brucellosis is one of the world 's major bacterial zoonosis and an important cause of a serious debilitating disease in humans ; further , it is the cause of abortion and sterility in animals .
brucella canis , a facultative , gram - negative , intracellular pathogen , is the etiologic agent of canine brucellosis .
canine brucellosis is widely distributed around the world and this is an important disease due to the economic losses in animal production and its risks for human health .
serological diagnosis is usually performed using a tube agglutination test ( tat ) , a rapid slide agglutination test , and a gel immunodiffusion test [ 24 ] .
however , agglutination tests often give false positive results because of cross - reactions with other pathogens .
a general strategy for eliminating cross - reactions is to use purified antigen with unique epitopes in the serological tests .
bacterial cell wall antigens of b. canis can be prepared by hot saline extraction and are reportedly useful in serological diagnosis . in a previous study , we reported the characterization of crude hot saline extracts and identified three antigens , namely , rhizopine - binding protein ( rbp , former assigned ribose abc transporter ) , cu - zn superoxide dismutase ( sod ) , and a hypothetical protein ( ag 4 ) .
this paper describes the development of latex beads coated with purified recombinant versions of these antigens and demonstrates the usefulness of these antigen - coated latex beads in the serological diagnosis of canine brucellosis .
the gene encoding each antigen was amplified from chromosomal dna isolated from b. canis   using pcr with the pair of primers shown in table 1 .
his - tagged antigens were expressed in the escherichia coli strain dh5 , and their purification was performed as described by the manufacturer ( novagen , darmstadt , germany ) .
serum samples ( n = 743 ) were collected from fleas- or ticks - infested dogs consecutively admitted to animal hospitals in japan by the hospital staff in 2012 and 2013 .
b. canis - infected control sera were collected from three pcr - positive dogs , and b. canis - free control sera were collected from three specific pathogen - free ( spf ) dogs .
covalent coupling of the proteins to carboxylated polystyrene latex beads was performed as described by the manufacturer ( polyscience inc . , warrington , pa , usa ) .
briefly , equal volumes of antigen solution ( protein concentration 300  g / ml , measured using lowry method ) and 2.5% latex beads ( diameter 1  m , polyscience inc . ) were mixed for 18  h at room temperature with stirring .
then , the latex beads were blocked with 0.2  m borate buffer containing 0.25  m ethanolamine for 30  min at room temperature with stirring and were washed three times with 0.2  m borate buffer containing 1% bovine serum albumin .
the latex beads were then adjusted to a final concentration of 2.5% ( w / v ) with storage buffer and were maintained at 4c .
this test was performed by mixing equal volumes ( 50  l ) of latex bead solution and canine serum diluted 50-fold with pbs in each well of a 96-well round - bottom microwell plate ( nunc , roskilde , denmark ) and incubating the plate at room temperature for 18  h without shaking .
this test was performed by adding equal volumes ( 0.5  ml ) of heat - inactivated b. canis qe-13 whole - cell antigens ( optical density of 0.8 at 450  nm , kitasato laboratories , saitama , japan ) , with serum that had been serially diluted 2-fold with pbs , and incubating the mixture at 50c for 24  h. agglutination titers were determined from the final dilution of serum showing 50% agglutination .
the antigen solution was separated using 10% sds - page and then transferred to immobilon - p membranes ( millipore , billerica , ma , usa ) .
the efficiency of transfer was determined using coomassie brilliant blue r-250 , and then the membranes were tested for reactivity with antibodies in the b. canis - infected control sera that were collected from three pcr - positive dogs [ 6 , 8 ] .
immunoreactions were visualized using the enhanced chemiluminescence detection system ( ge healthcare life science , little chalfont , uk ) .
to test antigenic reactivity , four recombinant antigens were subjected to western blotting along with the dog sera that had tested positive or negative in the tat .
all recombinant antigens showed strong reactivity with the positive sera , but not with the negative sera ( figure 1 ) . to identify b. canis - infected sera , we performed tat on canine serum samples ( n = 743 ) collected from dogs consecutively admitted to animal hospitals in japan by the hospital staff .
antibodies to b. canis were detected in 9 of the 743 ( 1.2% ) serum samples ( table 2 ) .
latex beads were coated with single or multiple recombinant b. canis antigens with the aim of achieving a serodiagnostic method that is faster and easier to perform .
sera from b. canis - infected dogs , in which infection had been confirmed by tat and pcr previously , were mixed with the antigen - coated latex beads , and then this mixture was incubated at room temperature for 18  h. agglutination was clearly observed in all antigens ( figure 2 ) .
in contrast , b. canis - free dog serum showed no agglutination ( figure 2 ) .
various results were obtained when mat was conducted using the 743 dog serum samples with each of the antigen - coated latex beads , as shown in table 2 .
sod reacted with 8 tat - positive sera ( 88.9% ) , but rbp , ag 4 , and hsp60 ( heat shock protein 60 ) reacted with 6 ( 66.7% ) , 6 ( 66.7% ) , and 7 ( 77.8% ) sera , respectively ( table 2 ) .
it is well known that hemolysis of serum samples induces false positive results in tat . to investigate
if this source of false positive results is eliminated in the mat , we performed the assay with antigen - coated latex beads using three artificial hemolytic sera from b. canis - free dogs .
indeed , hemolyzed serum showed a false positive result in tat ( figure 3 ) .
in contrast , mat with rbp - coated latex beads gave a negative result with the hemolyzed serum ( figure 3 ) .
the same results were obtained with latex beads coated with other antigens ( data not shown ) .
tat and mat , using whole b. canis antigens , are currently being used for serological diagnosis in dogs in japan [ 11 , 12 ] .
however , nonspecific reactions occur in the serological diagnoses using whole bacterial cell antigens . in a previous study , we reported a rapid slide agglutination test using latex beads that were coated with antigens extracted by hot saline for the serological diagnosis of canine brucellosis . to develop a serological diagnosis method that is faster and easier to perform
however , we observed nonspecific reaction in mat and , therefore , we used latex beads coated with purified recombinant antigens in this study .
analysis of the antigens contained in the crude hot saline extracts , performed in our previous study , identified three antigens , namely , rbp , sod , and ag 4 .
rhizopines are known to be nutritional mediators and play a role in nitrogen - fixing symbiotic associations between members of the rhizobiaceae and legume plants . because b. canis is not a plant symbiont
in addition , the function of ag 4 is currently unknown , and because sod is a known antigenic protein of b. abortus , its potential value as a vaccine for brucellosis prevention and as a diagnostic reagent for the disease has been investigated [ 9 , 16 , 17 ] .
the results from our previous study also showed that recombinant sod was useful for the detection of canine brucellosis using elisa .
hsp60 has also been known as an immunoreactive protein and virulence factor of b. abortus .
the present study showed that these four recombinant antigens can be used as a potential diagnostic marker for canine brucellosis .
in particular , because the sod antigen showed the highest reactivity to tat - positive sera in the mat , recombinant sod may be most useful for serological diagnosis .
usually , serum samples taken from dogs living in japanese household do not show a positive result for pcr but sometimes show a positive result for tat .
we think bacteremia of brucella may be rare case in dogs that are kept at home in japan [ 68 ] .
although nonspecific agglutination shows a relationship with hemoglobin concentration , its detailed mechanism is still unclear .
the purified recombinant antigen - coated latex beads developed in the present study will be useful tools for the serological diagnosis of canine brucellosis from hemolyzed serum specimens .
furthermore , mat can be used for very small serum samples and offers potential for high throughput testing ; therefore , it would be suitable for the screening of valuable samples such as those from wildlife or small animals .
 
 brucella canis , a facultative intracellular pathogen , is the causative agent of canine brucellosis . 
 the diagnosis of canine brucellosis is based on bacteriological examination and serological methods , including agglutination and gel diffusion tests . in this study , four recombinant antigens , heat shock protein 60 , rhizopine - binding protein , cu - zn superoxide dismutase , and hypothetical protein ( ag 4 ) , were constructed . 
 these antigens were coated on latex beads and their usefulness in the serological diagnosis of canine brucellosis was examined . 
 all recombinant antigens showed specific reaction with sera from b. canis - infected dogs in western blotting . in a microplate agglutination test , mixing sera from b. canis - infected dogs , but not sera from b. canis - free dogs , with single or multiple antigens - coated latex beads produced clear agglutination . 
 moreover , the antigen - coated latex beads did not show nonspecific agglutination in hemolyzed serum samples . 
 a survey of canine serum samples conducted by the microplate agglutination test using single antigen - coated latex beads showed that this method would be useful in the serological diagnosis of canine brucellosis . 
 further investigations using more serum samples are required to confirm the usefulness of our method . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion