Significance Statement
Diagnosis of canine brucellosis caused by B.canis involves both
direct and indirect methods, but these tests have low specificity.
Development of more sensitive and specific serological tests for the
diagnosis of infections caused by B. canis is needed.
In the present study a western blotting assay has been developed in
order to define proteomics pattern associated to B. canisinfection using a panel of sera from dogs naturally infected and
non-infected with B. canis . A combined immunoproteomics and
bioinformatics approach was used to identify a set of immunogenic
proteins. Immunogenic bands ranging from 7 to 30 kDa were submitted to
ESI–LC-MS/MS and a total of 398 B. canis proteins were
identified. These proteins were analyzed by bioinformatics and were
predicted a set of B. canis specific candidate antigens that
could be used for development of more efficient diagnostic tests. In the
view to improve the diagnosis of canine brucellosis due to B.
canis possible applications of project results are discussed.
1. Introduction
Brucellosis is a chronic bacterial disease affecting both animals and
humans caused by Gram-negative coccobacilli of the genusBrucella . This genus includes several species responsible for
infection in livestock (B. melitensis , B. abortus ,B. ovis , B. suis ) but also in companion animals (B.
canis ) [1]. Canine brucellosis is mainly caused by B. canis .
Brucellosis due to B. abortus or B. melitensis is
diagnosed sporadically in dogs living in contact with infected
ruminants, but in these cases it represents only an epiphenomenon of the
infection circulating in the affected farm, and the dog does not play
the role of reservoir of the disease. The same applies to dog
brucellosis due to B. suis , which is rarely identified in dogs
and, in any case, always in connection with a coexisting infection in
pig farms or after exposition to infected feral pigs and boars [2, 3,
4]. B. canis was first isolated in 1966 in USA from aborted
fetuses in a kennel experiencing several cases of abortion and
infertility [5]. Later, B. canis infection has been
demonstrated by isolation or serological investigations in several
countries worldwide [6]. In Italy, the presence of anti-B.
canis antibodies in dogs was reported occasionally for a long period in
the past [7-14] and, in one case [15] B. canis was
detected by PCR in a dog with prostatitis and discospondylitis. In 2020,
for the first time, B. canis was isolated in a commercial
breeding kennel in central Italy [16].
Dogs and wild canids are thought to be the only significant hosts forB. canis among domesticated animals, while cattle, sheep and
swine were found to be highly resistant to the infection. The natural
pathways of transmission of canine brucellosis are different, but the
most common is the contact with placenta, fetal tissues and vaginal
discharges resulting from abortion. Infected female may transmitB. canis through placenta, aborted infected fetuses, or vaginal
discharges following an abortion, through contact with the mucous
membranes of the host organism [2].
Common symptoms are infertility, abortions, neonatal mortality,
epididymitis, prostatitis, discospondylitis and uveitis [17].
However, infected but asymptomatic animals are frequently observed
[5].
Due to silent symptoms, the disease spreads uncontrollably, before being
diagnosed, causing big economic damages in breeding kennels and problem
in assuring proper animal welfare. In dogs, therapeutic treatment with
antibiotics is not encouraged and antibiotic therapy does not completely
eliminate the pathogen, resulting in high risk of disease transmission
to other dogs and humans [5].
Humans can get B. canis infection through direct contact with
infected dogs, in particular with aborted fetuses, and secretions and
blood [18, 19], and they can develop clinical disease. The disease
can be asymptomatic and chronic; the symptoms are nonspecific and may
vary from fever to severe manifestations such as endocarditis,
osteomyelitis, and septicemia [17]. Kennel employees, veterinarians,
laboratory technicians, children and elderly and immunocompromised
people have higher risk to be infected by B. canis [5].
Diagnosis of canine brucellosis involves both direct and indirect
methods. The isolation of B. canis , meanly from blood culture,
gives confirmation of the infection while use of serological tests may
represent a more cost-effective approach for disease surveillance.B. canis carries rough LPS, so serological tests currently
available for the diagnosis of brucellosis caused by smoothBrucellae (B. melitensis , B. abortus , B.
suis ) cannot be used for the diagnosis of the disease caused byB. canis [1, 5].
The first serological tests developed for canine brucellosis were the
rapid slide agglutination test (RSAT) and rapid slide agglutination test
with 2-Mercaptoethanol (2ME-RSAT) [20]. However, since the beginning
it was noted a lack of specificity of these tests, counting for false
positive rates that commonly range from 20% up to even 50% [21].
To increase efficacy of serological diagnosis, the use of more than one
test in parallel has been suggested such as reported 2ME-RSAT as
screening tests, and indirect ELISA (i-ELISA) as a confirmatory test.
These tests have sensitivity ranging from 40 to 90% and specificity
between 60 and 100% [17]. Other diagnostic tests reported are the
agar gel immunodiffusion (AGID), the tube agglutination test (TAT), the
microagglutination test (MAT) and the complement fixation test (CFT)
[22].
All these tests suffer from lack of knowledge in accuracy, with only
limited data available in the international literature. In addition,
non-specific reactions are known with haemolysed sera or due to
cross-reactions with other bacteria, such as Pseudomonas spp.,Bordetella bronchiseptica , Streptococcus spp.,Staphylococcus spp., Salmonella spp., Yersinia
enterocolitica , Escherichia coli and Actinobacillus
equuli [6, 22]. Finally, most of the serological tests are not
available as commercial kits, raising the issue of antigen production
and test standardization, especially due to the lack of international
reference sera for B. canis .
Serological tests for the diagnosis of smooth Brucellae (B.
abortus , B. melitensis and B. suis ) infection use the
O-polysaccharide (OPS), an immunodominant epitope in smooth
lipopolysaccharide (s-LPS), as antigen; consequently, cross-reactions
with other Gram-negative bacteria, such as Y. enterocolitica O:9,
which shows analogous OPS structures, can occur [23]. B.
canis , similarly to B. ovis and B. abortus strain RB51,
has rough lipopolysaccharides (r-LPS) on its bacterial wall. The
diagnosis of ovine brucellosis caused by B. ovis is performed
using the homologous rough-specific antigen, obtained by extraction with
the hot-saline method. This antigen is enriched in r-LPS [24]. SinceB. ovis , B. canis and B. abortus strain RB51 shares
similar antigenic components, each of the three species may be employed
as antigen for the serological diagnosis of brucellosis caused by roughBrucella species [25-27]. Numerous studies have been done on
smooth Brucella species as B. abortus and B.
melitensis to identify Brucella unique proteins suitable as
antigens for the development of more specific serological tests [17,
28-32]. Only a few studies were focused on the characterization of
immunogenic proteins of rough Brucellae . Recently, identification
of B. canis immunogenic proteins by proteomics and bioinformatics
analyses was reported. Two recombinant cytoplasmic proteins were
expressed, and tested as antigens in i-ELISA assay to detect human and
canine brucellosis, but they were not able to detect canine brucellosis
with high specificity and sensitivity [17].
All these considerations highlight the need for development of more
sensitive and specific serological tests, as well as new protocols for
the diagnosis of infections caused by B. canis.
In the present study a western blotting assay has been developed to
define the serum antibody patterns associated to B. canisinfection using a panel of sera from dogs naturally infected and
non-infected with B. canis . Then LC-ESI- -MS/MS analyses and
bioinformatics tools have been combined to identify a set of immunogenic
proteins predicted as Brucella specific. Finally, possible
applications of project results are discussed in the view to improve the
diagnosis of canine brucellosis due to B. canis .
2. Materials and Methods