewes. It is also possible that the titer decline may have

been due to a natural decrease in sera antibody levels, and the titer surge from

Dec. 7 to Dec. 14 represented a secondary immune response induced by a

natural P. multocida infection or exposure. This is supported by the fact that the

vaccinated ewes antibody titer on Dec. 24 was higher than that on Dec. 7. The

slight titer increase that we noted in sera from Dec. 7 to Dec. 14 in both

vaccinated and control rams, suggests that a natural infection or exposure to P.

multocida may have occurred on the farm around Dec. 7.

29
5. References

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(Pasteurella) haemolytica. Front Biosci., 6:D1128-50.
2. Confer AW, 1993. Immunogens of Pasteurella. Vet Microbiol., 37(3-4):353-68.
3. Ackermann MR, Brogden KA, 2000. Response of the ruminant respiratory
tract to Mannheimia (Pasteurella) haemolytica. Microbes Infect., 2(9):1079-88.
4. Rudolph K. M., Hunter D. L., Rimler R. B., Cassirer E. F., Foreyt W. J., DeLong
WJ, Weiser GC, Ward AC, 2007. Microorganisms associated with a
pneumonic epizootic in Rocky Mountain bighorn sheep (Ovis canadensis
canadensis). J Zoo Wild. Med., 38(4):548-58.
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the prevalence and onset of lung lesions and their impact on growth of lambs.
Am J Vet Res., 67(5):890-4.
6. Ewers C, Lbke-Becker A, Wieler LH, 2004. Pasteurella: insights into the
virulence determinants of a heterogenous bacterial type. Berl Munch Tierarztl
Wochenschr., 117(9-10):367-86.
7. N.J.L. Gilmour, W. Donachie, A.D. Sutherland, J.S. Gilmour, G.E. Jones and
M. Quirie, 1991. Vaccine containing iron-regulated proteins of Pasteurella
haemolytica A2 enhances protection against experimental pasteurellosis in
lambs. Vaccine, 137140.
8. W. Donachie, C. Burrels, A.D. Sutherland, J.S. Gilmour and N.J.L. Gilmour,
1986. Immunity of specific pathogen free lambs to challenge with an aerosol of
Pasteurella haemolytica biotype A serotype 2. Pulmonary antibody and cell
response to primary and secondary infections. Vet. Immunol. Immunopathol.,
265279.
9. Mehmet Akan, Taner ncel, Bar Sareyypolu, Rfk Hazrolu, Osman
Yaar Tel and Zafer Cantekin, 2006. Vaccination studies of lambs against
experimental Mannheimia (Pasteurella) haemolytica infection. Small Ruminant
Research, 65: 4450.
10. S. Srinand, S. L. Hsuan, H. S. Yoo, S. K. Maheswaran, T. R. Ames and R. E.
Werdin, 1996. Comparative evaluation of antibodies induced by commercial
Pasteurella haemolytica vaccines using solid phase immunoassays.
Veterinary Microbiology, 49: 181 195.
11. Chandrasekaran S, Hizat K, Saad Z, Johara MY, Yeap PC, 1991. Evaluation
of Combined Pasteurella vaccines in control of sheep pneumonia. Br. Vet.,
147 (5): 437-443.
12. E. F. Cassirer, K. M. Rudolph, P. Fowler, V. L. Coggins, D.L. Hunter, and M. W.
Miller, 2001. Evaluation of ewe vaccination as a tool for increasing bighorn

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 lamb survival following pasteurellosis epizootics. Journal of Wildlife Diseases,
37(1): 4957.
13. Jerry K. McVicker, M. S. Louisa B. Tabatabai, 2002. Isolation of immunogenic
outer membrane proteins from Mannheimia haemolytica serotype 1 by use of
selective extraction and immunoaffinity chromatography. American Journal of
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14. Mosier, D. A., Confer, A. W., Hall, S. M., Gentry, M. J., Panciera, R. J., 1986.
Enzyme-linked immunosorbent assay for detection of serum antibodies to
Pasteurella haemolytica cytotoxin (leukotoxin) in cattle. J. Clin. Microbiol., 24:
218222.
15. Diker, K.S., Akan, M., Kaya, O., 2000. Evaluation of immunogenicity of
Pasteurella haemolytica serotypes in experimental models. Turk. J. Vet. Anim.
Sci., 24: 139143.
16. A. W. Confer, S. Ayalew, M. Montelongo, D. L. Step, J. H. Wray, R.D. Hansen,
R. J. Panciera, 2009. Immunity of cattle following vaccination with a
Mannheimia haemolytica chimeric PlpELKT (SAC89) protein. Vaccine, 27:
17711776.
17. Carmen C.M, Bester F. J., 1983. The inefficacy of polivalent Pasteurella
multocida vaccines for sheep. Onderstepoort J. Vet. Res., 50(2):101-104.
18. I. M. Lisse, P. Aaby, H. Whittle, H. Jensen, M. Engelmann and L. B.
Christensen, 1997. T-lymphocyte subsets in West African children: impact of
age, sex and season. J Pediatr., 130: 7781.
19. A. Amadori, R. Zamarchi, G. de Silvestro, G. Forza, C. Cavatton and G.A.
Danieli et al., 1995. Genetic control of the CD4/CD8 T cell ratio in humans. Nat.
Med., 1: 12791283.
20. S. S. Uppal, S. Verma and P.S. Dhot, 2003. Normal values of CD4 and CD8
lymphocyte subsets in healthy Indian adults and the effects of sex, age,
ethnicity and smoking. Cytom. Part B: Clin. Cytom., 52B: 3236.
21. B. J. Rudy, C. M. Wilson, S. Durako, A.-B. Moscicki, L. Muenz and S. D.
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31
6. Appendix
6.1 Raw data from ELISA tests
Table 1 Absorbance of Control Ewes serum samples against M. haemolytica on Nov. 16
Animal
ID
Dilutions 1 2 5 9 10 12 15 19 24 35
40 0.1534 0.2076 0.1975 0.2411 0.2124 0.1607 0.1466 0.1588 0.2172 0.1311
80 0.1246 0.1956 0.2143 0.1809 0.1739 0.1694 0.1373 0.1215 0.1427 0.1271
160 0.1239 0.1522 0.1526 0.1521 0.1438 0.0711 0.1144 0.1371 0.1432 0.1405
320 0.0913 0.1399 0.121 0.1434 0.1267 0.1511 0.0918 0.1349 0.1616 0.0899
640 0.0888 0.1096 0.1111 0.1014 0.0939 0.1272 0.072 0.1096 0.1346 0.1125
1280 0.0768 0.0987 0.0822 0.0885 0.0829 0.0993 0.0712 0.0789 0.0941 0.0744
blank 0.0662 0.0632 0.0642 0.0602 0.0589 0.0997 0.0631 0.0602 0.0548 0.0561

Table 2 Absorbance of Vaccinated Ewes serum samples against M. haemolytica on Nov. 16

Animal
ID
Dilutions 19 20 21 23 25 26 27 30 34 37
40 0.1837 0.2083 0.2032 0.2074 0.2132 0.2304 0.2009 0.206 0.133 0.1498
80 0.1581 0.1847 0.176 0.1966 0.1401 0.1949 0.1583 0.151 0.1238 0.1569
160 0.1313 0.1666 0.1775 0.1687 0.1342 0.1353 0.1555 0.1364 0.113 0.1434
320 0.1762 0.1451 0.1232 0.138 0.1057 0.1051 0.1308 0.1147 0.0993 0.1044
640 0.1023 0.1778 0.1584 0.1428 0.0922 0.0979 0.1023 0.091 0.101 0.0924
1280 0.0946 0.1662 0.1397 0.0889 0.0805 0.0921 0.0922 0.0911 0.0752 0.0804
blank 0.1045 0.0807 0.0831 0.0679 0.0658 0.0724 0.0647 0.07 0.0627 0.0634

Table 3 Absorbance of Control Ewes serum samples against P. multocida on Nov. 16

Animal
ID
Dilutions 1 2 5 9 10 12 15 19 24 35
40 0.3599 0.3809 0.5181 0.3229 0.2511 0.4124 0.5713 0.2885 0.4469 0.2626
80 0.2304 0.2652 0.3546 0.2811 0.3662 0.3526 0.3335 0.2497 0.2938 0.277
160 0.1566 0.1677 0.2832 0.246 0.2155 0.2207 0.2044 0.184 0.2397 0.2254
320 0.1244 0.148 0.2188 0.2361 0.2297 0.1748 0.1354 0.1604 0.1539 0.1499
640 0.1443 0.1255 0.152 0.1348 0.1507 0.1397 0.1332 0.1472 0.0976 0.1232
1280 0.0872 0.0949 0.1086 0.1149 0.1115 0.1063 0.0954 0.0962 0.1007 0.063
blank 0.0642 0.0759 0.0656 0.0703 0.0823 0.0652 0.0659 0.0679 0.0695 0.0695

32
Table 4 Absorbance of Vaccinated Ewe serum samples against P. multocida on Nov. 16
Animal
ID
Dilutions 19 20 21 23 25 26 27 30 34 37
40 0.3718 0.4412 0.4122 0.3645 0.3798 0.4079 0.4261 0.3784 0.3081 0.2369
80 0.3283 0.2968 0.3264 0.382 0.3324 0.3328 0.34 0.2839 0.2394 0.2389
160 0.262 0.2472 0.2304 0.3401 0.245 0.2504 0.2475 0.2404 0.1953 0.2096
320 0.2368 0.2229 0.159 0.253 0.2548 0.2163 0.274 0.2306 0.2103 0.2115
640 0.1751 0.1695 0.126 0.2168 0.1342 0.1697 0.1793 0.1737 0.1556 0.1681
1280 0.122 0.1317 0.103 0.1544 0.1353 0.1517 0.1528 0.125 0.1159 0.1251
blank 0.0699 0.0686 0.0675 0.0689 0.0713 0.0713 0.0722 0.0729 0.0741 0.0708

Table 5 Absorbance of Control Ewe serum samples against M. haemolytica on Nov. 30

Animal
ID
Dilution 1 2 5 9 10 12 15 19 24 35
40 0.1053 0.1132 0.1133 0.0954 0.0896 0.0872 0.0891 0.0861 0.1028 0.0877
80 0.0894 0.0997 0.1012 0.092 0.0833 0.0902 0.0837 0.0813 0.091 0.0883
160 0.0828 0.0811 0.0826 0.0874 0.0839 0.0877 0.0824 0.0772 0.0866 0.0795
320 0.0715 0.0794 0.0822 0.0777 0.0728 0.0819 0.0713 0.0739 0.0839 0.0759
640 0.0678 0.0741 0.0754 0.079 0.0726 0.0776 0.0699 0.0712 0.0853 0.0795
1280 0.0673 0.0681 0.0732 0.0753 0.0671 0.0755 0.0695 0.0731 0.0744 0.067
blank 0.0645 0.0657 0.0591 0.0609 0.0653 0.0579 0.0726 0.0681 0.0729 0.0736

Table 6 Absorbance of Vaccinated Ewe serum samples against M. haemolytica on Nov. 30

Animal
ID
Dilution 17 20 21 23 25 26 27 30 34 37
40 0.3998 0.1339 0.1139 0.107 0.1084 0.111 0.1077 0.1417 0.1724 0.1843
80 0.1039 0.1391 0.1515 0.107 0.0937 0.1113 0.1052 0.2014 0.1323 0.1752
160 0.088 0.1044 0.107 0.1077 0.0942 0.0967 0.0973 0.1613 0.1297 0.1462
320 0.099 0.0967 0.1021 0.096 0.0938 0.0985 0.0858 0.1534 0.1097 0.1422
640 0.1112 0.1018 0.096 0.0957 0.0844 0.0862 0.0958 0.1079 0.0865 0.1093
1280 0.078 0.0788 0.0878 0.0904 0.0877 0.08 0.086 0.0985 0.0819 0.0989
blank 0.0607 0.0573 0.0587 0.0682 0.0687 0.0638 0.059 0.0676 0.0624 0.0618

33
Table 7 Absorbance of Control Ewe serum samples against P. multocida on Nov. 30
Animal
ID
Dilution 1 2 5 9 10 12 15 19 24 35
40 0.191 0.1993 0.2943 0.1975 0.222 0.2917 0.2117 0.217 0.2525 0.2209
80 0.1628 0.1668 0.2524 0.1798 0.1773 0.2121 0.1874 0.2005 0.2334 0.1466
160 0.1434 0.1267 0.1805 0.1433 0.1386 0.1551 0.1664 0.1254 0.1749 0.1687
320 0.1108 0.1002 0.15 0.1081 0.1064 0.1425 0.1438 0.1327 0.17 0.1412
640 0.096 0.0842 0.1148 0.095 0.0889 0.1181 0.1201 0.1172 0.1471 0.109
1280 0.0861 0.0864 0.102 0.0882 0.088 0.1183 0.1123 0.0846 0.1153 0.0889
blank 0.0647 0.0756 0.077 0.0784 0.0743 0.085 0.069 0.0615 0.0624 0.0592

Table 8 Absorbance of Vaccinated Ewe serum samples against P. multocida on Nov. 30

Animal
ID
Dilution 17 20 21 23 25 26 27 30 34 37
40 0.3251 0.3068 0.2824 0.3193 0.2613 0.2983 0.2829 0.2377 0.3471 0.2433
80 0.2759 0.3075 0.3027 0.3056 0.2357 0.2609 0.2459 0.25 0.2713 0.1869
160 0.2494 0.239 0.1882 0.2509 0.2025 0.2293 0.2166 0.2051 0.2188 0.2066
320 0.1698 0.2083 0.1684 0.1987 0.1775 0.1893 0.1618 0.1645 0.1804 0.1827
640 0.1714 0.1784 0.1472 0.1617 0.1523 0.1449 0.1415 0.1434 0.1446 0.1373
1280 0.1398 0.1307 0.121 0.141 0.1164 0.1229 0.1138 0.1208 0.1254 0.1301
blank 0.0669 0.0629 0.0647 0.0715 0.0674 0.0618 0.0628 0.0648 0.0671 0.0611

Table 9 Absorbance of Control Ewe serum samples against M. haemolytica on Dec. 7

Animal ID
Dilution 1 3 5 9 10 12 15 19 24 35
40 0.1049 0.1406 0.2397 0.1507 0.1679 0.1177 0.112 0.1322 0.1482 0.1418
80 0.0958 0.1118 0.1794 0.1215 0.1297 0.1683 0.1139 0.1352 0.1459 0.1066
160 0.0824 0.1171 0.1471 0.1245 0.108 0.1245 0.1088 0.1009 0.1104 0.0925
320 0.0811 0.1122 0.1524 0.1141 0.0956 0.1335 0.0945 0.0946 0.1168 0.0951
640 0.1035 0.1034 0.1211 0.1083 0.0933 0.1235 0.0904 0.1043 0.0995 0.0745
1280 0.0672 0.1027 0.1014 0.0911 0.0899 0.1072 0.0855 0.0904 0.1055 0.0766
blank 0.0625 0.0624 0.059 0.0577 0.056 0.0575 0.0634 0.0564 0.0575 0.0591

34
Table 10 Absorbance of Vaccinated Ewe serum samples against M. haemolytica on Dec. 7
Animal
ID
Dilution 17 20 21 23 25 26 27 30 34 37
40 0.1469 0.1841 0.2077 0.2717 0.118 0.1356 0.1275 0.155 0.1207 0.1353
80 0.1311 0.1876 0.1811 0.1606 0.12 0.1315 0.1228 0.1128 0.1089 0.1407
160 0.1252 0.1248 0.1498 0.1578 0.1145 0.1198 0.1267 0.1219 0.0998 0.1317
320 0.099 0.1258 0.1634 0.1742 0.1193 0.1096 0.1175 0.098 0.0904 0.1202
640 0.1024 0.1084 0.1512 0.1373 0.1313 0.1531 0.19 0.1043 0.0806 0.0909
1280 0.12 0.1158 0.1418 0.1413 0.125 0.1206 0.1122 0.1239 0.0809 0.0871
blank 0.0629 0.0731 0.0669 0.0504 0.061 0.0597 0.0573 0.0585 0.0576 0.0622

Table 11 Absorbance of Control Ewe serum samples against P. multocida on Dec. 7

Animal
ID
Dilution 1 2 5 9 10 12 15 19 24 35
40 0.1872 0.186 0.3527 0.1985 0.2294 0.2189 0.1966 0.2225 0.2386 0.219
80 0.1384 0.1754 0.3474 0.1944 0.184 0.1645 0.163 0.1781 0.2193 0.2114
160 0.1216 0.1634 0.3102 0.1706 0.1504 0.2049 0.159 0.1634 0.1851 0.1741
320 0.0999 0.1182 0.245 0.158 0.1354 0.1612 0.1365 0.1378 0.1672 0.1481
640 0.0908 0.1086 0.1878 0.1211 0.143 0.1408 0.1228 0.1208 0.1269 0.125
1280 0.0786 0.092 0.1467 0.1032 0.0903 0.128 0.107 0.113 0.1065 0.1171
blank 0.0615 0.0601 0.061 0.0703 0.0577 0.0566 0.0568 0.0561 0.059 0.058

Table 12 Absorbance of Vaccinated Ewe serum samples against P. multocida on Dec. 7

Animal
ID
Dilution 17 20 21 23 25 26 27 30 34 37
40 0.1919 0.2737 0.2689 0.2454 0.1627 0.2083 0.1801 0.2103 0.2078 0.1812
80 0.1277 0.2065 0.2553 0.1968 0.2031 0.1823 0.1721 0.1786 0.2019 0.1664
160 0.1263 0.169 0.1962 0.2056 0.176 0.1887 0.1483 0.1899 0.1461 0.1646
320 0.1287 0.1824 0.1555 0.1725 0.1587 0.1814 0.1417 0.1635 0.1272 0.1252
640 0.1134 0.1335 0.1313 0.142 0.1389 0.1373 0.1321 0.1185 0.1233 0.143
1280 0.1113 0.1377 0.1055 0.132 0.1356 0.1146 0.1139 0.1412 0.1059 0.129
blank 0.0659 0.0592 0.0592 0.0631 0.0582 0.0594 0.0595 0.0628 0.0606 0.0615

35
Table 13 Absorbance of Control ewe serum samples against M. haemolytica on Dec. 14
Animal
ID
Dilution 17 20 21 23 25 26 27 30 34 37
1:40 0.1855 0.2916 0.3401 0.248 0.215 0.2293 0.2756 0.286 0.4174 0.3026
1:80 0.155 0.231 0.4654 0.2192 0.232 0.2679 0.2327 0.2305 0.4113 0.247
1:160 0.1333 0.2183 0.1762 0.1719 0.1704 0.2423 0.1607 0.1929 0.3215 0.191
1:320 0.1056 0.1592 0.1422 0.1394 0.1513 0.1665 0.1567 0.1456 0.3517 0.1553
1:640 0.0903 0.149 0.1275 0.1123 0.122 0.1722 0.1259 0.1498 0.2355 0.1385
1:1280 0.0938 0.1127 0.0909 0.1038 0.0876 0.1518 0.1041 0.1117 0.1737 0.1119
Blank 0.0719 0.06 0.0636 0.06 0.0603 0.0603 0.0653 0.0644 0.0614 0.062

Table 14 Absorbance of Vaccinated Ewe serum samples against M. haemolytica on Dec. 14

Animal
ID
Dilution 17 20 21 23 25 26 27 30 34 37
1:40 0.3932 0.4006 0.3977 0.5145 0.5558 0.4808 0.3239 0.34 0.5196 0.3196
1:80 0.3503 0.3336 0.3348 0.3472 0.3604 0.4006 0.3122 0.3952 0.4041 0.3119
1:160 0.2944 0.3388 0.3855 0.3307 0.3092 0.2705 0.2861 0.2386 0.3374 0.2308
1:320 0.2553 0.2326 0.2756 0.2502 0.2732 0.2296 0.2532 0.205 0.3516 0.2069
1:640 0.2207 0.21 0.2027 0.1975 0.2633 0.2211 0.1957 0.1476 0.213 0.1445
1:1280 0.2107 0.1671 0.1636 0.1679 0.1638 0.164 0.1265 0.1192 0.1605 0.1396
Blank 0.1182 0.0612 0.0626 0.063 0.0676 0.0646 0.0608 0.0604 0.0664 0.0694

Table 15 Absorbance of Control Ewe serum samples against P. multocida on Dec. 14

Animal
ID
Dilution 1 2 5 9 10 12 15 19 24 35
1:40 0.2346 0.3487 0.5961 0.3806 0.3846 0.2895 0.3325 0.412 0.4374 0.341
1:80 0.1895 0.2771 0.4412 0.3081 0.3084 0.3145 0.3081 0.3465 0.2945 0.2606
1:160 0.1627 0.2413 0.2751 0.2205 0.225 0.2967 0.2316 0.2764 0.4374 0.231
1:320 0.1471 0.1962 0.1988 0.1719 0.1663 0.2516 0.2066 0.2747 0.2118 0.1845
1:640 0.0994 0.1474 0.1485 0.1581 0.1315 0.1345 0.1715 0.1904 0.1605 0.1783
1:1280 0.0834 0.1111 0.1184 0.1005 0.1114 0.1585 0.133 0.1361 0.139 0.1184
Blank 0.0638 0.0701 0.0635 0.0638 0.0637 0.0615 0.0641 0.062 0.0638 0.0618

36
Table 16 Absorbance of Vaccinated Ewe serum samples against P. multocida on Dec. 14
Animal
ID
Dilution 17 20 21 23 25 26 27 30 34 37
1:40 0.6754 0.6545 0.6587 0.5687 0.5977 0.6212 0.4785 0.3853 0.6574 0.4599
1:80 0.6479 0.6446 0.5406 0.4582 0.6354 0.4617 0.479 0.414 0.7461 0.4423
1:160 0.6011 0.5775 0.53 0.4598 0.3777 0.4067 0.4712 0.4353 0.5303 0.3934
1:320 0.318 0.4415 0.3686 0.4234 0.4204 0.3156 0.3321 0.4212 0.4804 0.3753
1:640 0.2771 0.3918 0.2423 0.3329 0.2851 0.2374 0.2705 0.2843 0.3117 0.3066
1:1280 0.4044 0.2493 0.2261 0.2644 0.2023 0.1728 0.2094 0.2118 0.2042 0.2003
Blank 0.0768 0.0725 0.0739 0.0909 0.0642 0.0648 0.0722 0.0628 0.0682 0.0703

Table 17 Absorbance of Control Ram serum samples against M. haemolytica on Nov. 16

Animal
ID
Dilution 3 7 13 14 28 29 31 33 38 40
40 0.1018 0.1158 0.1766 0.1006 0.1138 0.1408 0.1653 0.104 0.1014 0.0965
80 0.1063 0.1093 0.1393 0.0916 0.1167 0.1153 0.1574 0.0929 0.123 0.095
160 0.1058 0.0943 0.1189 0.0869 0.1059 0.1425 0.138 0.0863 0.0906 0.0802
320 0.0848 0.1077 0.1852 0.0727 0.0995 0.1322 0.1197 0.1792 0.113 0.0725
640 0.112 0.0901 0.0887 0.0757 0.0865 0.0965 0.0965 0.0758 0.0842 0.0758
1280 0.0647 0.0815 0.0748 0.0826 0.0809 0.0809 0.0868 0.0647 0.0689 0.07
blank 0.0594 0.0612 0.0572 0.0707 0.0603 0.066 0.0627 0.0561 0.059 0.058

Table 18 Absorbance of Vaccinated Ram serum samples against M. haemolytica on Nov. 16

Animal
ID
Dilution 4 6 8 11 16 18 22 32 36 39
40 0.2134 0.1379 0.1337 0.308 0.1319 0.1372 0.1209 0.1437 0.1284 0.1257
80 0.1791 0.1742 0.1174 0.1304 0.1343 0.146 0.1298 0.1066 0.1182 0.1027
160 0.1672 0.1197 0.1402 0.1393 0.1075 0.1203 0.1311 0.1124 0.1426 0.0832
320 0.1279 0.109 0.1277 0.1366 0.1052 0.1151 0.1669 0.1091 0.125 0.0995
640 0.1295 0.1089 0.1139 0.1986 0.0986 0.0947 0.0899 0.1034 0.0902 0.0826
1280 0.1153 0.2168 0.0929 0.0822 0.0851 0.0867 0.124 0.0879 0.0833 0.0627
blank 0.3601 0.1207 0.0738 0.0613 0.0676 0.0671 0.0762 0.0656 0.0958 0.084

37
Table 19 Absorbance of Control Ram serum samples against P. multocida on Nov. 16
Animal
ID
Dilution 3 7 13 14 28 29 31 33 38 40
40 0.1319 0.2144 0.1225 0.1681 0.1728 0.1897 0.157 0.1411 0.165 0.1594
80 0.1153 0.1409 0.1325 0.1305 0.12 0.1307 0.1426 0.1203 0.1288 0.1129
160 0.1122 0.1195 0.0997 0.1254 0.1279 0.1141 0.1257 0.1381 0.1331 0.0963
320 0.1234 0.0898 0.0892 0.0875 0.0922 0.1783 0.0881 0.0961 0.148 0.2139
640 0.0796 0.0827 0.0762 0.0804 0.0873 0.1011 0.0838 0.0818 0.1043 0.074
1280 0.0739 0.0837 0.0758 0.0723 0.0717 0.0785 0.0805 0.1275 0.0783 0.076
blank 0.0621 0.0611 0.0608 0.0617 0.0644 0.0722 0.0633 0.0625 0.1425 0.0614

Table 20 Absorbance of Vaccinated Ram serum samples against P. multocida on Nov. 16

Animal
ID
Dilution 4 6 8 11 16 18 22 32 36 39
40 0.2624 0.2581 0.3064 0.2391 0.2047 0.259 0.2056 0.2551 0.161 0.2184
80 0.2295 0.2677 0.2212 0.1873 0.1779 0.1745 0.1847 0.1922 0.175 0.2397
160 0.1738 0.1716 0.1905 0.1897 0.142 0.1498 0.134 0.183 0.1883 0.1202
320 0.1455 0.141 0.1542 0.1301 0.109 0.1326 0.1707 0.1482 0.1147 0.1037
640 0.1202 0.1072 0.1037 0.104 0.0911 0.1248 0.0951 0.1216 0.0961 0.0963
1280 0.0918 0.0834 0.0856 0.1653 0.0992 0.084 0.0903 0.1101 0.0769 0.0739
blank 0.0766 0.0754 0.0599 0.0613 0.0708 0.062 0.063 0.1832 0.0665 0.0682

Table 21 Absorbance of Control Ram serum samples against M. haemolytica on Nov. 30

Animal ID
Dilution 3 7 13 14 28 29 31 33 38 40
40 0.1291 0.202 0.1704 0.1414 0.1418 0.1773 0.1938 0.1325 0.1019 0.1084
80 0.1112 0.1459 0.1804 0.1302 0.1491 0.181 0.1608 0.1244 0.0989 0.0951
160 0.113 0.1194 0.1542 0.1142 0.129 0.1212 0.1537 0.1009 0.0965 0.081
320 0.1017 0.1086 0.1419 0.105 0.1146 0.145 0.1271 0.0972 0.0827 0.081
640 0.0862 0.103 0.1266 0.09 0.0954 0.0999 0.1058 0.089 0.0888 0.0799
1280 0.0767 0.0878 0.0942 0.096 0.0903 0.1015 0.0962 0.0864 0.0773 0.0844
blank 0.0771 0.0877 0.0714 0.0657 0.0763 0.062 0.0585 0.06 0.0619 0.0672

38
Table 22 Absorbance of Vaccinated Ram serum samples against M. haemolytica on Nov. 30
Animal ID
Dilution 4 6 8 11 16 18 22 32 36 39
40 0.6438 0.4355 0.4177 0.3714 0.4047 0.4053 0.4556 0.4926 0.2853 0.3761
80 0.6532 0.392 0.3481 0.3739 0.3191 0.2592 0.2805 0.3393 0.2723 0.2284
160 0.4124 0.3691 0.2947 0.3123 0.2958 0.201 0.2203 0.2605 0.2837 0.229
320 0.3862 0.2796 0.3069 0.2768 0.2382 0.2325 0.204 0.2561 0.2236 0.205
640 0.269 0.2512 0.2441 0.2367 0.1812 0.1645 0.1993 0.1931 0.2006 0.1585
1280 0.2184 0.1808 0.1908 0.1682 0.1698 0.1187 0.1968 0.1637 0.1676 0.1684
blank 0.0661 0.0641 0.0646 0.0822 0.0653 0.0685 0.0635 0.0661 0.0669 0.0652

Table23 Absorbance of Control Ram serum samples against P. multocida on Nov. 30

Animal ID
Dilution 3 7 13 14 28 29 31 33 38 40
40 0.5554 0.8746 1.0379 0.6051 0.702 0.9075 0.6673 0.5821 0.5397 0.4351
80 0.6629 0.6412 0.697 0.574 0.6434 0.7202 0.6848 0.5693 0.5606 0.3784
160 0.3854 0.4902 0.6222 0.5301 0.5758 0.5722 0.4914 0.4206 0.3592 0.3151
320 0.2945 0.3402 0.5639 0.3572 0.4178 0.457 0.3019 0.2932 0.2837 0.2903
640 0.2191 0.2634 0.3192 0.2607 0.2676 0.3363 0.226 0.2169 0.155 0.2242
1280 0.2147 0.1978 0.2753 0.1993 0.2076 0.2479 0.1575 0.179 0.1467 0.1493
blank 0.1357 0.1216 0.1257 0.0936 0.0888 0.0726 0.0769 0.0692 0.0759 0.0916

Table24 Absorbance of Vaccinated Ram serum samples against P. multocida on Nov. 30

Animal ID
Dilution 4 6 8 11 16 18 22 32 36 39
40 0.8409 0.7265 0.585 0.6084 0.5308 0.5909 0.4915 0.5819 0.7728 0.5639
80 0.6303 0.5472 0.5404 0.5123 0.587 0.4444 0.4236 0.5252 0.5485 0.3906
160 0.5566 0.437 0.4033 0.4484 0.4844 0.3297 0.3309 0.386 0.4464 0.3302
320 0.4214 0.3496 0.34 0.3729 0.37 0.2919 0.2632 0.3429 0.3379 0.3464
640 0.3131 0.263 0.2658 0.284 0.3203 0.2417 0.1942 0.2765 0.2451 0.226
1280 0.2578 0.1963 0.1879 0.2283 0.2566 0.1826 0.1628 0.2407 0.1787 0.1859
blank 0.077 0.0714 0.0757 0.0855 0.0714 0.0774 0.0738 0.0728 0.0749 0.0704

39
Table 25 Absorbance of Control Ram serum samples against M. haemolytica on Dec. 7
Animal ID
Dilution 3 7 13 14 28 29 31 33 38 40
40 0.2631 0.4252 0.3605 0.7418 0.4705 0.5321 0.8336 0.5602 0.9114 0.6342
80 0.4775 0.4112 0.3412 0.3734 0.5365 0.6271 0.7153 0.6743 0.458 0.6177
160 0.5302 0.4909 0.3758 0.3426 0.5467 0.6066 0.7457 0.537 0.7123 0.9117
320 0.2064 0.1695 0.2059 0.2908 0.251 0.2642 0.1892 0.226 0.3398 0.2535
640 0.1307 0.1684 0.1546 0.1468 0.1718 0.5141 0.1876 0.5998 0.619 0.9749
1280 0.1912 0.1734 0.2677 0.1314 0.2746 0.1382 0.1815 0.1405 0.1648 0.156
blank 0.187 0.0739 0.0623 0.0677 0.0737 0.211 0.1374 0.5964 0.1842 0.2333

Table 26 Absorbance of Vaccinated Ram serum samples against M. haemolytica on Dec. 7

Animal ID
Dilution 4 6 8 11 16 18 22 32 36 39
40 0.4943 0.3597 0.1477 0.1935 0.1848 0.2121 0.1565 0.1524 0.1578 0.0917
80 0.7141 0.217 0.2144 0.232 0.1994 0.1905 0.1681 0.1729 0.1934 0.0929
160 0.6764 0.1702 0.2662 0.241 0.2359 0.1824 0.148 0.2147 0.2046 0.088
320 0.4527 0.2267 0.2261 0.2442 0.2007 0.1683 0.148 0.2042 0.1605 0.0863
640 0.4771 0.1782 0.1881 0.1935 0.1562 0.1597 0.1373 0.164 0.1372 0.082
1280 0.321 0.1604 0.1772 0.1604 0.1172 0.1282 0.0981 0.1219 0.1352 0.084
blank 0.068 0.0615 0.0625 0.0588 0.0638 0.0649 0.0598 0.0617 0.0582 0.0612

Table 27 Absorbance of Control Ram serum samples against P. multocida on Dec. 7

Animal ID
Dilution 3 7 13 14 28 29 31 33 38 40
40 0.336 0.3275 0.3935 0.3048 0.3031 0.3127 0.266 0.2034 0.249 0.2888
80 0.2562 0.3189 0.3981 0.3365 0.327 0.3865 0.2837 0.1557 0.1646 0.2484
160 0.2124 0.2387 0.2613 0.317 0.2601 0.277 0.2294 0.144 0.1228 0.1824
320 0.1514 0.1348 0.2094 0.2148 0.2045 0.2604 0.1562 0.092 0.0996 0.1574
640 0.1299 0.112 0.1676 0.1261 0.1421 0.1852 0.1006 0.0835 0.0816 0.1051
1280 0.1104 0.1011 0.1418 0.1183 0.1275 0.1506 0.0924 0.0716 0.0738 0.128
blank 0.0731 0.0676 0.0751 0.0681 0.0692 0.0681 0.0566 0.0589 0.0572 0.0607

40
Table 28 Absorbance of Vaccinated Ram serum samples against P. multocida on Dec. 7
Animal ID
Dilution 4 6 8 11 16 18 22 32 36 39
40 0.3959 0.1856 0.2281 0.2015 0.1944 0.2111 0.1693 0.1962 0.1941 0.1138
80 0.4005 0.1668 0.2062 0.1711 0.2083 0.1838 0.153 0.1777 0.1749 0.1206
160 0.3124 0.1437 0.1866 0.1392 0.169 0.1525 0.1308 0.1984 0.1467 0.1048
320 0.2632 0.1432 0.1661 0.1395 0.1391 0.1234 0.1131 0.1602 0.1246 0.0911
640 0.2471 0.0985 0.1247 0.1084 0.1178 0.1253 0.0975 0.1445 0.1056 0.0793
1280 0.1723 0.0932 0.086 0.1163 0.1055 0.0954 0.0738 0.1146 0.0907 0.0662
blank 0.0695 0.0875 0.0832 0.0701 0.0708 0.0743 0.06 0.0593 0.0575 0.0588

Table 29 Absorbance of Control Ram serum samples against M. haemolytica on Dec. 14

Animal
ID
Dilution 3 7 13 14 28 29 31 33 38 40
40 0.7745 0.6469 1.0738 0.7327 0.9857 1.0421 1.1733 1.1153 0.824 0.7635
80 0.6013 0.4145 1.16 0.5485 0.8507 0.7376 1.4002 1.3259 0.6388 0.7001
160 0.6162 0.4185 0.869 0.4653 0.5901 0.5839 0.8041 0.6836 0.3719 0.3532
320 0.5148 0.3557 0.1812 0.3474 0.5967 0.4024 0.5642 0.4761 0.4436 0.2115
640 0.3897 0.2834 0.4486 0.2668 0.3187 0.335 0.3863 0.3776 0.3534 0.3702
1280 0.2902 0.2058 0.116 0.1807 0.3015 0.2699 0.3252 0.2649 0.2556 0.2549
Blank 0.1023 0.1204 0.1065 0.0767 0.1162 0.0838 0.0796 0.0828 0.103 0.1207

Table 30 Absorbance of Vaccinated Ram serum samples against M. haemolytica on Dec. 14

Animal
ID
Dilution 4 6 8 11 16 18 22 32 36 39
40 1.964 1.508 1.5559 1.3463 1.2775 1.1108 1.3841 1.5459 1.7291 1.1893
80 2.3022 1.2801 1.4221 1.0602 1.1611 1.1108 1.0877 1.4232 1.6097 1.3296
160 2.1495 1.0539 0.4214 1.0157 0.8489 0.805 0.9618 1.1427 1.2002 0.9208
320 1.6586 1.0122 0.9566 0.8185 0.8034 0.6575 0.7932 0.988 0.8617 0.7701
640 1.4999 0.6983 0.6765 0.5257 0.7791 0.379 0.7385 0.6958 0.7935 0.5248
1280 1.5604 0.5619 0.5002 0.6005 0.4291 0.3334 0.4733 0.5153 0.6436 0.4478
Blank 0.2416 0.0885 0.0803 0.0896 0.1031 0.078 0.0736 0.0715 0.0852 0.1026

41
Table 31 Absorbance of Control Ram serum samples against P. multocida on Dec. 14
Animal
ID
Dilution 3 7 13 14 28 29 31 33 38 40
40 1.3107 1.0641 1.7337 1.0809 1.9964 1.4608 1.4621 1.301 1.2415 1.0039
80 1.1777 0.924 1.4068 0.7102 1.1953 0.8609 1.0103 1.2667 2.2802 1.0328
160 0.6478 0.5935 1.0261 1.7613 0.9361 1.2545 1.1372 1.2805 0.9152 0.7598
320 0.6846 0.4133 0.959 0.5159 0.7422 0.6267 0.5758 0.6576 0.9117 0.5674
640 0.5421 0.3974 0.7146 0.3403 0.5581 0.3746 0.4716 0.4162 0.5144 0.3975
1280 0.4019 0.3047 0.5254 0.3326 0.4249 0.3841 0.3641 0.3338 0.5049 0.3718
Blank 0.6008 0.2237 0.2465 0.2371 0.1965 0.1281 0.1951 0.1946 0.2333 0.3524

Table 32 Absorbance of Control Ram serum samples against P. multocida on Dec. 14

Animal
ID
Dilution 4 6 8 11 16 18 22 32 36 39
40 1.1153 0.9333 0.9181 0.8121 0.7806 0.7111 0.7316 0.8749 0.8543 0.7951
80 1.0434 0.7195 0.8123 0.7158 0.8229 0.6105 0.6877 0.7489 0.8541 0.6509
160 1.0614 0.6025 0.6992 0.5976 0.6236 0.5831 0.5761 0.5047 0.7307 0.5624
320 0.873 0.4861 0.5524 0.5128 0.4804 0.4535 0.5367 0.4574 0.5841 0.4848
640 0.765 0.4429 0.4033 0.4301 0.4453 0.3052 0.4382 0.3419 0.414 0.3938
1280 0.702 0.3596 0.3496 0.315 0.3652 0.3222 0.3356 0.3193 0.3312 0.3208
Blank 0.1174 0.1295 0.1058 0.1296 0.1054 0.1096 0.1098 0.1139 0.1136 0.1333

42
6.2 Specific antibody titers

Table 33 Summary of titers for all animals

Specific Specific
antibody antibody
titers titers
Animal Dates of against M. again P.
Treatment ID Sex Sampling haemolytica. multocida
Control 1 Ewe Nov. 16 160 640
Control 2 Ewe Nov. 16 640 320
Control 5 Ewe Nov. 16 640 640
Control 9 Ewe Nov. 16 320 320
Control 10 Ewe Nov. 16 320 640
Control 12 Ewe Nov. 16 640 320
Control 15 Ewe Nov. 16 160 160
Control 19 Ewe Nov. 16 640 640
Control 24 Ewe Nov. 16 640 320
Control 35 Ewe Nov. 16 160 320
Vaccinated 17 Ewe Nov. 16 320 640
Vaccinated 20 Ewe Nov. 16 1280 640
Vaccinated 21 Ewe Nov. 16 1280 320
Vaccinated 23 Ewe Nov. 16 640 1280
Vaccinated 25 Ewe Nov. 16 320 320
Vaccinated 26 Ewe Nov. 16 320 1280
Vaccinated 27 Ewe Nov. 16 320 1280
Vaccinated 30 Ewe Nov. 16 320 640
Vaccinated 34 Ewe Nov. 16 160 640
Vaccinated 37 Ewe Nov. 16 320 640
Control 3 Ram Nov. 16 160 40
Control 7 Ram Nov. 16 80 80
Control 13 Ram Nov. 16 320 80
Control 14 Ram Nov. 16 40 80
Control 28 Ram Nov. 16 320 40
Control 29 Ram Nov. 16 320 80
Control 31 Ram Nov. 16 320 80
Control 33 Ram Nov. 16 40 40
Control 38 Ram Nov. 16 80 320
Control 40 Ram Nov. 16 40 40
Vaccinated 4 Ram Nov. 16 1280 320
Vaccinated 6 Ram Nov. 16 1280 320

43
Table 33
Continued
Vaccinated 8 Ram Nov. 16 640 320
Vaccinated 11 Ram Nov. 16 640 320
Vaccinated 16 Ram Nov. 16 640 160
Vaccinated 18 Ram Nov. 16 320 320
Vaccinated 22 Ram Nov. 16 320 320
Vaccinated 32 Ram Nov. 16 640 320
Vaccinated 36 Ram Nov. 16 320 160
Vaccinated 39 Ram Nov. 16 320 80
Control 1 Ewe Nov. 30 160 160
Control 2 Ewe Nov. 30 80 160
Control 5 Ewe Nov. 30 320 320
Control 9 Ewe Nov. 30 160 160
Control 10 Ewe Nov. 30 160 160
Control 12 Ewe Nov. 30 320 320
Control 15 Ewe Nov. 30 160 320
Control 19 Ewe Nov. 30 80 160
Control 24 Ewe Nov. 30 640 640
Control 35 Ewe Nov. 30 80 320
Vaccinated 17 Ewe Nov. 30 640 1280
Vaccinated 20 Ewe Nov. 30 640 1280
Vaccinated 21 Ewe Nov. 30 1280 640
Vaccinated 23 Ewe Nov. 30 1280 1280
Vaccinated 25 Ewe Nov. 30 1280 640
Vaccinated 26 Ewe Nov. 30 1280 1280
Vaccinated 27 Ewe Nov. 30 1280 640
Vaccinated 30 Ewe Nov. 30 1280 640
Vaccinated 34 Ewe Nov. 30 1280 1280
Vaccinated 37 Ewe Nov. 30 1280 1280
Control 3 Ram Nov. 30 40 160
Control 7 Ram Nov. 30 160 320
Control 13 Ram Nov. 30 640 640
Control 14 Ram Nov. 30 160 320
Control 28 Ram Nov. 30 320 320
Control 29 Ram Nov. 30 320 640
Control 31 Ram Nov. 30 320 160
Control 33 Ram Nov. 30 80 160
Control 38 Ram Nov. 30 40 160
Control 40 Ram Nov. 30 40 80
Vaccinated 4 Ram Nov. 30 1280 320

44
Table 33
Continued
Vaccinated 6 Ram Nov. 30 1280 320
Vaccinated 8 Ram Nov. 30 1280 320
Vaccinated 11 Ram Nov. 30 1280 320
Vaccinated 16 Ram Nov. 30 1280 640
Vaccinated 18 Ram Nov. 30 1280 160
Vaccinated 22 Ram Nov. 30 1280 160
Vaccinated 32 Ram Nov. 30 1280 320
Vaccinated 36 Ram Nov. 30 1280 320
Vaccinated 39 Ram Nov. 30 1280 320
Control 1 Ewe Dec. 7 40 40
Control 2 Ewe Dec. 7 40 80
Control 5 Ewe Dec. 7 640 640
Control 9 Ewe Dec. 7 160 160
Control 10 Ewe Dec. 7 80 80
Control 12 Ewe Dec. 7 640 160
Control 15 Ewe Dec. 7 40 40
Control 19 Ewe Dec. 7 80 80
Control 24 Ewe Dec. 7 80 320
Control 35 Ewe Dec. 7 40 160
Vaccinated 17 Ewe Dec. 7 160 40
Vaccinated 20 Ewe Dec. 7 320 160
Vaccinated 21 Ewe Dec. 7 1280 160
Vaccinated 23 Ewe Dec. 7 1280 320
Vaccinated 25 Ewe Dec. 7 1280 160
Vaccinated 26 Ewe Dec. 7 1280 320
Vaccinated 27 Ewe Dec. 7 640 80
Vaccinated 30 Ewe Dec. 7 160 160
Vaccinated 34 Ewe Dec. 7 40 80
Vaccinated 37 Ewe Dec. 7 320 160
Control 3 Ram Dec. 7 160 160
Control 7 Ram Dec. 7 160 160
Control 13 Ram Dec. 7 160 640
Control 14 Ram Dec. 7 160 320
Control 28 Ram Dec. 7 160 320
Control 29 Ram Dec. 7 160 640
Control 31 Ram Dec. 7 160 160
Control 33 Ram Dec. 7 160 40
Control 38 Ram Dec. 7 320 80
Control 40 Ram Dec. 7 160 160

45
Table 33
Continued
Vaccinated 4 Ram Dec. 7 640 1280
Vaccinated 6 Ram Dec. 7 40 80
Vaccinated 8 Ram Dec. 7 40 320
Vaccinated 11 Ram Dec. 7 40 80
Vaccinated 16 Ram Dec. 7 40 160
Vaccinated 18 Ram Dec. 7 40 80
Vaccinated 22 Ram Dec. 7 40 40
Vaccinated 32 Ram Dec. 7 40 160
Vaccinated 36 Ram Dec. 7 40 80
Vaccinated 39 Ram Dec. 7 40 40
Control 1 Ewe Dec. 24 40 80
Control 2 Ewe Dec. 24 160 320
Control 5 Ewe Dec. 24 80 320
Control 9 Ewe Dec. 24 80 160
Control 10 Ewe Dec. 24 80 160
Control 12 Ewe Dec. 24 160 320
Control 15 Ewe Dec. 24 80 320
Control 19 Ewe Dec. 24 160 640
Control 24 Ewe Dec. 24 640 320
Control 35 Ewe Dec. 24 80 320
Vaccinated 17 Ewe Dec. 24 1280 1280
Vaccinated 20 Ewe Dec. 24 640 1280
Vaccinated 21 Ewe Dec. 24 640 1280
Vaccinated 23 Ewe Dec. 24 640 1280
Vaccinated 25 Ewe Dec. 24 640 1280
Vaccinated 26 Ewe Dec. 24 640 640
Vaccinated 27 Ewe Dec. 24 640 1280
Vaccinated 30 Ewe Dec. 24 320 1280
Vaccinated 34 Ewe Dec. 24 640 1280
Vaccinated 37 Ewe Dec. 24 320 1280
Control 3 Ram Dec. 24 320 640
Control 7 Ram Dec. 24 40 160
Control 13 Ram Dec. 24 640 640
Control 14 Ram Dec. 24 160 160
Control 28 Ram Dec. 24 320 640
Control 29 Ram Dec. 24 160 320
Control 31 Ram Dec. 24 320 320
Control 33 Ram Dec. 24 320 320
Control 38 Ram Dec. 24 320 320

46
Table 33
Continued
Control 40 Ram Dec. 24 80 320
Vaccinated 4 Ram Dec. 24 1280 1280
Vaccinated 6 Ram Dec. 24 1280 160
Vaccinated 8 Ram Dec. 24 1280 320
Vaccinated 11 Ram Dec. 24 1280 160
Vaccinated 16 Ram Dec. 24 1280 160
Vaccinated 18 Ram Dec. 24 320 160
Vaccinated 22 Ram Dec. 24 1280 320
Vaccinated 32 Ram Dec. 24 1280 80
Vaccinated 36 Ram Dec. 24 1280 320
Vaccinated 39 Ram Dec. 24 1280 160

47