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Pesquisa Veterinária Brasileira

Print version ISSN 0100-736XOn-line version ISSN 1678-5150

Pesq. Vet. Bras. vol.40 no.2 Rio de Janeiro Feb. 2020  Epub Apr 09, 2020

https://doi.org/10.1590/1678-5150-pvb-6481 

LIVESTOCK DISEASES

Pathogenic potential of Brucella ovis field isolates with different genotypic profile and protection provided by the vaccine strain B. ovis ΔabcBA against B. ovis field isolates in mice

Vacina viva atenuada Brucella ovis Δ abcBA encapsulada protege camundongos frente a desafios de B. ovis isoladas de campo

Thaynara P. Carvalho2 

Noelly Q. Ribeiro2 

Juliana P.S. Mol2 

Fabíola B. Costa2 

Camila Eckstein2 

Nayara F. Paula2 

Tatiane A. Paixão3 

Renato L. Santos2  * 
http://orcid.org/0000-0002-4830-0470

2Departamento de Clínica e Cirurgia Veterinária, Escola de Veterinária, Universidade Federal de Minas Gerais (UFMG), Campus Pampulha, Av. Pres. Antônio Carlos 6627, Belo Horizonte, MG 31270-901, Brazil.

3Departamento de Patologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG), Campus Pampulha, Av. Pres. Antônio Carlos 6627, Belo Horizonte, MG 31270-901.


ABSTRACT:

Brucella ovis causes economic and reproductive losses in sheep herds. The goal of this study was to characterize infection with B. ovis field isolates in a murine model, and to evaluate protection induced by the candidate vaccine strain B. ovis ΔabcBA in mice challenged with these field isolates. B. ovis field strains were able to colonize and cause lesions in the liver and spleen of infected mice. After an initial screening, two strains were selected for further characterization (B. ovis 94 AV and B. ovis 266 L). Both strains had in vitro growth kinetics that was similar to that of the reference strain B. ovis ATCC 25840. Vaccination with B. ovis ΔabcBA encapsulated with 1% alginate was protective against the challenge with field strains, with the following protection indexes: 0.751, 1.736, and 2.746, for mice challenged with B. ovis ATCC25840, B. ovis 94 AV, and B. ovis 266 L, respectively. In conclusion, these results demonstrated that B. ovis field strains were capable of infecting and inducing lesions in experimentally infected mice. The attenuated vaccine strain B. ovis ΔabcBA induced protection in mice challenged with different B. ovis field isolates, resulting in higher protection indexes against more pathogenic strains.

INDEX TERMS: Pathogeny; Brucella ovis; isolates; genotypic profile; protection; vaccine strain ΔabcBA; mice; immunization; field isolated strains; brucellosis

RESUMO:

Brucella ovis é responsável por perdas econômicas e reprodutivas em rebanhos ovinos. O objetivo deste trabalho foi caracterizar a infecção com as cepas isoladas de campo de B. ovis em modelo murino e avaliar a eficiência vacinal da mutante B. ovis ΔabcAB para proteção contra desafio com as cepas isoladas de campo. Foram utilizadas sete cepas isoladas de campo foram capazes de colonizar e provocar lesões no fígado e no baço de camundongos após sete dias pós-infecção. Após triagem, duas cepas foram selecionadas para a melhor caracterização (B. ovis 94 AV and B. ovis 266L). Ambas apresentaram crescimento em placa de cultivo semelhante ao da cepa de referência B. ovis ATCC 25840. A vacinação com a cepa de Brucella ovis ΔabcBA encapsulada com alginato a 1% foi capaz de proteger camundongos desafiados com as cepas isoladas de campo, com os seguintes índices de proteção: 0,751, 1,736 e 2,746, para camundongos desafiados com B. ovis ATCC 25840, B. ovis 94 AV e B. ovis 266 L, respectivamente. Estes resultados demonstraram que as cepas isoladas de campo de B. ovis são capazes de infectar e induzir lesão em camundongos experimentalmente infectados. O uso da cepa mutante atenuada B. ovis ΔabcBA para vacinação de fêmeas C57BL/6 desafiados com diferentes cepas de B. ovis induziu proteção nos camundongos desafiados com diferentes cepas de B. ovis. Deste modo, mostrando-se eficiente na proteção das cepas de campo de B. ovis.

TERMOS DE INDEXAÇÃO: Vacina viva; Brucella ovis; ΔabcBA; camundongos; imunização; brucelose ovina; mutante encapsulada; cepas isoladas de campo

Introduction

Brucellosis is a group of infectious diseases caused by facultative, intracellular, Gram-negative coccobacillary bacteria of the genus Brucella that affects domestic and wild animals and causes zoonotic infections in man (Olsen et al. 2011). B. ovis is considered a non-zoonotic species, and it is responsible for economic and reproductive losses in sheep herds (Poester et al. 2013).

Brucellosis in rams is clinically characterized by unilateral or bilateral granulomatous epididymitis and seminal vesiculitis. These changes result in poor sperm quality with increased defects of the tail of the spermatozoa, presence of inflammatory cells in the ejaculate, and consequent subfertility or infertility (Carvalho Júnior et al. 2012, OIE 2015). In ewes, the disease is usually asymptomatic, but endometritis and, more rarely, abortions, stillbirths, and weak offsprings may be observed (Grilló et al. 1999).

Currently, the vaccine available in some countries for B. ovis prevention is the Rev-1 strain, a live attenuated Brucella melitensis vaccine (Ridler & West 2011). However, the Rev-1 strain can interfere with serological tests, it can induce abortions when administered to pregnant animals, and it is capable of infecting and causing disease in humans (Blasco & Díaz 1993, Blasco 1997).

Brucella spp., as well as other intracellular bacteria has several strategies to achieve a safe replication niche within the host cell (Gorvel & Moreno 2002). Intracellular survival of Brucella spp. requires a functional virB-encoded type IV secretion system (T4SS). Brucella strains lacking a functional T4SS cannot evade degradation in lysosomes so they do not replicate or survive within the host cell (Celli et al. 2003). A previous study demonstrated that a B. ovis specific ABC transporter is required for B. ovis survival in vivo and evasion from phagosome/lysosome fusion (Silva et al. 2011b, Macedo et al. 2015). Additionally, B. ovis-specific ABC transporter is required for normal expression of the virB-encoded T4SS since in the absence of this ABC transporter there is a post-transcriptional impairement of expression of virB-encoded proteins (Silva et al. 2014). Indeed, B. ovis mutant strains lacking a functional B. ovis-specific ABC transporter (Silva et al. 2011b) or the virB-encoded T4SS (Sá et al. 2012) have similar phenotypes.

ABC transporters have various substrates including polyamines (Igarashi et al. 2001), peptides (Detmers et al. 2001), and amino acids (Hosie & Poole 2001, Danese et al. 2004). Brucella spp. genome encodes several ABC transporters, whereas B. ovis has 29 pseudogene-forming mutations in coding sequences for ABC-like carrier systems, so B. ovis cannot transport some substances such as polyamines, erythritol, and glycine (Jenner et al. 2009). A B. ovis-specific genomic island (Tsolis et al. 2009), named BOPI-1 for B. ovis pathogenicity island 1 (Silva et al. 2011b), encodes an ABC transporter that is essential for pathogenesis since the B. ovis ΔabcBA strain is strongly attenuated in vitro and in vivo so this genomic island has been named BOPI-1 for B. ovis pathogenicity island 1 (Silva et al. 2011b). However, the substrates of this particular ABC transporter are still unkown (Silva et al. 2014). In spite of its attenuation, B. ovis ΔabcBA triggers humoral and cellular immune responses in rams that are indistinguishable from those triggered by the wild-type parental strain (Silva et al. 2013). Therefore, B. ovis ΔabcBA has been tested as an experimental candidate vaccine strain and provided protection in a mouse model of infection (Silva et al. 2015a). Furthermore, when tested in the natural host, this vaccine strain prevented any clinical sign of disease, macro- and microscopic lesions, and induced sterile immunity in experimentally challenged rams (Silva et al. 2015b).

There is relatively low genetic variability within the genus Brucella, which has even supported the proposition of a monospecific genus (Verger et al. 1985). However, there are striking differences in host specificity and pathogenicity among different Brucella species (Chain et al. 2005). Therefore, the Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA) has been used as a tool for genetic and epidemiologic characterization of Brucella spp. (Whatmore 2009). The analysis of fourteen B. ovis Brazilian field isolates demonstrated some degree of genetic diversity (Dorneles et al. 2014). Considering the molecular differences identified by MLVA-16 among B. ovis field isolates, the aim of this study was to characterize field isolates of B. ovis in a murine model of infection and to evaluate the efficiency of the B. ovis ΔabcBA vaccine strain to protect mice challenged with B. ovis field isolates.

Materials and Methods

Bacterial strains. As detailed in Table 1, this study included seven field Brucella ovis strains isolated from semen of naturally infected rams, which have been previously genotypically characterized by MLVA-16 (Dorneles et al. 2014), the reference wild-type strain B. ovis ATCC 25840, and the candidate vaccine strain B. ovis ΔabcBA (Silva et al. 2011b, 2015a, 2015b). Bacteria were grown on the tryptic soy agar (TSA) plates supplemented with 1% hemoglobin, for 3 days at 37°C with 5% CO2. For the vaccine strain (B. ovis ΔabcBA), TSA was supplemented with 1% hemoglobin and 100 μg/mL of kanamycin. Bacteria were suspended in phosphate-buffered saline (PBS) (pH 7.4) and bacterial concentration was estimated by spectrophotometry (Smart Spec, Bio-Rad, Hercules, CA) at the optical density of 600nm (OD 600).

Table 1. Brucella strains used in this study 

Strain City Country Year of isolation
B. ovis ATCC 25840 ___ Australia 1960
B. ovis ΔabcBA ___ ___ 2011
B. ovis 94 AV Livramento/MS Brazil 1995
B. ovis 266 L Livramento/MS Brazil 1995
B. ovis 0204 Uruguaiana/MS Brazil 1997
B. ovis 286 L Livramento/MS Brazil 1995
B. ovis 252 L Livramento/MS Brazil 1995
B. ovis 100 V Livramento/MS Brazil 1995
B. ovis 203 L Livramento/MS Brazil 1995

Animals. The experimental protocol used in this study has been approved by the Animal Experimentation Ethics Committee at the Universidade Federal de Minas Gerais (CEUA-UFMG protocols 41/2018 and 107/2015). Mice were maintained in cages under controlled temperature and humidity (25°C, 70%), fed commercial feed and water ad libitum. Mice were intraperitoneally infected with 1 x 106 colony forming units (CFU) of B. ovis suspended in 100μL of sterile PBS. Euthanasia was performed at 1, 7, or 30 days post-infection (dpi).

In vitro growth of Brucella ovis ATCC 25840 and field isolated strains. In vitro growth of B. ovis ATCC 25840 and field isolates (94 AV and 266L) was evaluated on solid media as follows: bacterial suspensions were prepared in PBS to a concentration of 103 CFU/mL. 100μL of each suspension were then plated on TSA medium with 1% hemoglobin and without antibiotics. Plates were incubated at 37°C in 5% CO2 and at 0, 12, 24, 48, 72, 96, and 120 hours post-inoculation colonies were harvested and suspended in either 1mL (0 to 48h) or 2mL (72 to 120h) of sterile PBS. Bacterial suspensions were serially diluted (10-fold dilutions) and plated on TSA plus 1% hemoglobin by using the drop plate method. The experiment was performed in duplicate, and the total numbers of CFU per milliliter were determined at each time point.

In vitro infection of RAW 264.7 murine macrophages. The murine macrophage cell line RAW 264.7 was cultured in RPMI medium (Gibco; Invitrogen) supplemented with 10% fetal bovine serum (FBS). Cells were seeded in 96-well culture plates (5 × 105 macrophages/well) and incubated at 37°C with 5% CO2. Macrophages were infected with the B. ovis ATCC 25840 or field isolates (B. ovis 94 AV or 266 L) at a multiplicity of infection (MOI) of 100. Plates were centrifuged at 1,000 × g for 5min at 15°C and incubated at 37°C for 30min. Macrophages were washed once with sterile PBS and then incubated at 37°C for 1h with RPMI solution supplemented with 10% FBS and 50μg/mL of gentamicin (Invitrogen, São Paulo, Brazil). Next, each well was washed once with sterile PBS, and macrophages were lysed with sterile distilled water for 20min at 0, 4, 24, and 48hpi. Intracellular bacteria recovered from lysed macrophages were serially diluted (10-fold dilutions) in PBS and plated in duplicate on TSA medium with 1% hemoglobin for 3-6 days of incubation at 37°C with 5% CO2 for CFU counting. Two independent experiments were performed in triplicates.

Vaccine experiments. Encapsulation of the B. ovis ΔabcBA vaccine strain was performed as previously described (Silva et al. 2015a). Briefly, 2 x 1010 CFU of B. ovis ΔabcBA were resuspended in 2mL of 1% alginate solution (Sigma-Aldrich) and dripped in 10mL of polymerization solution (0.5 mM CaCl2), using a 0.23mm x 4mm needle, followed by homogenization for 15 minutes. Capsules were washed twice with 10mM MOPS solution with 0.85% NaCl (pH 7.4) for 5min. Capsules were then shaken with 0.05% alginate solution for 5min. The encapsulated vaccine strain was subcutaneously inoculated in fifteen female C57BL/6 mice with a final dose of 1 x 108 CFU per mouse. Other fifteen mice were inoculated with sterile PBS by the same route. The size of the capsules has previously been described by Silva et al. (2015a). Four weeks after immunization, mice were intraperitoneally challenged with 106 CFU of wild-type B. ovis (ATCC 25840) or fields strains (B. ovis 94 AV and 266 L). Two weeks later, mice were euthanized, and samples of liver and spleen were aseptically collected, weighed, and homogenized in 2mL of sterile PBS. Serial 10-fold dilutions of the homogenates were plated for the CFU counting. Briefly, organs were homogenized in sterile PBS and plated on the TSA plates with 1% hemoglobin. Bacterial colonies were counted at 3-6 days after plating.

Histopathology. Liver, spleen, superficial cervical lymph node, and the subcutaneous site of vaccination were sampled, fixed by immersion in 10% buffered formalin for 24 hours, and embedded in paraffin. Four μm tissue sections were stained with hematoxylin and eosin. Lesions (inflammation and necrosis) were scored from 0 to 3, being 0-absent, 1-mild, 2-moderate, and 3-severe, with a total score ranging from 0 to 6.

Statistical analysis. Statistical analyses were performed using the Graph Pad Prism version 5.0 software. CFU values were logarithmically transformed prior to analysis of variance (ANOVA). Means were compared by the Tukey’s test. Histopathological scores were compared using the non-parametric Mann-Whitney test.

Results

Brucella ovis field isolates were capable of infecting mice

Infectivity of field isolates was assessed in BALB/c mice (n=3 per group) that were inoculated with 106 CFU of each Brucella ovis strain (100 V, 203 L, 266 L, 204, 286 L, 252 L, and 94 AV). At 7 days post-infection, strains 94 AV and 252 L had higher numbers of CFU/g in the spleen when compared to the other strains (p<0.05), with differences of more than one log of CFU (Fig.1A). In the liver, B. ovis 94 AV was recovered in higher numbers when compared to other strains (Fig.1B). The spleen and liver (Fig.1C,D) from all infected mice had multifocal microgranulomas characterized by a histiocytic and neutrophilic inflammatory infiltrate with epithelioid macrophages. There were no significant differences in histopathology scores between different strains (data not shown). These results indicate that all B. ovis field isolates were capable of colonizing and cause lesions in the liver and spleen of BALB/c mice.

Fig.1. Experimental infection of BALB/c mice with different field isolated strains of Brucella ovis. Mice were infected with 1×106CFU/mouse from different field strains (100 V, 203, 266 L, 204, 252 L, 286 L, and 94 AV). Each data point represents the number of CFU/g recovered from the (A) spleen and (B) liver of each mouse at 7dpi. The line in each group indicates the mean. All data were logarithmically transformed prior to ANOVA, and the means were compared by the Tukey’s test. Statistically significant differences are indicated by asterisks (** p<0.01). All infected mice developed microgranulomas (arrows) in the (C) spleen and (D) liver. (C,D) HE, bar = 100μm, bar = 200μm. 

Brucella ovis field isolates had variable growth kinetics in RAW 264.7 cells

Considering our initial results (Fig.1), two strains were selected for further characterization: B. ovis 94 AV, which was recoverd in higher numbers in the liver and spleen; and B. ovis 266 L, which had a phenotype similar to the other isolates. Initially, in vitro growth of B. ovis field isolates was compared to that of the reference strain B. ovis ATCC 25840 in TSA medium with 1% hemoglobin. Both field isolates had in vitro growth curves similar to the reference stain. All strains had an exponential growth phase between 24 and 72h of incubation at 37°C with 5% CO2, and then entered the stationary growth phase (Fig.2A). The kinetics of intracellular growth of these strains was then assessed by infecting RAW 264.7 murine macrophage cells. At 0 and 4 hours after infection, significantly higher CFU numbers of B. ovis ATCC 25840 were recovered when compared to the field isolates (p<0.05), with more than one log difference at time 0, indicating that the reference strain B. ovis ATCC 25840 had higher levels of internalization in RAW cells when compared to field strains (Fig.2B). At 24 hours after inoculation, the opposite was observed with significantly higher CFU numbers of field isolates recovered from the intracellular compartment of macrophages (approximately one log difference) when compared to the reference strain (p<0.05), demonstrating that the reference strain underwent a decrease in its intracellular population before it started growing intracellularly, whereas the field isolates, although less invasive, grew steadily from the beginning of the time course. At 48 hours after inoculation, both field isolates were recovered in higher numbers when compared to the previous time points indicating they were all able to survive and grow intracellularly in RAW cells (Fig.2B). These results clearly demonstrated a different kinetics of internalization and intracellular survival between the reference strain and field isolates. Although less invasive, field isolates were able to start multiplying intracellularly at very early time points, when compared to the reference strain, which had an initial decline before start multiplying within macrophages (Fig.2B).

Fig.2. (A) In vitro growth curve of different Brucella ovis strains. B. ovis ATCC 25840, 94 AV, and 266 L were grown on TSA plates with 1% hemoglobin at 37oC with 5% CO2. (B) In vitro infection of murine macrophage cell line RAW 264.7 (MOI 100) with the reference B. ovis strain (ATCC 25840) or field isolates (94 AV and 266 L). Macrophages were grown in 96-well plates and infected with B. ovis strains with a MOI of 1:100. Intracellular bacteria were recovered at 0, 4, 24, and 48hpi. Time zero represents the number of CFUs of bacteria after 1 hour of incubation with gentamycin. Data points represent the mean and standard deviation of two independent experiments performed in triplicates. Data were logarithmically transformedprior to ANOVA, and means were compared by the Tukey’s test. Statistically significant differences are indicated by asterisks (* p<0.05; ** p<0.01). 

Colonization of spleen and liver of mice infected with Brucella ovis field isolates

Considering the differences in intracellular growth, we investigated the kinetics of infection of B. ovis 94 AV and 266 L in the mouse model. BALB/c mice (n=5 per group) were intraperitoneally infected with 106 CFU/mice of the reference strain B. ovis ATCC 25840 or the two field isolates. Mice were sampled at 1, 7, and 30dpi. At 1dpi, bacterial loads in the spleen and liver were significantly higher (nearly 2 log difference) in mice infected with the reference strain (p<0.05), when compared to both field isolates (94 AV and 266 L). At 7dpi, all strains had similar bacterial loads in the spleen (Fig.3A), whereas B. ovis 266 L was recovered in lower numbers from the liver when compared to the reference strain (Fig.3B). At 30dpi, mice challenged with B. ovis 266 L had higher bacterial loads in the spleen and liver (p<0.05) when compared to mice infected with the other field isolate (94 AV) and the reference strain (Fig.3A,B).

Fig.3. Kinetics of Brucella ovis infection in BALB/c mice. Mice were intraperitoneally infected with 1×106CFU of B. ovis ATCC 25840, 94 AV, or 266 L. Samples of (A) spleen and (B) liver were collected for bacterial counts at 1, 7, and 30dpi. Each data point represents the mean and standard deviation (n=5). Data were logarithmically transformedprior to ANOVA, and means were compared by the Tukey’s test. Statistically significant differences are indicated by asterisks (* p<0.05; ** p<0.01). Differences between the groups in the liver at 7dpi are indicated by different letters (p<0.05). 

Histological changes were similar in the liver and spleen from mice infected with different strains. At 1dpi, there were no inflammatory changes, whereas at 7 and 30dpi there were moderate multifocal microgranulomas in the liver and spleen. There were no significant differences in histopathology scores attributed to histological lesions in the spleens and livers from mice infected with different strains.

Immunization with encapsulated Brucella ovis ΔabcBA induces protection of experimentally challenged mice with field isolates

Previous studies demonstrated that the attenuated mutant strain B. ovis ΔabcBA induces protection in mice and in rams (Silva et al. 2015a, 2015b). Here we assessed whether vaccination with B. ovis ΔabcBA protects mice challenged with field isolated B. ovis strains, which is relevant since all previous studies evaluated protection against the reference strain, and in here we demonstrated differences in the kinetics of intracellular and in vivo infection and growth when comparing field isolates with the reference strain. As expected, immunized mice had significant reduction in bacterial loads in the liver and spleen when compared to non-immunized mice (p<0.001). Protection indexes are described in Table 2. Interestingly, protection indexes were higher in mice challenged with field strains, particularly mice challenged with B. ovis 266 L, with reductions in splenic bacterial loads close to 1, 2 or 3 logs of CFU in mice challenged with B. ovis ATCC 25840, B. ovis 94 AV, or B. ovis 266 L, respectively.

Table 2. Protection indexes induced by Brucella ovis ΔabcBA encapsulated with alginate in C57BL/6 mice experimentally challenged with diferente strains of B. ovis 

Challenge strain (1x 106 per mouse) CFU/spleen immunized mice CFU/spleen non-immunized mice Protection index
B. ovis ATCC 25840 4.668 ± 0.383 5.419 ± 0.219 0.751*
B. ovis 266 L 3.786 ± 0.276 6.532 ± 0.649 2.746**
B. ovis 94 AV 4.846 ± 0.599 6.596 ± 0.355 1.736**

* Statistically significant difference (p<0.05), ** statistically significant difference (p<0.01)

Bacterial colonization in the liver was also significantly lower in vaccinated mice, with decreases in bacterial loads in the range of 0.5, 1, and 1 for mice challenged with B. ovis ATCC 25840, B. ovis 94 AV, and B. ovis 266 L, respectively.

Immunized mice challenged with different B. ovis strains did not develop splenomegaly, while non-immunized mice developed evident splenomegaly after infection. In the liver of all non-immunized mice, there were multifocal coalescent firm white nodular lesions of approximately 0.1 to 0.4cm in diameter. In contrast, immunized mice developed less severe lesions (Fig.4). Histologically, non-immunized mice had a moderate to severe, multifocal, inflammatory infiltrate composed of epithelioid macrophages and neutrophils with mild accumulation of fibrin in the marginal zone and red pulp in the spleen, characterizing a moderate to severe, multifocal pyogranulomatous splenitis. Immunized mice had milder similar microscopic lesions. Histopathology scores were significantly lower in immunized mice when compared to non-immunized controls (p<0.01). Histological changes in the liver of nonimmunized mice were characterized by a mild to moderate, multifocal, randomly distributed, inflammatory infiltrated composed of epithelioid macrophages, neutrophils, and lymphocytes, associated with moderate multifocal necrosis and thrombosis. Immunized mice developed only a few mild microgranulomas in the liver (Fig.4). Histopathology scores for hepatic lesions in groups immunized with encapsulated B. ovis ΔabcBA were significantly lower when compared to non-immunized mice (p<0.01) regardless of the challenge strain (Fig.4).

Fig.4. Protection induced by the vaccine strain Brucella ovis ΔabcBA encapsulated with alginate in C57BL/6 mice experimentally challenged with diferente B. ovis strains. (A) Number of of B. ovis CFU recovered from the liver. Each column represents the mean and standard deviation (n=5). Raw data were logarithmically transformed prior to ANOVA, and means were compared by Tukey’s test. Significant differences between bacterial strains are indicated by asterisks (* p<0.05; ** p<0.01; *** p<0.001). (B) Score for lesions in the liver of mice. Means were compared by the Kruskal-Wallis nonparametric test. Representative histological changes in the liver of (C) non-immunized mice with with extensive microgranulomas associated with necrosis; or (D) immunized mice with very mild changes. Mice challenged with B. ovis 266 L. (C,D) HE, bar = 100μm. 

Vaccination sites were initially swollen, but this change regressed significantly until the day of euthanasia. Histopathologically, there were small granulomas at the site of vaccination (data not shown).

Discussion

This study characterized in vivo and in vitro behavior of Brucella ovis field isolates. There were clear differences in pathogenic potential among B. ovis field isolates based on intracellular growth as well as in vivo infection in the mouse model. This is a relevant finding considering the fact that Brucella spp. have little genetic variability (Tsolis 2002). Interestingly, protection indexes induced by the candidate vaccine strain B. ovis ΔabcBA were higher for strains with higher virulence. These results indicate that the vaccine strain protects against different strains of B. ovis, and protection is even more evident against more pathogenic strains, demonstrating a robust immunogenicity of this experimental vaccine formulation.

Variable pathogenicity among field isolates should not be considered unexpected since Brucella, like other bacteria, is able to undergo spontaneous mutations or metabolic adaptations depending on the environmental conditions to which it is exposed, including temperature, humidity, host cell defense, and intracellular environment. Minimal genomic mutations may result in major phenotypic changes affecting survival and virulence of bacteria. For instance, the vaccine strain B. abortus S19, isolated in 1923 from the milk of a Jersey cow (Buck 1930), that after being accidentally left out at room temperature for one year spontaneously developed an attenuated phenotype (Graves 1943). Importantly, there were no previous studies comparing the pathogenicity of B. ovis strains with different genotypes based on MLVA-16 (Dorneles et al. 2014).

All seven B. ovis strains included in this study were directly isolated from the semen of naturally infected rams (Dorneles et al. 2014), indicating that these rams likely had clinical changes and were sources of infection to other sheep within their herds (Burgess 1982). Although there is no information regarding clinical signs associated with these isolates, MLVA16 demonstrated different genotypes (Dorneles et al. 2014). Therefore, in order to assess possible differences in pathogenic potential of these strains, we used the mouse, which has been extensively employed as an infection model for Brucella spp. (Silva et al. 2011a) being a suitable model for B. ovis infection (Silva et al. 2011b). In this study, mice infected with 106 CFU of B. ovis field isolates (100 V, 203 L, 266 L, 204, 286 L, 252 L, and 94 AV) became experimentally infected. All strains were capable of causing lesions in the liver and spleen at 7dpi, although there were significant differences in their ability to multiply intracellularly and colonize and survive in vivo.

B. ovis strains included in this study were isolated directly from the natural host (Dorneles et al. 2014), where the bacteria face harsh intracellular conditions including exposure to reactive oxygen species, low pH, and low nutrient levels. Brucella spp. can adapt to the intracellular environment upon activation of expression of certain virulence factors (Kohler et al. 2002). Different strains of a given Brucella species may exhibit different intracellular kinetics (Harmon et al. 1988, Kohler et al. 2002, Silva et al. 2014). This may explain the differences in intracellular and in vivo survival and multiplication observed in this study, whereas in vitro growth on solid medium was remarkably similar among these isolates. Adaptation and attenuation of Brucella reference strains handled frequently in the laboratory conditions to in vitro and in vivo models is described (Bosseray 1991, Grilló et al. 2012). Although it may warrant different phenotype of reference strain from field isolates, it does not explain in vivo difference between field isolates.

BALB/c and C57BL/6 are suitable models for B. ovis infection since they develop a systemic infection that results in lesions in the liver and spleen. BALB/c mice are more susceptible to B. ovis than C57BL/6 mice, and under experimental conditions, mice do not develop B. ovis-induced genital lesions as observed in the natural host, which makes the mouse a useful model of infection although they do not mimic the natural disease (Silva et al. 2011b). In general, the mouse model is useful for comparing different strains. In this study, strains 94 AV and 266 L were able to colonize the spleen and liver and persist for up to 30 dpi. Virulent Brucella strains are capable of colonizing the liver and spleen of mice and persist for a long period (Silva et al. 2011b, Grilló et al. 2012). Our results indicate that the two field isolates tested were fully virulent since they were capable to establish systemic infection and persist in the mouse, and survive intracellularly in cultured macrophages. Importantly, the field strain B. ovis 266 L had the best fitness both intracellularly in cultured macrophages as well as in vivo in mice.

Recent studies have demonstrated that the candidate vaccine strain B. ovis ΔabcBA induces a protection in mice (Silva et al. 2015a), while it promotes sterile immunity in experimentally challenged rams (Silva et al. 2015b). In mice, higher protection indexes were induced by the vaccine strain B. ovis ΔabcBA in C57BL/6 mice when compared to BALB/c mice (Silva et al. 2015a). This study demonstrated that the vaccine strain B. ovis ΔabcBA also provided protection for mice challenged with different field isolates. Interestingly, the highest protection index was observed in the group challenged with strain 266 L, which had the best adaptation to intracellular survival in cultured macrophages and in vivo colonization and persistence. Importantly, vaccinal protection was not restricted to lower colonization by the virulent strain, but also by prevention of lesions since histopathology scores were lower in the vaccinated mice. These results are quite encouraging since the vaccine strain performed even better when vaccinated mice were challenged with more pathogenic field strains, which likely activate virulence factors more efficiently for adaptation to the host (Kohler et al. 2002). These results also support the dogma in brucellosis vaccinology that live vaccines are more efficient compared to the other vaccine categories (Carvalho et al. 2016). Considering the efficiency of this vaccine strain under experimental conditions, and the absence of a commercially available and specific B. ovis vaccine, this vaccinal protocol may potentially be an efficient tool for preventing reproductive losses caused by B. ovis (Carvalho Júnior et al. 2012, Poester et al. 2013). Furthermore, unlike the B. melitensis Rev1 vaccine strain used in several countries, B. ovis ΔabcBA does not have zoonotic potential, thus eliminating the occupational risks due to accidental human vaccine exposure (Xavier et al. 2009).

Conclusion

In conclusion, there were significant differences in pathogenicity among Brucella ovis field isolates. Importantly, the B. ovis ΔabcBA vaccine strain induced protection against field isolates with protections indexes that were higher for mice challenged with more pathogenic strains.

Acknowledgements

Work in RLS lab is supported by “Conselho Nacional de Desenvolvimento Científico e Tecnológico” (CNPq), “Fundação de Amparo a Pesquisa do Estado de Minas Gerais” (FAPEMIG), and “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior” (CAPES), Brazil. T.A.P. and R.L.S. have fellowships from CNPq.

References

Blasco J.M. 1997. A review of the use of B. melitensis Rev 1 vaccine in adult sheep and goats. Prev. Vet. Med. 31(3-4):275-283. <http://dx.doi.org/10.1016/S0167-5877(96)01110-5> <PMid:9234451> [ Links ]

Blasco J.M. & Díaz R. 1993. Brucella melitensis Rev 1 vaccine as a cause of human brucellosis. Lancet 342(8874):805. <http://dx.doi.org/10.1016/0140-6736(93)91571-3> <PMid:8103891> [ Links ]

Bosseray N. 1991. Brucella melitensis Rev 1 living attenuated vaccine: stability of markers, residual virulence and immunogenicity in mice. Biologicals 19(4):355-363. <http://dx.doi.org/10.1016/S1045-1056(05)80025-9> <PMid:1797046> [ Links ]

Buck J.M. 1930. Studies on vaccination during calfhood to prevent bovine infectious abortion. J. Agric. Res. 41(9):667-689. [ Links ]

Burgess G.W. 1982. Ovine contagious epididymitis: a review. Vet. Microbiol. 7(6):551-575. <http://dx.doi.org/10.1016/0378-1135(82)90049-9> <PMid:6762755> [ Links ]

Carvalho Júnior C.A., Moustacas V.S., Xavier M.N., Costa E.A., Costa L.F., Silva T.M.A., Paixão T.A., Borges A.M., Gouveia A.M.G. & Santos R.L. 2012. Andrological, pathologic, morphometric, and ultrasonographic findings in rams experimentally infected with Brucella ovis. Small Rumin. Res. 102(2/3):213-222. <http://dx.doi.org/10.1016/j.smallrumres.2011.08.004> [ Links ]

Carvalho T.F., Haddad J.P., Paixão T.A. & Santos R.L. 2016. Meta-Analysis and advancement of brucellosis vaccinology. PLos One 11(11):1-28. <http://dx.doi.org/10.1371/journal.pone.0166582> <PMid:27846274> [ Links ]

Celli J., de Chastellier C., Franchini D.M., Pizarro-Cerda J., Moreno E. & Gorvel J.P. 2003. Brucella evades macrophage killing via VirB-dependent sustained interactions with the endoplasmic reticulum. J. Exp. Med. 198(4):545-556. <http://dx.doi.org/10.1084/jem.20030088> <PMid:12925673> [ Links ]

Chain P.S.G., Comerci D.J., Tolmasky M.E., Larimer F.W., Malfatti S.A., Vergez L.M., Aguero F., Land M.L., Ugalde R.A. & Garcia E. 2005. Whole-genome analyses of speciation events in pathogenic Brucellae. Infect. Immun. 73(12):8353-8361. <http://dx.doi.org/10.1128/IAI.73.12.8353-8361.2005> <PMid:16299333> [ Links ]

Danese I., Haine V., Delrue R., Lestrate P., Stevaux O., Mertens P., Paquet J.Y., Godfroid J., De Bolle X. & Letesson J.J. 2004. The Ton system, an ABC transporter, and a universally conserved GTPase are involved in iron utilization by Brucella melitensis 16M. Infect. Immun. 72(10):5783-5790. <http://dx.doi.org/10.1128/IAI.72.10.5783-5790.2004> <PMid:15385478> [ Links ]

Detmers F.J.M., Lanfermeijer F.C. & Poolman B. 2001. Peptides and ATP binding cassette peptide transportes. Res. Microbiol. 152(3-4):245-258. <http://dx.doi.org/10.1016/S0923-2508(01)01196-2> <PMid:11421272> [ Links ]

Dorneles E.M.S., Freire G.N., Dasso M.G., Poester F.P. & Lage A.P. 2014. Genetic diversity of Brucella ovis isolates from Rio Grande do Sul, Brazil, by MLVA16. BMC Res. Notes 7(1):447. <http://dx.doi.org/10.1186/1756-0500-7-447> <PMid:25015223> [ Links ]

Graves R.R. 1943. The story of John M. Buck’s and Matilda’s contribution to the cattle industry. J. Am. Vet. Med. Assoc. 102:193-195. [ Links ]

Grilló M.J., Marín C.M., Barberán M. & Blasco J.M. 1999. Experimental Brucella ovis infections in pregnant ewes. Vet. Rec. 144(20):555-558. <http://dx.doi.org/10.1136/vr.144.20.555> <PMid:10371013> [ Links ]

Grilló M.J., Blasco J.M., Gorvel J.P., Moriyón I. & Moreno E. 2012. What we have to learn from bruccelosis in the mouse model? Vet. Res. 43(1):1-72. <http://dx.doi.org/10.1186/1297-9716-43-29> <PMid:22500859> [ Links ]

Gorvel J.P. & Moreno E. 2002. Brucella intracellular life: from invasion to intracellular multiplication. Vet. Microbiol. 90(1-4):281-297. <http://dx.doi.org/10.1016/S0378-1135(02)00214-6> <PMid:12414149> [ Links ]

Harmon B.G., Adams L.G. & Frey M. 1988. Survival of rough and smooth strains of Brucella abortus in bovine mammary gland macrophages. Am. J. Vet. Res. 49(7):1092-1097. <PMid:3138931> [ Links ]

Hosie A.H.F. & Poole P.S. 2001. Bacterial ABC transporters of amino acids. Res. Microbiol. 152(3-4):259-270. <http://dx.doi.org/10.1016/S0923-2508(01)01197-4> <PMid:11421273> [ Links ]

Igarashi K., Ito K. & Kashiwagi K. 2001. Polyamine uptake systems in Escherichia coli. Res. Microbiol. 152(3-4):271-278. <http://dx.doi.org/10.1016/S0923-2508(01)01198-6> <PMid:11421274> [ Links ]

Jenner D.C., Dassa E., Whatmore A.M. & Atkins H.S. 2009. ATP binding cassette systems of Brucella. Comp. Funct. Genomics 2009:1-17. <http://dx.doi.org/10.1155/2009/354649> <PMid:20169092> [ Links ]

Kohler S., Foulongne V., Ouahrani-Bettache S., Bourg G., Teyssier J., Ramuz M. & Liautard J.P. 2002. The analysis of the intramacrophagic virulome of Brucella suis deciphers the environment encountered by the pathogen inside the macrophage host cell. Proc. Natl. Acad. Sci. 99(24):15711-15716. <http://dx.doi.org/10.1073/pnas.232454299> <PMid:12438693> [ Links ]

Macedo A.A., Silva A.P., Mol J.P., Costa L.F., Garcia L.N., Araújo M.S., Martins Filho O.A., Paixão T.A. & Santos R.L. 2015. The abcEDCBA-encoded ABC transporter and the virB operon-encoded type IV secretion system of Brucella ovis are critical for intracellular trafficking and survival in ovine monocyte-derived macrophages. PLoS One 10(9):1-23. <http://dx.doi.org/10.1371/journal.pone.0138131> <PMid:26366863> [ Links ]

OIE. 2015. Ovine epididymitis (Brucella ovis), p.1467-1479. In: Ibid (Ed), Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. OIE, Paris. Available at <Available at http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/3.07.07_OVINE_EPID.pdf > Access on January 17, 2020. [ Links ]

Olsen S.C., Thoen C.O. & Cheville N.F. 2011. Brucella, p.429-438. In: Gyles C.L., Prescott F.J., Songer G.J. & Thoen C.O. (Eds), Pathogenesis of Bacterial Infections in Animals. Wiley-blackwell, Iowa. [ Links ]

Poester F.P., Samartino L.E. & Santos R.L. 2013. Pathogenesis and pathobiology of brucellosis in livestock. Rev. Sci. Tech. 32(1):105-115. <http://dx.doi.org/10.20506/rst.32.1.2193> <PMid:23837369> [ Links ]

Ridler A.L. & West D.M. 2011. Control of Brucella ovis infection in sheep. Vet. Clin. N. Am. Food Anim. Pract. 27(1):61-66. <http://dx.doi.org/10.1016/j.cvfa.2010.10.013> <PMid:21215890> [ Links ]

Sá J.C., Silva T.M., Costa E.A., Silva A.P., Tsolis R.M., Paixão T.A., Carvalho Neta A.V. & Santos R.L. 2012. The virB-encoded type IV secretion system is critical for establishment of infection and persistence of Brucella ovis infection in mice. Vet. Microbiol. 159(1-2):130-140. <http://dx.doi.org/10.1016/j.vetmic.2012.03.029> <PMid:22483850> [ Links ]

Silva A.P.C., Macêdo A.A., Silva T.M., Ximenes L.C., Brandão H.M., Paixão T.A. & Santos R.L. 2015a. Protection provided by an encapsulated live attenuated ΔabcBA strain of Brucella ovis against experimental challenge in a murine model. Clin. Vaccine Immunol. 22(7):789-797. <http://dx.doi.org/10.1128/CVI.00191-15> <PMid:25947146> [ Links ]

Silva A.P.C., Macêdo A.A., Costa L.F., Rocha C.E., Garcia L.N., Farias J.R., Gomes P.P., Teixeira G.C., Fonseca K.W., Maia A.R., Neves G.G., Romão E.L., Silva T.M., Mol J.P., Oliveira R.M., Araújo M.S., Nascimento E.F., Martins-Filho O.A., Brandão H.M., Paixão T.A. & Santos R.L. 2015b. Encapsulated Brucella ovis lacking a putative ATP-binding cassette transporter (ΔabcBA) protects against wild type Brucella ovis in Rams. PLoS One 10(8):1-23. <http://dx.doi.org/10.1371/journal.pone.0136865> <PMid:26317399> [ Links ]

Silva T.M.A., Costa E.A., Paixão T.A., Tsolis R.M. & Santos R.L. 2011a. Laboratory animal models for brucellosis research. J. Biomed. Biotechnol. 2011:518323. <http://dx.doi.org/10.1155/2011/518323> <PMid:21403904> [ Links ]

Silva T.M.A., Paixão T.A., Costa E.A., Xavier M.N., Sá J.C., Moustacas V.S., den Hartigh A.B., Carvalho Neta A.V., Oliveira S.C., Tsolis R. & Santos R.L. 2011b. Putative ATP-Binding cassette transporter is essential for Brucella ovis pathogenesis in mice. Infect. Immun. 79(4):1706-1717. <http://dx.doi.org/10.1128/IAI.01109-10> <PMid:21300772> [ Links ]

Silva A.P., Macêdo A.A., Costa L.F., Turchetti A.P., Bull V., Pessoa M.S., Araújo M.S., Nascimento E.F., Martins-Filho O.A., Paixão T.A. & Santos R.L. 2013. Brucella ovis lacking a species-specific putative ATP-binding cassette transporter is attenuated but immunogenic in rams. Vet. Microbiol. 167(3-4):546-553. <http://dx.doi.org/10.1016/j.vetmic.2013.09.003> <PMid:24075357> [ Links ]

Silva T.M.A., Mol J.P.S., Winter M.G., Atluri V., Xavier M.N., Pires S.F., Paixão T.A., Andrade H.M., Santos R.L. & Tsolis R.M. 2014. The predicted ABC transporter AbcEDCBA is required for type IV secretion system expression and lysosomal evasion by Brucella ovis. Plos One 9(12):1-27. <http://dx.doi.org/10.1371/journal.pone.0114532> <PMid:25474545> [ Links ]

Tsolis R.M. 2002. Comparative genome analysis of the alpha-proteobacteria: relationships between plant and animal pathogens and host specificity. Proc. Natl. Acad. Sci. USA 99(20):12503-12505. <http://dx.doi.org/10.1073/pnas.212508599> <PMid:12271145> [ Links ]

Tsolis R.M., Seshadri R., Santos R.L., Sangari F.J., Lobo J.M., de Jong M.F., Ren Q., Myers G., Brinkac L.M., Nelson W.C., Deboy R.T., Angiuoli S., Khouri H., Dimitrov G., Robinson J.R., Mulligan S., Walker R.L., Elzer P.E., Hassan K.A. & Paulsen I.T. 2009. Genome degradation in Brucella ovis corresponds with narrowing of its host range and tissue tropism. PLoS One 4(5):1-28. <http://dx.doi.org/10.1371/journal.pone.0005519> <PMid:19436743> [ Links ]

Verger J.M., Grimont F., Grimont P.A.D. & Grayon M. 1985. Brucella, a monospecific genus as shown by deoxyribonucleic acid hybridization. Int. J. Syst. Evol. Microbiol. 35(3):292-295. [ Links ]

Whatmore A.M. 2009. Current understanding of the genetic diversity of Brucella, an expanding genus of zoonotic pathogens. Infect. Genet. Evol. 9(6):1168-1184. <http://dx.doi.org/10.1016/j.meegid.2009.07.001> <PMid:19628055> [ Links ]

Xavier M.N., Paixão T.A., Poester F.P., Lage A.P. & Santos R.L. 2009. Pathological, Immunohistochemical and Bacteriological Study of Tissues and Milk of Cows and Fetuses Experimentally Infected with Brucella abortus. J. Comp. Pathol. 140(2-3):149-15. <http://dx.doi.org/10.1016/j.jcpa.2008.10.004> <PMid:19111839> [ Links ]

Received: August 13, 2019; Accepted: September 01, 2019

*Corresponding author: rls@ufmg.br

Conflict of interest statement.- The authors have no conflicts of interest to declare.

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