Virulence of Brucella abortus isolated from cattle and water buffalo.

Abstract

Brucellosis has been documented in domestic water buffalo (Bubalus bubalis) but published literature is limited despite the importance of this species in tropical agricultural systems. The objective of this study was to compare the virulence of Brucella abortus isolates recovered from cattle and water buffalo. Nineteen strains of B. abortus from cattle and domestic water buffalo in Trinidad were intraperitoneally inoculated into BALB/c mice. Spleens were cultured for B. abortus and histopathological severity scores were calculated based on lymphoid depletion, lymphoid necrosis, splenitis, and macrophage accumulation. A general linear model approach was used to estimate the effect of isolate source (cattle versus water buffalo) on virulence. Isolates of water buffalo origin were significantly less virulent in the mouse model based on recovered B. abortus from splenic tissues, spleen/weight ratio, and lymphoid necrosis but not overall histopathological severity scores. Further investigation of isolates recovered from water buffalo might provide the key to the development of procedures for brucellosis control in tropical environments.

Full text

Page 1

1
Virulence of Brucella abortus isolated from cattle and water buffalo

A.A. Adesiyun1, DVM, MPH, PhD; G.T. Fosgate2,*, DVM, PhD; R. Seebaransingh1, DVM, MS;
G. Brown1, DVM; S. Stoute1, DVM; A. Stewart-Johnson1, BSC, MSc

1School of Veterinary Medicine, Faculty of Medical Sciences, University of the West Indies, St.
Augustine, Trinidad and Tobago
2Department of Production Animal Studies, Faculty of Veterinary Science, University of
Pretoria, Onderstepoort, South Africa

*Correspondence to:
G. T. Fosgate
Tel: +27 12 529 8257
Fax: +27 12 529 8315
E-mail: geoffrey.fosgate@up.ac.za

Page 2

2
Abstract
Brucellosis has been documented in domestic water buffalo (Bubalus bubalis) but
published literature is limited despite the importance of this species in tropical agricultural
systems. The objective of this study was to compare the virulence of B. abortus isolates
recovered from cattle and water buffalo. Nineteen strains of Brucella abortus from cattle and
domestic water buffalo in Trinidad were intraperitoneally inoculated into BALB/c mice. Spleens
were cultured for B. abortus and a histopathological severity scores were calculated based on
lymphoid depletion, lymphoid necrosis, splenitis, and macrophage accumulation. A general
linear model approach was used to estimate the effect of isolate source (cattle versus water
buffalo) on virulence. Isolates of water buffalo origin were significantly less virulent in the
mouse model based on recovered B. abortus from splenic tissues, spleen:weight ratio, and
lymphoid necrosis but not overall histopathological severity scores. Further investigation of
isolates recovered from water buffalo might provide the key to the development of procedures
for brucellosis control in tropical environments.

Page 3

3
Introduction
Infection with Brucella abortus has been recognized in domestic water buffalo (Bubalus
bubalis) of Trinidad (Fosgate et al. 2002a) and other regions of the world (Dhand et al. 2005;
Samaha et al. 2008; Capparelli 2009). Reported differences in successful isolation of B. abortus
from seropositive cattle versus water buffalo (Fosgate et al. 2002a) have been hypothesized to be
due to different sensitivity and specificity of serological assays (Fosgate et al. 2002b).
Alternative hypotheses include water buffalo being more resistant to infection with Brucella
abortus (Ramnanan 2010) and differing virulence of isolates circulating within the infected
populations of cattle and water buffalo. Mouse models have been used to study the virulence of
Brucella spp. (Miyoshi et al. 2007) and the BALB/cByJ strain has been determined to be most
susceptible to B. abortus infection (High et al. 2007). The purpose of this study was to compare
the virulence of B. abortus isolates recovered from cattle and water buffalo.

Materials and methods
BALB/c mice aged 9 weeks were used in the study after obtaining approval from the
Ethics Committee of the Faculty of Medical Sciences, University of the West Indies. A total of
19 strains of Brucella abortus were selected to represent all confirmed1 isolates from cattle and
domestic water buffalo in Trinidad in addition to vaccine strains 19 (S19)2 and RB51 (SRB51).3
The 21 strains of B. abortus were subcultured onto blood agar plates and growth was harvested
with 1.0 ml of phosphate buffered saline (pH 7.4). Bacterial suspensions were adjusted
spectrophotometrically and each of 20 mice per group was inoculated with 0.1 ml
intraperitoneally. Five mice were randomly selected at 3, 6, 9 and 12 weeks post-inoculation.
Each selected mouse was anaesthesized and the spleen was aseptically removed. Spleens were

Page 4

4
macerated and serially diluted in phosphate buffered saline to determine the number of B.

abortus per gram of splenic tissues. Portions of spleens from 2 randomly selected mice (out of
the total of 5 per time period) were sampled for histopathological studies.

Splenic tissues were fixed in 10% buffered formalin, embedded in paraffin, sectioned at 5
microns, and stained using a routine haematoxylin and eosin stain. Pathology of the white and
red pulp sections was scored. Lymphocyte depletion was graded as 1 for loss of lymphocyte

density in at least one follicle and 2 for loss of lymphocyte density in more than one follicle.
Lymphoid necrosis was graded as 1 for the presence of lymphocyte necrosis in at least one
follicle and 2 for the presence of lymphocyte necrosis in more than one follicle. Splenitis was
graded as: 1) neutrophils within splenic tissue or within splenic capsule, 2) neutrophils within
splenic tissue and within splenic capsule, 3) granulomas present with or without neutrophils
within splenic tissue and capsule, and 4/5) severe manifestations of 1-3. Macrophage
accumulation around follicles was graded as 0-5 for normal to severe accumulation.

Data were described by calculating medians, ranges, means, and standard deviations.
The number of colony forming units (CFU) per gram of splenic tissue was log10 transformed for

presentation and statistical analysis. A histopathological severity score was calculated as the
sum of the individual scores for lymphoid depletion, lymphoid necrosis, splenitis, and
macrophage accumulation and natural log transformed for statistical analysis. A general linear
model approach was used to estimate the effect of isolate source (cattle versus water buffalo)

while adjusting for the effects of inoculating dose, week of sampling, and whether or not the
isolate was recovered from an animal with clinical signs of brucellosis. Evaluated outcomes
included CFU per gram of splenic tissue, spleen:weight ratio, histopathological severity score,
and inoculating dose. Mann-Whitney U tests were used to estimate the effect of isolate source

Page 5

5
on individual histopathological lesion scores. Statistical analyses were performed in
commercially available software4 and interpreted at the 5% level of significance.

Results
Thirteen isolates of water buffalo origin and 6 isolates from cattle were evaluated in
addition to the 2 vaccine strains. Three of the evaluated isolates from water buffalo were
recovered from aborted tissues and 3 others from skin lesions of naturally infected animals. All
remaining isolates (including all those from cattle) were recovered from apparently healthy
seropositive animals sampled at slaughter. Animals yielding isolates originated from 2 herds
each of cattle and water buffalo. However, 3 of the herds were originally managed by the same
corporation and the other cattle farm was the location of the only water buffalo abattoir in
Trinidad. All cattle isolates and 1 isolate from an apparently health water buffalo were recovered
in 1999. Three isolates from apparently healthy water buffalo were isolated in 2001 and the
remaining was recovered over the subsequent years.
The CFU/gram of splenic tissue, spleen:weight ratio, and lymphoid necrosis scores were
significantly lower for isolates from water buffalo (Table 1). Descriptively, the CFU/gram did
not change over time for isolates from water buffalo compared to isolates originating from cattle
(Fig. 1). Spectrophotometrical adjustment of bacterial suspensions was not precise and
inoculating dose varied by isolate but the mean dose was not significantly different for mice
receiving water buffalo and cattle origin isolates.

Page 6

6
Discussion
The water buffalo (Bubalus bubalis) is an important domestic species in many tropical
countries but there is limited peer-reviewed literature concerning brucellosis in this species. This
report provides evidence suggesting that B. abortus isolates recovered from domestic water
buffalo of Trinidad have lower virulence compared to cattle. The reason for this lower virulence
is uncertain and it is unknown if the population of isolates available for study accurately reflects
the population of all isolates circulating among livestock of the island. However, this
recognition could have theoretical and practical implications. Brucella abortus infection might
be more host-adapted to water buffalo and passage within this species could be the cause of the
lower virulence. It is also theoretically possible that the original source of infection was different
between cattle and water buffalo and management might limit transmission between species.
The strains of B. abortus circulating within populations have importance when designing
control programs. Vaccination studies have not been able to identify protocols that protect water
buffalo from infection with field strains of B. abortus (Diptee et al. 2007). However, a recent
report (Caporale et al. 2010) identified a similar vaccination strategy that protects water buffalo
from infection with a laboratory strain of B. abortus. An unanswered hypothesis is that water
buffalo have greater natural resistance to brucellosis and further investigation of isolates
recovered from water buffalo could address this question and provide other important
information for better understanding and controlling brucellosis within tropical regions.

Page 7

7
Acknowledgements
The authors are grateful to the Campus Research Funds Committee, St. Augustine Campus for
funding the research project. We are grateful to Drs. Michael Diptee and Anil Ramnanan for
allowing the strains of B. abortus isolated from their studies available for the current
investigation. The technical support provided by Kirk Williams, Sabita Singh and Elliot
Neptune is appreciated.

Footnotes
1Confirmed as B. abortus biovar 1 by the Central Veterinary Laboratory, Weybridge, UK
2Kindly provided by Drs. J. Payeur and B. Martin, National Veterinary Services Laboratories,
Ames, IA, USA
3Colorado Serum Company, Denver, CO, USA
4SPSS version 17.0, SPSS Inc, Chicago, IL, USA

Competing interests
The authors declare that there are no competing interests.

Page 8

8
References
Caporale, V., Bonfini, B., Di Giannatale, E., Di Provvido, A., Forcella, S., Giovannini, A.,
Tittarelli, M. and Scacchia, M., 2010. Efficacy of Brucella abortus strain RB51 compared
to the reference vaccine Brucella abortus strain 19 in water buffalo, Veterinaria Italiana
46, 13–19
Capparelli, R., Parlato, M., Iannaccone, M., Roperto, S., Marabelli, R. and Iannelli, D. 2009.
Heterogeneous shedding of Brucella abortus in milk and its effect on the control of
animal brucellosis, Journal of Applied Microbiology 106, 2041–2047
Dhand, N. K., Gumber, S., Singh, B. B., Aradhana, Bal, M. S., Kumar, H., Sharma, D. R.,
Singh, J. and Sandhu, K. S. 2005. A study on the epidemiology of brucellosis in Punjab
(India) using Survey Toolbox, Revue Scientifique et Technique 24, 879–-885
Diptee, M. D., Asgarali, Z., Cambell, M., Fosgate, G. and Adesiyun, A. A., 2007.
Post-exposure serologic and bacteriologic response of water buffalo (Bubalus bubalis) to
Brucella abortus biovar 1 following vaccination with Brucella abortus strain RB51,
Revue Scientifique et Technique 26, 669–678
Fosgate, G. T., Adesiyun, A. A., Hird, D. W., Hietala, S. K. and Ryan, J., 2002a.
Isolation of Brucella abortus biovar 1 from cattle and water buffalo of Trinidad,
Veterinary Record 151, 272–273
Fosgate, G. T., Adesiyun, A. A., Hird, D. W., Johnson, W. O., Hietala, S. K.,
Schurig, G. G. and Ryan, J., 2002b. Comparison of serologic tests for detection of
Brucella infections in cattle and water buffalo (Bubalus bubalis), American Journal of
Veterinary Research 63, 1598–1605
High, K. P., Prasad, R., Marion, C. R., Schurig, G. G., Boyle, S. M. and

Page 9

9
Sriranganathan, N., 2007. Outcome and immune responses after B. abortus infection in
young adult and aged mice, Biogerontology 8, 583–593
Miyoshi, A., Rosinha, G. M. S., Carmago, I. L. B. C., Trant, C. M. C., Cardoso,
F. C., Azevedo, V. and Oliveira, S. C., 2007. The role of the vacB gene in the
pathogenesis of Brucella abortus, Microbes and Infection 9, 375–381
Ramnanan, A., 2010. Response of water buffalo (Bubalus bubalis) to experimental
challenge with Brucella abortus biovar 1 following vaccination with two dosages of
Brucella abortus strain RB51 (unpublished M.Phil. thesis, University of the West Indies)
Samaha, H., Al-Rowaily, M., Khoudair, R. M. and Ashour, H. M. 2008. Multicenter study of
brucellosis in Egypt, Emerging Infectious Diseases 14, 1916–1918

Page 10

10
Table 1. Descriptive statistics and comparisons of virulence measures using an experimental mouse model for Brucella abortus
isolates from cattle (n = 6) and domestic water buffalo (n = 13) from Trinidad.
Cattle isolates Water buffalo isolates
Outcome Mean (SD) Median (range) Mean (SD) Median (range) P value
Log10 CFU recovered 4.89 (1.22) 5.11 (<2, 7.78) 3.98 (1.91) 4.70 (<2, 7.53) <0.001a
Spleen : weight ratio 7.7E-3 (4.6E-3) 6.0E-3 (1.9E-3, 2.4E-2) 6.1E-3 (3.1E-3) 5.1E-3 (2.4E-3, 2.0E-2) <0.001a
Lymphoid depletion 0.77 (0.88) 1 (0, 4) 0.59 (0.76) 0 (0, 2) 0.296b
Lymphoid necrosis 0.54 (0.74) 0 (0, 3) 0.27 (0.53) 0 (0, 2) 0.033b
Spleen inflammation 2.03 (0.99) 2 (0, 4) 2.15 (0.86) 2 (1, 4) 0.528b
Macrophage accumulation 1.26 (0.70) 1 (0, 3) 1.12 (0.68) 1 (0, 3) 0.302b
Overall (sum) histopathological
score
Log10 CFU inoculating dose
4.60 (1.93) 4 (2, 9) 4.14 (1.84) 4 (1, 10) 0.273a
3.93 (1.59) 4.17 (<2, 6) 3.90 (0.61) 4.11 (2, 5) 0.715a
SD = standard deviation
aBased on general linear model
bBased on Mann-Whitney U tests

Page 11

Fig. 1. Mean colony forming units (CFU)/gram recovered from the spleens of experimentally
inoculated mice with Brucella abortus vaccine strains (circles) and isolates from cattle (triangles;
n = 6) and domestic water buffalo (squares; n = 13) from Trinidad. Error bars correspond to the
standard error of the mean.


6
5
=> 4
LL
U - o 3
.... - Cl
0
...J
2
1
0
0 3 6
9
12
Weeks

Page 12

6
5
:J 4
u..
U -
o 3
~ -
.3 2
C>
1
o
o
3
6
Weeks
9
12