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Direct fluorescent antibody test and bacteriological culture for detection of Brucella suis in swine tissues.

INTRODUCTION

Brucellosis is caused by gram-negative Brucella sp and is one of the most widespread zoonoses and an economically important disease (1). Direct diagnoses include isolation of these bacteria by culture and identification by biochemical tests, or detection of DNA sequences by polymerase chain reaction (PCR).

Cultural examination takes a long time and is not always suitable even on selective media, because of overgrowth by contaminating organisms (3). PCR is an extremely powerful technique but requires DNA extraction from samples and the use of specific equipment.

The direct fluorescent antibody test (DFAT) appears to offer a specific and quick alternative to the Gram or stamp stains onto smears from tissues or suspicious colonies (2). DFAT is a common laboratory technique, which is based on the use of specific antibodies chemically conjugated to fluorescent dyes. The fluorescence can be visualized by a fluorescence microscope used for the routine diagnosis(e.g. diagnosis of campylobacteriosis) (7).

The aim of this work was evaluate the gammaglobulin fraction of polyclonal anti-Brucella abortus serum labeled with fluorescein iso-tio-cyanate (FITC-labeled antibody): 1) against different smooth and rough Brucella sp; 2) against bacterium of other genus; and 3) to compare DFAT results with bacteriological culture for the detection of B. suis in different tissues from seropositive pigs.

MATERIAL AND METHODS

Serological tests. Serum samples from aborted sow and boar were analyzed by Buffer Plate Agglutination test (BPAT), Bengal Rose Test (RBT) and Fluorescense Polarization Assay (FPA) following supplier instructions (Laboratorio Biologico de Tandil, Argentina). Results were interpreted according to the procedures recommended by Servicio Nacional de Sanidad y Calidad Agroalimentaria (SENASA) (8).

Tissue samples, bacteriological culture. Material from fetal tissues, aborted sow and boar from brucellosis endemic farm were submitted for routine diagnosis and were used in this study. Samples were taken of 4 liver and spleen fetal tissues; placenta, spleen and liver from an aborted sow; and spleen, liver, testes, seminal glands, prostate and bulbouretral glands, epididymis (head, body and tails), right and left testes and cervical and inguinal lymph nodes from a boar. Each sample was homogenized in saline solution and was seeded onto skidrow and tryptone soy agar added with yeast (TSAYE) media. Plates were incubated in 10% of C[O.sub.2] at 37[degrees]C for ten days. Suspected colonies were identified by Gram staining, catalase, oxidase, urease, nitrate reductase tests and S[H.sub.2] production (4).

Smears. Impression smears from all tissue samples were made onto glass slides and allowed to air-dry. Procedure to staining was detailed in "determination of conjugate specificity" using the optimal dilution of the fluorescein conjugate determined by check board titration against different smooth Brucella sp (1:200). Only the presence of individual or clumping yellowish-green fluorescent organisms with the morphological characteristics of Brucella sp was considered positive. Control smears of smooth B. abortus suspensions were included in each series of tests. A suspension of B. abortus strain was included, as positive control of the reaction.

Polyclonal anti-Brucella abortus serum. Polyclonal anti-B. abortus serum was produced by laboratorio biologico de Tandil (Biotandil SRL). Briefly, specific antibodies against B. abortus were purified from the serum of hyperimmunized goat. Goat was immunized with inactivated B. abortus S19 suspension four times by intramuscular via. The gamma globulin fraction from the serum was precipitated by the addition of ammonium sulphate. This fraction was conjugated to FITC as previously described by other investigators (9).

Determination of conjugate specificity. To determine the specificity of the fluorescent conjugate, it was diluted in Phosphate Buffered Saline (PBS) pH 8 at the following 10 dilutions: 1/25, 1/50, 1/100, 1/150, 1/200, 1/300, 1/400, 1/500 and 1/600. Ten microliters of each bacterial suspension was smeared in each one of 12 wells printed with ink per glass slide and was air-dried at 37[degrees]C. Smears were fixed with absolute ethanol at 37[degrees]C and then rinsed with distilled water. Twenty microliters of each conjugate dilution were added to each reaction site on the slides. Reaction was allowed to proceed in a moist atmosphere at 37[degrees]C for 1 hour. Conjugate was rinsed off with PBS three times and then with distilled water once. Cover slips were mounted with buffered glycerol (pH 8) and were examined by fluorescence microscopy with incident lamination at 100x (Zeiss--Primo Star). The highest conjugate dilution at which fluorescence was detected in each bacterial smear was taken as a final end point.

Bacteria. To check cross-reactivity of the fluorescent conjugate, individual suspensions of different smooth (Brucella abortus 544, B. melitensis H38, B. suis 1330) and rough Brucella strains (B. canis RM6/66, B. canis (M-), B. ovis REO 198), other Gram negatives (Actinobacillus seminis, Campylobacter fetus, Escherichia coli, Histophilus somni, Mannheimia haemolytica, Proteus vulgaris) and Gram positives (Corynebacterium pseudotuberculosis, Staphylococcus aureus, Trueperella pyogenes (ex-Arcanobacterium) bacteria were inactivated and were prepared in phenol saline solution. After that, density of each bacterial suspension was adjusted to match turbidity standard of 0.5 McFarland units (approximately 1.5 x [10.sup.8] bacteria).

Statistical analysis. The kappa index of concordance between bacteriological culture and DFAT was determined by EPIDAT 4.2

RESULTS

Serum samples from aborted sow and boar were positive in the three techniques used to evaluate the presence of anti-Brucella antibodies. Tissue samples were seeded in a base and selective media. Bacteriological results and DFAT assayed onto tissue smears are shown in Table 1.

The results obtained after incubation of FITC-labeled anti-B. abortus conjugate with different bacterial suspensions are shown in Table 2. Extremely positive and strongly positive reactions were observed in high dilutions of conjugate against all tested smooth B. abortus, B. suisand, and B. melitensis reference strains (Figure 1: A, B, and C).

However, fluorescent conjugate reacted with strong to weak intensity and in low dilutions against B. canis or B. ovis reference strains. No fluorescent bacteria were seen in preparations from other Gram positive or Gram negative bacteria that can cause serological cross reaction with Brucella sp in different domestic animals.

Substantial concordance was observed between bacteriological culture (gold standard) and DFAT (kappa=0.6923; (IC 95% 0.47-1)). Tissue smears from pigs infected with Brucella sp showed the presence of yellowish-green fluorescent organisms of brucella morphology located in clumps or individual particles (Figure 1: D and E). Occasionally, in all types of preparation, isolated particles showing yellowish fluorescence or indistinct patches showing similar fluorescence were observed.

DISCUSSION

Although a presumptive diagnosis of brucellosis can be made by demonstrating high or rising antibody titers to Brucella antigens, isolation of the organism from fluids or tissue cultures is the only irrefutable proof of the disease (4).

On the basis of the actual work we confirm that DFAT with this FITC-labeled anti-B.abortus conjugate allows safe and quick detection of Brucella sp onto smears of specimen or tissue from suspicious animals or from isolated colonies of brucella.

In addition, the direct binding of the polyclonal antibody to specific epitopes reduces the number of steps in the procedure, saving time and reducing non-specific background signal (6). This also limits the possibility of antibody cross-reactivity and possible mistakes throughout the process.

Since already it has been demonstrated by other authors, the use of this technique as a first step for diagnostic would allow much quicker results than bacteriological culture whose time is extensive and where isolation is subject to viability of the bacterium and to the employment of selective culture media that prevent the growth of contaminants (5).

Acknowledgements. We thank Drs. N.Guida (UBA) and F.Paolicchi (INTA Balcarce) for the strains provided, and J.Garcia, B.Riccio, G.Yaniz, P.Dominguez and M. Indart by the technical help in the necropsies.

REFERENCES

(1.) Adone R, Pasquali P. 2013. Epidemiosurveillance of brucellosis. Rev Sci Tech 32: 199-205.

(2.) Ajai CO, Cook JE, Dennis SM. 1980. Diagnosing ovine epididymitis by immunofluorescence. Vet Rec 107: 421-424.

(3.) Al Dahouk S, Tomaso H, Nockler K, Neubauer H, Frangoulidis D. 2003. Laboratory-based diagnosis of brucellosis-a review of the literature. Part I: Techniques for direct detection and identification of Brucella sp Clin Lab 49: 487-505.

(4.) Alton GG, Jones LM, Angus RD, Verger JM. 1988. Serological methods. Techniques for the Brucellosis laboratory. Ed. Institut National de la Recherche Agronomique, Paris, p. 157-167.

(5.) Corbel MJ. 1973. The direct fluorescent antibody test for detection of Brucella abortus in bovine abortion material. J Hyg 71:123-129.

(6.) Fritschy J, Hartig W. 2001. Immunofluorescence. doi:10.1038/npg.els.0001174. https://onlinelibrary.wiley.com/doi/10.1038/npg.els.0001174

(7.) Mellick PW, Winter AJ, McEntee K. 1965. Diagnosis of vibriosis in the bull by the use of the fluorescent antibody technique. Corn Vet 55: 280-294.

(8.) Nicola A, Elena S. 2009. Manual de diagnostico serologico de la brucelosis bovina, Ed. SENASA, Buenos Aires, p. 95.

(9.) Soto P, Di Rocco MJ. 1984. Campylobacteriosis bovina. Prevalencia en diversas zonas de la Republica Argentina. Rev Investig Agropec 19: 273-279.

Estein, S.M. (1,6); Bence, A.R. (2,5); Cacciato, C.S. (3,5); Echavarria, H.M. (4); Soto, P. (3,4)

(1) Lab.Inmunologia CIVETAN-CONICET, (2) Dep.Fisiopatologia, (3) Lab.Microbiologia, Fac.Cs.Vet.UNCPBA, Tandil, Buenos Aires, Argentina. (4) Lab.Tandil. (5) Comision Investig.Cientif. (CICPBA). (6) Consejo Nac.Investig.Cientif.Tecn. (CONICET).

E-mail: silmares@vet.unicen.edu.ar

Recibido: 2 noviembre 2018 / Aceptado: 20 diciembre 2018. Proyectos 03/H278-C(SeCAT UNCPBA) y PIP0569 (CONICET)
Table 1. Bacteriological culture and DFAT in different tissues from
fetal swine tissues, aborted sow and boar with positive serology.

origin  tissues              DFAT    bact.culture

        liver of F1        positive    positive
        spleen of F1       positive    positive
        liver of F2        positive    positive
fetal   spleen of F2       positive    positive
        liver of F3        positive    positive
        spleen of F3       positive    positive
        liver of F4        positive    positive
        spleen of F4       positive    positive
        placenta           positive    positive
        cervical lymph.    negative    negative
female  inguinal lymph.    negative    positive
        spleen             positive    positive
        liver              negative    negative
        right teste        positive    positive
        left teste         negative    negative
        spleen             negative    positive
        liver              negative    positive
        seminal glands     negative    negative
male    prostate gland     positive    positive
        left head of ep.   negative    negative
        right head of ep.  positive    positive
        leftbody of ep.    negative    negative
        rightbody of ep.   positive    positive
        lefttail of ep.    negative    negative
        righttail of ep.   positive    positive

DFAT: direct fluorescent antibody test; bact.: bacteriological; ep:
epididymis; F1 to F4: fetal organs.

Table 2. Fluorescence intensities against different bacterium after
incubation with fluorescein-labeled anti-Brucella globulin.

bacteria                   anti-Brucella fluorescent conjugate dilutions
                           1/25  1/50  1/75  1/100  1/150  1/200  1/300

Brucilla abortus 544 (S)   +++   +++   +++    +++    +++    +++    +++
B. melitensis H38 (S)      +++   ++ |  ++ |  ++ |   ++ |   ++ |    ++
B. suis 1330 (S)           +++   +++   +++    +++    +++    +++    +++
B. canis RM6/66 (R)        ++ |  ++ |  ++ |  ++ |    + |    + |     +
B. canis M- (R)            + |   + |    +      +      |      -      -
B. ovis REO 198(R)         + |   + |    +      |      -      -      -
Actinobacillus seminis      -     -     -      -      -      -      -
Campylobacter fetus         |     -     -      -      -      -      -
Escherichia coli            -     -     -      -      -      -      -
Histophilus somni           -     -     -      -      -      -      -
Mannheimia haemolytica      -     -     -      -      -      -      -
Proteus vulgaris            -     -     -      -      -      -      -
Corynebacterium pseudoTBC   |     |     |      |      -      -      -
Staphylococcus aureus       -     -     -      -      -      -      -
Trueperella pyogenes        ||    |     -      -      -      -      -

bacteria                   anti-Brucella fluorescent conjugate dilutions
                                         1/400  1/500  1/600

Brucilla abortus 544 (S)                  +++    +++    +++
B. melitensis H38 (S)                     + |    + |    + |
B. suis 1330 (S)                          +++    +++    +++
B. canis RM6/66 (R)                        +      +      +
B. canis M- (R)                            -      -      -
B. ovis REO 198(R)                         -      -      -
Actinobacillus seminis                     -      -      -
Campylobacter fetus                        -      -      -
Escherichia coli                           -      -      -
Histophilus somni                          -      -      -
Mannheimia haemolytica                     -      -      -
Proteus vulgaris                           -      -      -
Corynebacterium pseudoTBC                  -      -      -
Staphylococcus aureus                      -      -      -
Trueperella pyogenes                       -      -      -

Fluorescence intensity of conjugate: (+++) extremely positive reaction,
(++ |) strongly positive reaction, (++) mild reaction, (+) moderate
reaction, (+ |) weakly positive reaction, (|) weak reaction, (-)
negative reaction.
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Author:Estein, S.M.; Bence, A.R.; Cacciato, C.S.; Echavarria, H.M.; Soto, P.
Publication:Revista Veterinaria
Date:Jul 1, 2019
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