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14 of 92 15 had changes that were suggestive of Mycoplasma bovis infection as a primary cause of pneumonia; and,
6 of the 92 7 had changes that were suggestive of bacterial infection in combination with viral infection.
A large body of descriptive information was generated on histologic changes in tissue samples collected during the project. The information was used in conjunction with all other
available information relevant to disease before death, at necropsy and in pathologic examination of tissues or swabs to determine the most likely cause of death. Further
descriptions of histologic changes are not provided here because the range of changes presented in isolation is likely to be relatively uninformative.
In some cases histologic evidence in other tissues was noteworthy because it identified unusual potential diseases or was suggestive of specific pathogens of interest.
For example, there were 16 cases where examination of heart tissue identified pathology involving the heart. These included 10 animals with myocarditis lesions and in these 10
animals histologic changes were suggestive of specific conditions:
Acute myocarditis lesions were observed in five animals and in three of these there were concurrent lesions in other tissues that were considered consistent with systemic
infection with Histophilus somni, also called Haemophilus septicaemia or Haemophilus somnus disease complex; and
Chronic myocarditis lesions were observed in five animals and three of these had sarcocysts present in the heart muscle, indicating infection with Sarcocystis species of
sporozoan parasite.
7.8 Molecular results
Molecular analyses involved the application of specific quantitative polymerase chain reaction qPCR assays to lung tissue and swabs nasal swabs or lung tissue swabs to
detect the presence of genetic material indicative of respiratory disease pathogens including the four bacterial pathogens and five viral pathogens of particular interest for this project.
Presence alone of the infectious agents of interest based on qPCR tests is difficult to interpret, because healthy animals may carry these agents without necessarily suffering from
disease. A decision was made to examine qPCR results in conjunction with results from histologic examination of tissue samples from the same animals in order to allow better
interpretation of the qPCR results in light of the presence or absence of lung disease based on histologic examination of lung tissue samples.
Samples suitable for qPCR assay were collected from 159 animals but eight of these animals did not have matched lung tissue samples for histology examination.
There were 151 animals that had matched lung andor nasal samples allowing qPCR tests for viral agents to be completed and compared to histology findings from lung tissues
collected on the same animals.
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There were 128 animals that had matched lung andor nasal samples allowing qPCR tests for bacterial agents to be completed and compared to histology findings from lung tissues
collected from the same animals.
Results are presented in Table 7. For the 151 samples that had both viral qPCR tests and histology, the histology results were
used to classify animals as either having pneumonia n=102 or not having pneumonia n=49.
A qPCR positive test result indicates presence of genetic material in the sample from the specific pathogen being tested. The presence of a qPCR positive test result does not
necessarily mean that the tissue contained live, active infective material from the specific pathogen at the time the animal died. It means that at some time in the period before the
animal died the specific pathogen almost certainly had been present in that tissue. In some cases even after an animal has been infected and recovered there may still be small
amounts of residual genetic material present in tissues that is no longer viable but that may still be detected using a qPCR test. In this report we are classifying a qPCR positive test
result as indicating that the animal had recently been infected with that pathogen.
Results from the qPCR tests detecting presence of viral genetic material can then be directly compared between those animals with histologic pneumonia abnormal lung and those
without histologic pneumonia normal lung. The odds ratio provides a statistical test to compare the strength of association between qPCR status positive vs negative and
histologic status pneumonia vs no pneumonia. When the odds ratio is greater than one it is indicating that animals with pneumonia were more likely to have positive qPCR results
compared to animals without pneumonia. When the odds ratio is less than one it is indicating that animals with pneumonia were less likely to have positive qPCR results compared to
animals without pneumonia. The p-value and confidence intervals can be used to determine if the odds ratio is statistically significant. If the p-value is less than 0.05 or if the confidence
interval for the odds ratio does not include one, then the comparison is significant.
The prevalence of the viruses of interest in lung samples was relatively low and there was not a lot of difference between the group with histologic pneumonia and the group without
histologic pneumonia. In fact none of the statistical comparisons were statistically significant, indicating that qPCR positive test results for viral pathogens were just as likely to occur in
animals without pneumonia as they were in animals with pneumonia.
There was one comparison that was almost significant, involving Bovine Viral Diarrhoea Virus BVDV. Animals with histologic pneumonia had a 4.1-fold higher odds of being qPCR
positive for BVDV compared to animals that had no histologic evidence of pneumonia. The p-value for this comparison was 0.053. All other comparisons were not significant p0.05.
The prevalence of bacterial pathogens in animals with histologic pneumonia ranged from 35 to 60 and was significantly higher than the presence of the same bacteria in samples from
animals with no histologic evidence of pneumonia prevalence ranging from 2 to 14.
For each specific bacterial pathogen, the statistical comparisons were highly significant Table 7:
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Animals with histologic pneumonia had a 4.1-fold higher odds of being qPCR positive for H. somni compared to animals that had no histologic evidence of pneumonia p0.001;
Animals with histologic pneumonia had a 63.2-fold higher odds of being qPCR positive for M. bovis compared to animals that had no histologic evidence of pneumonia
p0.001; Animals with histologic pneumonia had a 11.1-fold higher odds of being qPCR positive
for M. haemolytica compared to animals that had no histologic evidence of pneumonia p0.001; and
Animals with histologic pneumonia had a 9.3-fold higher odds of being qPCR positive for P. multocida compared to animals that had no histologic evidence of pneumonia
p0.001.
In the previous section of this report, histologic changes in lung tissues from those animals with pneumonia were used to tentatively identify specific pathogens bacteria andor viruses
that were considered likely to have been involved in the underlying pneumonia. These results were then compared to the findings from qPCR detection of pathogens in lung
samples.
Where histology results indicated that bacterial infection was likely to have been involved in the pneumonia, qPCR tests were positive for one or more of the bacterial agents in 75 of
cases.
Where histology results indicated that viral infection was likely to have been involved in the pneumonia, qPCR results were positive for one or more viral pathogens in 45 of cases.
There were a number of cases where multiple pathogens were present in the same tissue samples indicating concurrent infection with more than one pathogen. Simple correlations
were performed as a crude measure of whether some combinations of pathogens were more likely to have occurred together than as a result of chance alone.
The presence of H. somni in lung tissue was significantly correlated with the presence of M. bovis, P. multocida, and viral pathogens p0.001, in particular BCoV and BVDV p 0.01.
The presence of M. bovis and M. haemolytica were significantly correlated with the presence of P. multocida p0.001.
Page 58 of 201 Table 7: Summary of results of qPCR detection of specific genetic material in lung samples for viral pathogens from 151 animals and for bacterial
pathogens from 128 animals. Each row provides a count of the number of animals tested from two groups with and without histologic evidence of pneumonia, the number of animals with qPCR positive test results for each pathogen, and the odds ratio comparing the odds of qPCR positive
results in animals with histologic pneumonia to the odds of being qPCR positive in animals without histologic pneumonia. OR=odds ratio, CI=confidence interval.
Pathogens detected in
lung samples Animals with histologic evidence of
pneumonia Animals with no histologic evidence of
pneumonia Odds ratio
95 CI for OR p-value
qPCR + No. tested
Prevalence qPCR +
No. tested Prevalence
n N
of N n
N of N
OR Viruses
BCoV 11
102 10.8
4 49
8.2 1.4
0.4 to 6.2 0.6
BoHV-1 3
102 2.9
49 0.0
2 0.2 to 100.4
0.5 BRSV
2 102
2.0 4
49 8.2
0.23 0.02 to 1.7
0.07 BVDV
15 102
14.7 2
49 4.1
4.1 0.9 to 37.7
0.053 BPIV-3
102 0.0
49 0.0
0.5 0.01 to 38.9
0.6
Bacteria
H. somni 40
84 47.6
6 44
13.6 5.8
2.1 to 18.2 0.001
M. bovis 50
84 59.5
1 44
2.3 63.2
9.5 to 2604.2 0.001
M. haemolytica 29
84 34.5
2 44
4.5 11.1
2.5 to 99.6 0.001
P. multocida 34
84 40.5
3 44
6.8 9.3
2.6 to 49.9 0.001
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Nasal swabs were collected at necropsy from 84 animals. Matched nasal swab samples and lung histology results were available for 82 animals.
Histology findings from microscopic examination of lung tissue samples were used to classify the 82 animals into those animals with histologic evidence of pneumonia n=57 and
those animals with no histologic evidence of pneumonia n=25.
The qPCR tests of nasal swabs from the same animals were used to classify animals as positive or negative for each specific viral and bacterial pathogen.
The prevalence for each pathogen in animals with histologic pneumonia was then compared to the prevalence for each pathogen in animals with no histologic evidence of pneumonia. A
statistical test based on the odds ratio was used to test whether animals with histologic pneumonia were more or less likely to have pathogens detected in nasal swabs.
Results are presented in Table 8. Respiratory viruses were detected in nasal swabs of animals with and without histologic
evidence of pneumonia. The prevalence of qPCR positive results from nasal swabs in animals with histologic pneumonia appeared numerically higher than the prevalence in nasal
swabs from animals with no histologic evidence of pneumonia but statistical comparisons of these associations returned non-significant results for all the viruses that were tested
p0.05. The results indicate that the presence of a viral pathogen in a nasal swab has little relationship with the likelihood of that animal having histologic evidence of pneumonia.
Bacterial pathogens were detected in nasal swabs of animals with and without histologic evidence of pneumonia. The prevalence of bacterial pathogens in nasal swabs appeared to
be higher in animals with histologic evidence of pneumonia compared to those animals with no histologic evidence of pneumonia. Statistical testing of the association between presence
of nasal qPCR positive result and presence or absence of histologic evidence of pneumonia returned significant results for three of the four bacteria tested Table 8:
Animals with histologic pneumonia had a 1.7-fold higher odds of being qPCR positive for H. somni in nasal swabs compared to animals without pneumonia p=0.3;
Animals with histologic pneumonia had a 15-fold higher odds of being qPCR positive for M. bovis in nasal swabs compared to animals without pneumonia p0.001;
Animals with histologic pneumonia had a 7.2-fold higher odds of being qPCR positive for M. haemolytica in nasal swabs compared to animals without pneumonia p=0.01; and,
Animals with histologic pneumonia had a 8.4-fold higher odds of being qPCR positive for P. multocida in nasal swabs compared to animals without pneumonia p0.001.
Page 60 of 201 Table 8: Summary of results of qPCR detection of specific genetic material in nasal swab for pathogens in nasal swabs from 82 animals and for
histology findings from lung tissues from the same animals. Each row provides a count of the number of animals tested from two groups with and without histologic evidence of pneumonia, the number of animals with qPCR positive test results for each pathogen, and the odds ratio
comparing the odds of qPCR positive results in animals with histologic pneumonia to the odds of being qPCR positive in animals without histologic pneumonia. OR=odds ratio, CI=confidence interval.
Pathogens detected in
nasal swabs Animals with histologic evidence of
pneumonia Animals with no histologic evidence of
pneumonia Odds ratio
95 CI for OR p-value
qPCR + No. tested
Prevalence qPCR +
No. tested Prevalence
n N
of N n
N of N
OR Viruses
BCoV 9
57 15.8
2 25
8.0 2.2
0.4 to 21.9 0.3
BoHV-1 9
57 15.8
1 25
4.0 4.5
0.6 to 205.3 0.1
BRSV 2
57 3.5
25 0.0
1.4 0.1 to 75.9
0.8 BVDV
4 57
7.0 1
25 4.0
1.8 0.2 to 92.9
0.6 BPIV-3
1 57
1.8 25
0.0 0.9
0.05 to 56.9 0.9
Bacteria
H. somni 30
57 52.6
10 25
40.0 1.7
0.6 to 4.9 0.3
M. bovis 34
57 59.6
2 25
8.0 15
3.1 to 139 0.001
M. haemolytica 22
57 38.6
2 25
8.0 7.2
1.5 to 67.9 0.01
P. multocida 35
57 61.4
4 25
16.0 8.4
2.3 to 36.9 0.001
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There were 79 animals that had qPCR test results available from both nasal swab samples and from lung samples.
Results are presented in Table 9. The results suggest that detection of a pathogen in a nasal swab is associated with an
increased risk of that same pathogen being detected in lung tissue and that this risk varies between pathogens.
Caution is urged in interpreting these findings because of the low number of samples in some combinations. The sparse sample size in some cells is reflected in the wide
confidence intervals for odds ratio estimates.
However, the results do suggest that nasal detection of pathogens may be a marker of risk for presence of the same pathogens in lung. Results presented earlier in this section
indicated that there is an association between detection of a pathogen in lung tissue and the presence of pneumonia.
Page 62 of 201 Table 9: Summary of results of qPCR detection of viruses and bacteria in matched samples from nasal swabs and lung tissue. Odds ratios are
presented as a measure of the association between the odds of returning a positive lung test in animals with a positive nose test compared to the odds of returning a positive lung test in those animals with a negative nose test.
Pathogens No. of
animals tested
qPCR results for each combination of nose and lung samples Nose +
Nose + Lung + as
of all nose + Nose -
Nose - Lung + as
of all nose -
Odds ratio
95 CI for OR
p-value Lung -
Lung + Lung +
Lung -
N
n n
n n
OR Viruses
BCoV 79
7 4
36.4 2
66 2.9
18.9 2.1 to 226
0.003 BoHV-1
79 7
3 30.0
69 0.0
34.5 2.7 to 1721
0.001 BRSV
79 1
1 50.0
1 76
1.3 76
0.5 to 6024 0.05
BVDV 79
2 3
60.0 2
72 2.7
54 3.4 to 871
0.001 BPIV-3
79 1
0.0 78
0.0 39.5
0.3 to 3132 0.07
Bacteria
H. somni 79
18 21
53.8 4
36 10.0
10.5 2.9 to 46.9
0.001 M. bovis
79 11
23 67.6
5 40
11.1 16.7
4.6 to 66.8 0.001
M. haemolytica 79
14 9
39.1 7
49 12.5
4.5 1.2 to 16.8
0.01 P. multocida
79 20
17 45.9
3 39
7.1 11.1
2.7 to 63.7 0.001
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Swabs were collected from a small number of animals presenting with diseases other than pneumonia.
There were nine swabs collected from diseased joints or from peripheral cellulitis lesions. Of these samples two 22 returned a positive qPCR test for Mycoplasma bovis M. bovis.
There were a further five animals that had returned a positive qPCR test result for Mycoplasma bovis on lung andor nasal swabs and that were also noted as having been
lame in the clinical category record. None of these animals had swabs collected from the site of the lameness so it was not possible to explore qPCR testing to look for presence of M.
bovis or any other pathogen in musculoskeletal lesions.
7.9 Final diagnosis on cause of death
