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