Swine brucellosis due to brucella suis biovar 2: development of diagnostic tests and control strategies
- Autores: Lucia Dieste Perez
- Directores de la Tesis: Pilar María Muñoz Díaz (dir. tes.), José María Blasco Martínez (dir. tes.)
- Lectura: En la Universidad de Zaragoza ( España ) en 2015
- Idioma: español
- Tribunal Calificador de la Tesis: Juan José Badiola Díez (presid.), Mª Jesús Grilló Dolset (secret.), Joaquim Segalés Coma (voc.)
- Materias:
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- Resumen
Swine brucellosis due to Brucella suis biovar 2 is an emerging disease in the European Union whose control is based on serological testing of pigs and the subsequent culling of those found seropositive or, most commonly, the full stamping out of infected herds (EFSA, 2009). It is widely accepted that current serological tests used for the diagnosis of this infection in swine have important deficiencies (Rogers et al., 1989; Ferris et al., 1995; EFSA, 2009). In addition to intrinsic problems or defects in validation that limit the diagnostic performance of brucellosis tests (Mc Given et al., 2012), many pig farms in brucellosis-free countries are currently affected by false positive serological reactions (FPSR) (Thibodeau et al., 2001; EFSA, 2009). Regarding with control and eradication, strategies for minimising the impact of swine brucellosis in infected herds are scanty (Olsen et al., 2012). At the present time, no suitable vaccines have been developed as an effective tool to control B. suis infection in pigs (Olsen et al., 2012). And, on the other hand, when the affected farms are very large industrial holdings or belong to outdoor breeding systems based on endangered pig breeds, full depopulation is unfeasible. Having in consideration these two issues, the final objective of this Thesis was to develop tests of improved performance for diagnosing swine brucellosis caused by B. suis biovar 2 as well as cost-effective strategies to minimise its impact in infected herds.
The memory of this work is presented as a compendium of 5 scientific publications. The first 4 publications deal with the development of in vivo and in vitro indirect diagnostic tests of improved specificity, based essentially on the use of genus specific cytosolic protein (CP) extracts from genetically defined rough (R) Brucella mutants devoid of the O-polysaccharide (O/PS) moiety of the Brucella smooth lipopolysaccharide (S-LPS). The last publication deals with the development of cost-effective antibiotic therapies for treating swine brucellosis, with the objective of minimising the impact of the disease in infected herds. The respective summaries are as follows:
Publication 1 In this study we assessed the performance of the most widely used Brucella S-LPS based tests for diagnosing swine brucellosis caused by B. suis biovar 2: Rose Bengal Test (RBT), indirect ELISA (i-ELISA), blocking ELISA (b-ELISA) and two competitive ELISAs (c-ELISA). RBT, i-ELISA and b-ELISA tests resulted in maximum diagnostic sensitivity (Se) and specificity (Sp) when they were analysed with a frequentistic analysis using sera from known gold standard populations. In contrast, the c-ELISAs resulted in lower diagnostic Se when cut-off values were set up to result in perfect Sp. A Bayesian analysis of the tests yielding the best diagnostic performance (RBT, i-ELISA and b-ELISA), using a large collection of field sera, resulted in similar Se among tests, but results were markedly lower than the values obtained after frequentistic analysis. This work proves the availability of adequate diagnostic tests for brucellosis in swine, but emphasises the need of a suitable validation before their application under field conditions.
Publication 2 Current serological tests detect antibodies to the O/PS moiety of the Brucella S-LPS. This fact causes a lack of specificity when infections by Yersinia enterocolitica O:9 and other gram-negative bacteria carrying cross-reacting O/PS occur, being these bacteria considered as the major cause of false positive serological reactions (FPSR) in pigs. The only strategy considered fully specific for discriminating FPSR from true Brucella infections is based on the in vivo skin test with the protein-rich brucellin extract obtained from the B. melitensis B115 spontaneous R mutant. However, B115 mutant, although unable to synthesise S-LPS, accumulates O/PS internally, which could originate diagnostic problems. Since the brucellin skin test has been seldom used in pigs and FPSR are very common in these animals, we assessed its diagnostic performance for swine brucellosis using CP extracts obtained from both genetically defined B. abortus R mutants in manBcore and per genes (critical for O/PS biosynthesis) and also from B. melitensis B115 spontaneous R mutant. The diagnostic sensitivity and specificity were determined in B. suis biovar 2 culture positive and brucellosis free sows, and the apparent prevalence was estimated in sows of unknown individual bacteriological and serological status but belonging to B. suis biovar 2 naturally infected herds. Moreover, the specificity for discriminating brucellosis from FPSR was assessed in brucellosis free boars showing cross-reactions. The skin test with allergens obtained from genetically defined O/PS free R B. abortus mutants performed similarly to the classical B. melitensis B115 brucellin, and resulted in 100 % specificity when testing Brucella-free animals, even when they showed FPSR in S-LPS based serological tests (i-ELISA, RBT and complement fixation [CFT]). As a conclusion, we demonstrated that the O/PS-free genetically defined R mutants are an appropriate alternative to obtain cytosolic protein extracts of improved diagnostic performance for diagnosing swine brucellosis even in presence of FPSR.
Publication 3 Skin test with Brucella CP extracts (widely known as brucellin) is the most specific immunoassay to differentiate true B. suis infections from FPSR in pigs. Since brucellin has been seldom used in swine, the pathological features of skin test responses in B. suis infected animals have not been the objective of deep studies nor described with detail. In this work the kinetics of skin test pathological events taking place in B. suis biovar 2 naturally infected pigs inoculated intradermally with O/PS free CP extracts from the genetically defined B. abortus per mutant was determined. A similar heterologous extract from O. intermedium was also tested for comparative purposes. No relevant differences were evidenced among homologous and heterologous allergens, and the main clinical features evidenced in infected pigs were clearly visible elevated areas of the skin showing different induration degrees. Moreover, an important vascular reaction resulting in an evident erythema was produced in most infected sows at 24-48 h after inoculation. These evident clinical features facilitate the macroscopical interpretation of positive reactions. Histologically, a combined immediate (type III) and delayed (type IV) hypersensitivity reactions was identified as the most relevant feature of the inflammatory responses produced. As a conclusion, the skin test with CP extracts from the O/PS-free genetically defined B. abortus¿per rough mutant is a practical and effective alternative of improved specificity for diagnosing swine brucellosis.
Publication 4 Current serological tests for swine brucellosis were developed for detecting antibodies to the O/PS moiety of the Brucella S-LPS. Thus, their diagnostic specificity is seriously compromised by FPSR when bacteria carrying cross-reacting O/PS infect pigs. FPSR occur very commonly throughout Spain and Europe, and the only tool available for a fully specific B. suis diagnosis is the in vivo skin test with O/PS free Brucella CP extracts. However, this test is somewhat cumbersome and expensive to be performed at field level, and a suitable serological screening of FPSR would be advisable. Using sera of sows naturally infected by B. suis biovar 2, brucellosis-free sows and pigs from brucellosis-free herds affected by FPSR, we assessed the diagnostic performance of an i-ELISA with R LPS (thus devoid of O/PS) and of gel immunodiffusion (GD), counterimmunoelectrophoresis (CIE), latex agglutination (LAT) and i-ELISA with O/PS free CP extracts in comparison with several S-LPS based tests (RBT, CFT, GD and i-ELISA). When Sp was adjusted to 100 %, the Se of the R-LPS i-ELISA was very low (30 %), and the adoption of other cut-offs resulted in poor specificity/sensitivity ratios. Although their Sp was 100 %, the Se of CP based tests (i-ELISA, LAT, CIE, and GD) was only low to moderate (45, 58, 61 and 63 %, respectively). Among the S-LPS based tests, GD was the unique resulting in acceptable Se and Sp (68 and 100 %, respectively). Despite these shortcomings, and when the purpose is to screen out of FPSR at herd level, GD tests may offer a technically simple and practical in vitro alternative to skin testing performed in vivo.
Publication 5 Despite the existence of sporadic outbreaks, the EU is considered officially as free from swine brucellosis. According to this situation, the control of B. suis biovar 2 outbreaks is based solely on the full depopulation of infected holdings. However, culling of very large herds or outdoor farming systems rearing endangered pig breeds is either economically unfeasible or impracticable. Thus, an antibiotic treatment could be cost-effective in these particular circumstances, and therefore suitable therapeutic alternatives should be worthy of study. The aim of this work was to develop effective treatments against B. suis biovar 2 infection in pigs. Minimum inhibitory concentration (MIC) for antibiotics used in the classical brucellosis treatment and two new generation macrolides (tulathromycin and tildipirosin) was determined for a representative collection of B. suis biovar 2 field and B. suis reference strains. MIC90 values for the drugs tested ranged from 0.01 to 0.25 µg / mL. The best potential candidates were then evaluated in a mouse model of infection, given alone or combined. Ten groups of 7 BALB/c mice each were inoculated (1 x 106 CFU / mouse) with a virulent B. suis biovar 2 field strain representative of genotypes found in Spain. All groups, excepting that composed by untreated control mice, were treated during 14 days, 15 days after the experimental infection (moment showing the highest splenic infection) with doxycycline, dihydrostreptomycin, tulathromycin (1 or 2 doses) or tildipirosin (1 or 2 doses) given alone, and doxycycline combined with dihydrostreptomycin, tulathromycin or tildipirosin, respectively. Combined tildipirosin treatment was the most effective in this mouse model of infection and it was selected for the study in pigs. Then 16 B. suis biovar 2 naturally infected sows were treated with oxytetracycline (20 mg / kg BW / daily) given orally for 21 days. Eight of them received also tildipirosin (4 mg / kg BW) given intramuscularly in 2 doses with 10 days interval. An extensive bacteriological study conducted ten days after ceasing treatments proved the excellent efficacy of the combined oxytetracycline / tildipirosin therapy for the clearance of the infection. This therapy could be a cost-effective strategy for treating brucellosis in pigs with the objective of minimising the impact of brucellosis in the infected holdings.
As a general conclusion, basing on the results obtained from works included in this Thesis, we can conclude that, whenever properly validated, currently available serological tests are suitable for the screening of swine brucellosis. In addition, diagnostic immunoassays of improved diagnostic performance developed in this work result successful to solve the FPSR, a huge problem in pigs in the EU. Finally, we have developed an effective antibiotic therapy to be used as a cost-effective strategy for minimising the impact of brucellosis in the infected herds.
References:
EFSA, 2009. Opinion of the Panel of Animal Health and Welfare (AHAW) on a request from the commission on porcine brucellosis (Brucella suis). EFSA J. 1144:1-112.
Ferris RA, Schoenbaum MA, Crawford RP. 1995. Comparison of serologic tests and bacteriologic culture for detection of brucellosis in swine from naturally infected herds. JAVMA. 207: 332-1333.
Mc Given JA, Nicola A, Commander NJ, Duncombe L, Taylor AV, Villari S, Dainty A, Thirlwall R, Bouzelmat N, Perrett LL, Brew SD, Stack JA. 2012. An evaluation of the capability of existing and novel serodiagnostic methods for porcine brucellosis to reduce false positive serological reactions. Vet Microbiol. 160:378-386.
Olsen SC, Garin-Bastuji B, Blasco JM, Nicola AM, Samartino L. 2012. Brucellosis In: Zimmerman JJ, Karriker LA, Ramírez A, Schwartz KJ, Stevenson GW (Eds.) Diseases of Swine, 10th ed., Wiley-Blackwell, USA, pp. 697-708.
Rogers RJ, Cook DR, Ketterer PJ, Baldock FC, Blackall PJ, Stewart RW. 1989. An evaluation of three serological tests for antibody to Brucella suis in pigs. Aus Vet J. 66(3): 77-80.
Thibodeau V, Frost EH, Quessy S. 2001. Development of an ELISA procedure to detect swine carriers of pathogenic Yersinia enterocolitica. Vet Microbiol. 82:249-259.
