Monocyte-derived macrophages from Zebu (Bos taurus indicus) are more efficient to control Brucella abortus intracellular survival than macrophages from European cattle (Bos taurus taurus)

Veterinary Immunology and Immunopathology 151 (2013) 294–302

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Monocyte-derived macrophages from Zebu (Bos taurus indicus) are more efficient to control Brucella abortus intracellular survival than macrophages from European cattle (Bos taurus taurus) A.A. Macedo a , E.A. Costa a , A.P.C. Silva a , T.A. Paixão b , R.L. Santos a,∗ a b

Departamento de Clínica e Cirurgia Veterinárias, Escola de Veterinária, Universidade Federal de Minas Gerais, Brazil Departamento de Patologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Brazil

a r t i c l e

i n f o

Article history: Received 12 November 2012 Received in revised form 1 December 2012 Accepted 3 December 2012 Keywords: Brucella abortus Cattle Macrophage Innate immunity

a b s t r a c t Brucellosis is one of the most important zoonotic diseases in the world. Considering its strict zoonotic nature, understanding of the pathogenesis and immunity of Brucella spp. in natural animal hosts is essential to prevent human infections. Natural resistance against brucellosis has been demonstrated in cattle, and it is associated with the ability of macrophages to prevent intracellular replication of Brucella abortus. Identification of breeds that are resistant to B. abortus may contribute for controlling and eradicating brucellosis in cattle. This study aimed to compare macrophages from Nelore (Bos taurus indicus) or Holstein (Bos taurus taurus) regarding their resistance to B. abortus infection. Macrophages from Nelore were significantly more efficient in controlling intracellular growth of B. abortus when compared to Holstein macrophages even under intralysosomal iron restricting conditions. Furthermore, Nelore macrophages had higher transcription levels of inducible nitric oxide synthase (iNOS) and TNF-␣ at 12 h post-infection (hpi) and higher levels of IL-12 at 24 hpi when compared to Holstein macrophages. Conversely, Holstein macrophages had higher levels of IL-10 transcripts at 24 hpi. Macrohages from Nelore also generated more nitric oxide (NO) in response to B. abortus infection when compared to Holstein macrophages. In conclusion, cultured Nelore macrophages are more effective in controlling intracellular replication of B. abortus, suggesting that Nelore cattle is likely to have a higher degree of natural resistance to brucellosis than Holstein. © 2012 Elsevier B.V. All rights reserved.

1. Introduction

Abbreviations: cDNA, complementary DNA; Ct , threshold cycle; DFO, desferrioxamine; DHBA, dihydroxybenzoic acid; hpi, hours postinfection; IL, interleukin; iNOS, inducible nitric oxide synthase; LAMP, lysosomal-associated membrane protein; Nramp, Protein Associated with Macrophage Natural Resistance; NO, oxide nitric; RT-qPCR, reverse transcription-quantitative real-time polymerase chain reaction; TSA, tryptic soy agar. ∗ Corresponding author at: Departamento de Clínica e Cirurgia Veterinária, Escola de Veterinária, Universidade Federal de Minas Gerais, Av. Presidente Antônio Carlos, 6627 – CEP 30161-970, Belo Horizonte, MG, Brazil. Tel.: +55 31 3409 2239; fax: +55 31 3409 2230. E-mail address: [email protected] (R.L. Santos). 0165-2427/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetimm.2012.12.001

Brucellosis is a globally relevant zoonosis that is caused by intracellular pathogens of the genus Brucella (Young, 1983). B. abortus is the most important etiological agent of bovine brucellosis, but this organism can also infect buffalo, camels, deer, horses, goats, sheep, and man (Enright et al., 1984; Kudi et al., 1997). Brucellosis in cattle is associated with decreased milk production, increased number of somatic cells in milk, abortion at the middle or late gestation, birth of weak calves and postpartum metritis (Emminger and Schalm, 1943; Xavier et al., 2009). In bulls, B. abortus may cause orchitis that is often associated with epididymitis and vesiculitis (Eaglesome and Garcia, 1992).

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Natural resistance can be defined as the ability of the host to avoid the development of disease upon infection without previous exposure to the infectious agent (Templeton et al., 1988). In brucellosis, several factors including the number of cell surface receptors involved in recognition of molecular patterns associated with Brucella, maturation and activation of macrophages and dendritic cells, antimicrobial peptides, and cytokine production may influence natural resistance (Adams and Schutta, 2010). In this context, the ability of macrophages to prevent intracellular growth of B. abortus has been extensively used as an indicator of the resistant phenotype against brucellosis (Campbell and Adams, 1992; Qureshi et al., 1996; Martínez et al., 2010; Rossetti et al., 2011). Natural resistance in cattle has been associated with the natural resistance-associated macrophage protein 1 (Nramp1), which is a divalent cation transporter that is thought to decrease availability of iron in the phagolysosome (Forbes and Gros, 2003). Thus, in this study we employed a dhbC mutant strain that is defective in iron acquisition (Bellaire et al., 2003a). Previous studies have comparatively addressed natural resistance in Zebu (Bos taurus indicus) and European (Bos taurus taurus) breeds. Thus, it has been demonstrated that Zebu cattle are more resistant to babesiosis (Parker et al., 1985; Bock et al., 1999), ticks (Rechav and Kostrzewski, ˜ et al., 1991; Wambura et al., 1998), and nematodes (Pena 2000), when compared to European breeds. Aditionally, the Zebu breed Nelore is more resistant to Rhipicephalus (Boophilus) microplus and Babesia bovis when compared to other Zebu breeds (Utech et al., 1978). Possible differences in resistance against brucellosis in these two subspecies have not been reported, which prompted us to perform a comparative evaluation of intracellular survivial of B. abortus in macrophages from these cattle. Control of bovine brucellosis is based on vaccination, serologic surveys, culling of infected cattle, and stringent sanitary management. However, these measures combined may not be sufficient for eradicating the disease (Rogers et al., 1989; Martínez et al., 2010). Furthermore, several studies attempted to identify genetic markers of resistance against bovine brucellosis, but results are controversial. The usefulness of putative genotypic markers of resistance against bovine brucellosis remains unconclusive (Adams and Templeton, 1998; Barthel et al., 2001; Paixão et al., 2007, 2012; Martínez et al., 2010). Considering that brucellosis is one of the most important zoonotic diseases in the world as well as its strict zoonotic nature, understanding of the pathogenesis and immunity of Brucella spp. in natural animal hosts is essential to prevent human infections. Therefore, the aim of this study was to investigate natural resistance against B. abortus in European (Holstein) and Zebu (Nelore) breeds. 2. Materials and methods

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the siderophore 2,3-dihydroxybenzoic acid (2,3-DHBA) in response to iron limitation (Bellaire et al., 2003a,b). Briefly, the coding region of the operon dhbCEBA (Gene Bank accession number AF302798) was initially amplified by polymerase chain reaction (PCR) and inserted into pCR2.1TOPO-TA vector using TOPO cloning kit (Invitrogen, Brazil). The insert was removed from the TOPO-TA by double digestion, using the enzymes BamHI and XhoI, and cloned into the vector pBluescript KS. A 381 bp internal fragment of the coding region dhbC was replaced by a kanamycin resistance gene KIXX (1.6 kb) digestion from pUC4-KIXX (Amersham Pharmacia Biotech, USA) with SmaI, followed by pBluescript vector digested with the enzyme EcoRV. The plasmid generated were transformed into Escherichia coli, extracted by a commercial kit (plasmid midi-kit; Qiagen, Brazil), and transformed into electrocompetent B. abortus 2308 by electroporation as previously described (Tatum et al., 1992). Immediately after electroporation, 1 mL of medium SOCB (2% tryptona, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2 , 10 mM MgSO4 and 20 mM glucose) was added and incubated at 37 ◦ C for 16 h under agitation. Then, 100 ␮L of each sample was plated on tryptic soy agar (TSA) (Difco, Invitrogen, Brazil) with 100 ␮g/mL of kanamycin (Gibco, Invitrogen, Brazil) and incubated for 4–7 days at 37 ◦ C and 5% CO2 . Colonies that grew on TSA plate were recovered, suspended in 100 ␮L of sterile phosphate-buffered saline (PBS) and plated on TSA with kanamycin or TSA with 200 ␮g/mL of ampicillin (Gibco, Invitrogen, Brazil) and incubated for 4–7 days at 37 ◦ C and 5% CO2 . Finally, mutant colonies that were resistant to kanamycin and sensitive to ampicillin were selected and the mutation was confirmed by PCR. Both parental and mutant strains were grown on TSA for 72 h at 37 ◦ C in an atmosphere of 5% CO2 . Bacterial suspensions were adjusted to a given final concentration by spectrophotometry. All procedures involving live B. abortus were carried out in a biosafety level 3 laboratory. 2.2. Animals Monocytes were isolated from peripheral blood of 16–18-month-old Nelore and Holstein bulls. A total of 14 cattle (n = 7 for each breed) was used for assessing intracellular survival, whereas a total of 20 cattle (n = 10 for each breed) was used for evaluation of transcription, and 12 cattle (n = 6 for each breed) were used for evaluation of nictic oxide production. Considering that our experimental protocol required non vaccinated cattle, we elected to use males instead of females since vaccination of female calves is mandatory in the area of this study. These cattle were offspring of distinct sires and dams, and they were all from Brucella-free herds. This experiment was approved by the Ethics Committee on Animal Experimentation of Universidade Federal de Minas Gerais (CETEA/UFMG, protocol 143/2010).

2.1. Bacterial strains and culture conditions B. abortus strain 2308 was used in all experiments in this study. A B. abortus mutant strain was generated through deletion of the dhbC gene, thus inactivating the dhbCEBA operon that is responsible for biosynthesis of

2.3. Monocyte-derived macrophage isolation, culture, and infection The protocol used for monocyte isolation has been previously (Campbell and Adams, 1992). Monocytes

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were isolated from Nelore (n = 7) and Holstein (n = 7) bulls. Blood (52 mL) was collected from the jugular vein into a 60 mL syringe containing 8 mL anticoagulant (acid–citrate–dextrose). Blood samples were diluted in an equal volume of PBS (pH 7.4) containing 13 mM sodium citrate (PBS–citrate), and layered over a Percoll (GE Healthcare, Sweden) column with a specific density of 1.077 (mixture of the following solution: 10:1 Percoll, 1.5 M NaCl in 12% NaH2 PO4 , 130 mM trisodium citrate, 5% bovine serum albumin, and PBS [adjusted for a final refractive index of 1.3460]). After centrifugation at 1000 × g for 20 min, interface mononuclear cells were transferred to a clean polypropylene tube. Cells were washed three times in PBS–citrate and resuspended in 8 mL of RPMI medium (Gibco, Invitrogen, Brazil) supplemented with 4 mM lglutamine (Gibco, Invitrogen, Brazil), 1 mM nonessential aminoacids (Gibco, Invitrogen, Brazil), 1 mM sodium pyruvate (Gibco, Invitrogen, Brazil), 2.9 mM 7.5% sodium bicarbonate (Gibco, Invitrogen, Brazil), and 4% inactivated fetal bovine serum (FBS) (Gibco, Invitrogen, Brazil). Cell suspensions were transferred into 50-mL Teflon Erlenmeyer flasks (Nalgene Company, USA), and incubated at 37 ◦ C with 5% CO2 for 24 h. Nonadherent cells were removed by replacing the culture medium with 8 mL of supplemented RPMI medium with 12.5% inactivated FBS per flask, which were incubated at 37 ◦ C in 5% CO2 for additional 10 days, changing the medium every 3 days. After 11 days in culture, monocyte-derived macrophages were resuspended by chilling the Teflon flasks on ice for 20–30 min followed by agitation. Viable cells were counted in a hemocytometer chamber to trypan blue exclusion and resuspended to a concentration of 5 × 105 cells/ml in supplemented RPMI medium with 12.5% inactivated FBS, and 5 × 104 cells per well were seeded in triplicates into 96-well plates and incubated overnight at 37 ◦ C in 5% CO2 . Inocula were quantified by spectrophotometer and diluted to a final concentration of 5 × 107 colony-forming units (CFU) of B. abortus strain 2308 per mL. Medium from each well was replaced with 100 ␮l of this suspension (multiplicity of infection of 100:1). Plates were then centrifuged for 5 min and incubated for 30 min at 37 ◦ C in 5% CO2 . Cells were washed once with medium, 100 ␮L of a 50 ␮g/mL solution of gentamicin (Gibco, Invitrogen, Brazil) in supplemented RPMI with 12.5% inactivated FBS was added to each well, and incubated for 1 h (37 ◦ C in 5% CO2 ) to kill extracellular bacteria. Macrophages were then washed twice with sterile PBS and lysed with 100 ␮L of 0.01% Triton X-100 or incubated for further 24 or 48 with supplemented RPMI medium with 12.5% inactivated containing 25 ␮g/mL of gentamicin and lysed at these time points. Ten fold serial dilutions were plated on TSA to verify bacterial counts. CFU numbers obtained at 1 (considered time 0), 24, or 48 h post-infection (hpi) were compared between two breeds. The concentration of inocula was confirmed by plating. The inocula were also incubated with medium containing gentamicin for 1 h to confirm the activity of the antibiotic. 2.4. Depletion of intralysosomal iron To verify whether the intracellular survival of B. abortus is dependent on the availability of intralysosomal

iron, in vitro infection experiments as described above were repeated under conditions of intralysosomal iron depletion. Macrophages were treated with 1 mM desferrioxamine (DFO) (Sigma–Aldrich, USA), which is a lysossomotropic iron chelator (Persson et al., 2003), for 1 h prior to inoculation with B. abortus. Macrophages and B. abortus were cultured separately in medium containing DFO to ensure that the compound had no toxicity to either the macrophages or the bacteria.

2.5. Kinectics of B. abortus dhbC in Zebu and European macrophages To further characterize iron acquisition B. abortus within phagolysosomes of macrophages from Zebu and European cattle the dhbC mutant strain was used for macrophage infections with an experimental design similar to that described above for the wild type strain. Ten fold serial dilutions were plated on TSA with 100 ␮g/mL of kanamycin to ensure the resistance phenotype of the mutant.

2.6. Total RNA extraction, complementary DNA (cDNA) synthesis, and reverse transcription quantitative PCR (RT-qPCR) To analyze the transcription of pro-inflammatory and anti-inflammatory cytokines during B. abortus infection, macrophages from Nelore (n = 10) and Holstein (n = 10) cattle were seeded in 6-well plates at a concentration of 1 × 106 macrophages per well under the same conditions described above and inoculated with B. abortus strain 2308 (multiplicity of infection of 100:1). Total RNA was extracted at 12 and 24 hpi using the Trizol reagent (Invitrogen, USA), according to the manufacturer’s recommendations, and stored at −80 ◦ C until cDNA synthesis. Purity and quantity of RNA were assessed spectrophotometrically and by electrophoresis in 1% agarose gel/formaldehyde. For cDNA synthesis, 1 ␮g of RNA was used with a commercial kit (SuperScript First-Strand Synthesis System for RT-qPCR; Invitrogen, USA) following the manufacturer’s instruction. cDNA was stored at −20 ◦ C until further processing. RT-qPCR, was performed with 2.5 ␮L of cDNA, 1 ␮L of foward and 1 ␮L reverse primers, both at a concentration of 10 mM and 12.5 ␮L of Platinum SYBR Green qPCR Supermix (Platinum SYBR Green qPCR Supermix – UDG with ROX, Invitrogen) in a final volume of 25 ␮L per reaction. Cycling parameters were 50 ◦ C for 2 min, 95 ◦ C for 10 min, 40 cycles of 95 ◦ C for 15 s and 60 ◦ C for 1 min using an ABI 7500 thermal cycler (Applied Biosystems, USA). Data were analyzed by the comparative Ct (threshold cycle) method, as described by Livak and Schmittgen (2001). Transcript levels were calculated from Ct values normalized to the reference gene GAPDH (Ct = Ct GAPDH − Ct cytokine ). Transcript levels relative to uninfected controls of Nramp1, inducible nitric oxide synthase (iNOS), tumor necrosis factor ␣ (TNF-␣), interleukin 10 (IL-10), IL-12, and IL-4 were calculated using the Ct method and then compared between Zebu and European breeds. Primers used in this study are described in Table 1.

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Table 1 Primers used for RTqPCR. Genea

Primers

Gene Bank accession number

Nramp1

F: 5 -AAGATCCCCATTCCGGATAC-3 R: 5 -AGCCTGAAGATCCGACTCAA-3

NM 174652.2

iNOS

F: 5 - AGCGGAGTGACTTTCCAAGA-3 R: 5 - TTTTGGGGTTCATGATGGAT-3

NM 001076799.1

TNF␣

F: 5 -AACATCCTGTCTGCCATCAAG-3 R: 5 -GGAAGACTCCTCCCTGGTAGAT-3

EU276079.1

IL-12

F: 5 -CAAAAAGGAAGATGGAATTTGG-3 R: 5 -CCAGAATAATCCTTTGCCTCAC-3

EU276076.1

IL-10

F: 5 - CCAAGCCTTGTCGGAAATGA-3 R: 5 - GTTCACGTGCTCCTTGATGTCA-3

EU276074.1

IL-4

F: 5 -TTGGAATTGAGCTTAGGCGTAT-3 R: 5 -CCAAGAGGTCTTTCAGCGTACT-3

EU276069.1

GAPDH

F: 5 -ATGGTGAAGGTCGGAGTGAACG-3 R: 5 -TGTAGTGAAGGTCAATGAAGGGGTC-3

NM 001034034.1

a Nramp1: Natural Resistance Associated Macrophage Protein 1; iNOS: inducible nitric oxide synthase; TNF-␣: Tumor Necrose Factor Alpha; IL-12: Interleukin 12; IL-10: Interleukin 10; IL-4: Interleukin 4; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.

2.7. Measurement of oxide nitric (NO) production

2.9. Statistical analysis

Production of NO in macrophages from both breeds during B. abortus infection was evaluated based on accumulation of nitrite (NO2 − ) in the supernatant, a stable end product of NO. NO2 was detected using the Griess reagent (Promega, USA) according to the manufacturer’s recommendations. Macrophages from Nelore (n = 6) and Holstein (n = 6) were seeded in 96-well plates and infected under the same conditions as described above. Supernatant was collected at 0, 6, 12, and 24 hpi. Samples were stored at −80 ◦ C until further processing. Heat-killed (HK) Salmonella enterica serotype Typhimurium (S. Typhimurium) was included in this experiment as a positive control.

CFU data underwent logarithmic transformation prior to analysis of variance (ANOVA). Means were compared by Tukey’s test (Graphpad Prism 5.0, USA). Normalized Ct values were analyzed according to 2−Ct and the data obtained were logarithmically transformed and then subjected to ANOVA, and means were compared between groups by Student’s t-test (Graphpad Prism 5.0, USA). Differences NO levels were analyzed by Tukey’s test (Graphpad Prism 5.0, USA). All values were considered significant when p < 0.05. All experiments were performed in triplicates.

2.8. Transmission electron microscopy (TEM) Macrophages from one Nelore and one Holstein were seeded in a 24-well plate and infected under the same conditions as described above. At 48 hpi the supernatant was replaced by a solution of 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2), and incubated at room temperature for 24 h. Cells were then removed and centrifuged at 1000 × g for 10 min. The precipitate was resuspended in 0.1 M cacodylate buffer, pH 7.2 and processed for TEM. Briefly, cells were rinsed with 0.1 M cacodylate buffer, and post-fixed with a 1% osmium tetroxide and 0.8% potassium ferrocyanide solution for 1 h. Cells were then rinsed in 0.1 M cacodylate buffer and dehydrated in increasing concentrations of acetone (i.e. 50%, 70%, 90%, and 100% twice). The cells were slowly impregnated in Epon resin for 12 h diluted 1:1 with 100% acetone, and then embedded in 100% Epon resin for 6 h, and polymerized at 60 ◦ C for 48 h. Ultrathin sections were cut using an ultramicrotome (Leica EM UC6, Austria), contrasted with 5% uranyl acetate and lead citrate, and examined under a transmission electron microscope (Tecnai G2-12-120 kV – FEI SpiritBiotwin, USA).

3. Results 3.1. Macrophages from Holstein are more permissive to B. abortus replication than macrophages from Nelore independently of iron availability CFU numbers of B. abortus recovered at 0 hpi was similar and decreased by 24 hpi in macrophages from both breeds (i.e. Nelore and Holstein). These results indicated that internalization and initial intracellular control of B. abortus is similar in both breeds. However, at 48 hpi CFU numbers of B. abortus were significantly higher within macrophages from Holstein when compared to Nelore macrophages (P < 0.0001) (Fig. 1A). Considering that natural resistance has been linked to Nramp1, which is a divalent cation transporter that is thought to favor intracellular killing of bacteria by transporting iron from the vacuole into the cytosol (Forbes and Gros, 2003), we performed experiments under conditions of defective iron acquisition by the bacteria (i.e. dhbC B. abortus) and intralysosomal iron deprivation. The dhbC mutant strain was internalized at similar levels as the wild type parental strain in both breeds, but it was significantly attenuated in macrophages from both breeds when compared to the parental wild type B. abortus strain 2308 (P < 0.0001) at 24 and 48 hpi (Fig. 1A). Importantly, Nelore macrophages

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Fig. 2. Transmission electron micrographs of Brucella abortus-infected monocyte-derived bovine macrophages from Holstein (A) and Nelore (B) cattle at 48 h post infection. Bacteria are located in membrane bound vacuoles with an electron dense vacuolar membrane, and digitiform projections of the vacuolar membrane into the vacuole. Magnifications are indicated in the right bottom side.

Fig. 1. Internalization and intracellular survival of wild type Brucella abortus (strain 2308) and B. abortus dhbC in bovine peripheral blood monocyte-derived macrophages with or without iron intralysosomal chelation: Hostein (Bos taurus taurus) and Nelore (Bos taurus indicus) macrophages were challenged with wild type or dhbC B. abortus under standard culture conditions (A) or under iron deprivation (B and C). CFU data underwent logarithmic transformation prior to analysis of variance (ANOVA). Means were compared by Tukey’s test. Data points represent mean and standard error (n = 7). *Indicates statistically significant differences between different treatments in the same breed (P < 0.0001). # Indicates statistically significant differences between different breeds in the same treatment (P < 0.0001).

were more effective in controlling replication of the dhbC mutant strain at 24 hpi (P < 0.01) and 48 hpi (P < 0.0001) when compared to Holstein macrophages (Fig. 1A). Additionally, under intralysosomal iron chelation conditions, B. abortus intracellular survival decreased in macrophages of both breeds at 48 hpi (P < 0.001), although Nelore macrophages were more effective for controlling intracellular bacterial replication at 48 hpi (P < 0.001) when compared to Holstein (Fig. 1B). As expedted, the dhbC strain also had a decreased capacity to survive within macrophages with intralysosomal iron depletion at 24 and 48 hpi (P < 0.01) in Nelore macrophages when to compared to macrophages from Holstein (Fig. 1C). Ultrastructural analysis of infected bovine macrophages revealed that B. abortus was located within membrane bound vacuoles (Fig. 2A), and most vacuoles at 48 hpi had a vacuolar membrane that was noticeably electon dense (Fig. 2B). Importantly, no ultrastructural differences were observed when macrophages from Nelore and Holstein were compared.

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Fig. 4. Production of NO by bovine monocyte-derived macrophages infected with Brucella abortus. Nitrite concentrations were determined using the Griess reagent in the culture supernatants from cultured Nellore (n = 6) or Hostein (n = 6) macrophages either uninfected negative controls (ni), or inoculated with B. abortus. Heat-killed Salmonella enterica serotype Typhimurium was used as positive control. Columns represent means and standard deviations. Statistically significant differences between Nellore and Hostein are indicated by asterisks (*P < 0.01, **P < 0.001).

3.3. B. abortus-infected macrophages from Nelore generate higher levels of NO when compared to Holstein macrophages

Fig. 3. Relative changes in transcript levels of Nramp1, iNOS, TNF-␣, IL10, IL-12, and IL-4 in monocyte-derived macrophages from Nelore (n = 10) and Hostein (n = 10) cattle infected with Brucella abortus at 12 (A) or 24 hpi (B) compared to uninfected controls. Columns represent geometric mean and standard error. Data underwent logarithmic transformation prior to analysis of variance. Means were compared by Student’s t-test. Statistically significant differences between Nelore and Hostein are indicated by asterisks (**P < 0.001, ***P < 0.0001).

3.2. Transcription profile of macrophages from Nelore differ from Holstein macrophages during B. abortus infection Transcription of proinflammatory and antiinflammatory cytokines was evaluated using total RNA extracted from infected macrophages from Nelore or Holstein at 12 and 24 hpi. B. abortus-infected Nelore macrophages had higher levels of iNOS and TNF-␣ transcripts when compared to Hostein macrophages (P < 0.0001) at 12 hpi (Fig. 3A). In contrast, transcription levels of IL-4 were higher in Holstein macrophages when compared to Nelore macrophages (P < 0.001) at 12 hpi (Fig. 3A). At 24 hpi, Holstein macrophages had higher levels of IL-10 transcripts when compared to Nelore macrophages (P < 0.001), whereas levels of IL-12 transcripts were significantly higher in B. abortus-infected Nelore macrophages (P < 0.0001) (Fig. 3B). Similar levels of Nramp1 transcripts were observed in both breeds at 24 and 48 hpi. Together theses results demonstred that B. abortus infection induced higher levels of transcription of pro-inflammatory cytokines, which should favor control of infection by the host, in B. abortus-infected Nelore macrophages, whereas B. abortus-infected Holstein macrophages had higher transcription levels of anti-inflammatory cytokines, thus favoring infection.

To further characterize mechanisms that may be associated with controlling intracellular replication of B. abortus in macrophages from Nelore or Hostein cattle, and considering the profile of iNOS transcription (Fig. 3A), NO production was measured in culture supernate at 0, 6, 12, and 24 hpi. Higher production of NO was observed in Nelore macrophages at 6, 12, and 24 hpi (P < 0.001, P < 0.001 and P < 0.01, respectively) when compared to Holstein macrophages (Fig. 4). 4. Discussion In this study we have demonstrated for the first time that peripheral blood monocyte-derived macrophages from Nelore cattle control the replication of B. abortus more efficiently than macrophages from Holstein cattle. B. abortus is phagocytosed by professional phagocytes, such as macrophages and it is able to survive and replicate within these host cells establishing persistent infection (Samartino and Enright, 1992; Campbell et al., 1994; Carvalho Neta et al., 2008, 2010; Pizarro-Cerdá et al., 2000; Heller et al., 2012). Thus, bactericidal activity of macrophages is a surrogate of natural resistance against bovine brucellosis (Qureshi et al., 1996; Price et al., 1990; Martínez et al., 2010). Indeed, the ability of bovine macrophages to control B. abortus intracellular replication in vitro has been demonstrated to be a reliable phenotypic marker of natural resistance against brucellosis (Harmon et al., 1989; Price et al., 1990; Campbell and Adams, 1992; Campbell et al., 1994; Qureshi et al., 1996; Rossetti et al., 2011). However there is no evidence that natural resistance to brucelose linked to bovine breeds. Our results suggest that Nelore, a Zebu breed, may be more resistant to brucellosis than Holstein cattle. The intracellular fate of B. abortus has been studied in mouse macrophages (Salcedo et al., 2008; Starr

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et al., 2012). Upon infection of phagocytes, B. abortus localizes withing the Brucella-containing vacuole (BCV), which traffics from the endocytic compartment to the endoplasmic reticulum (ER), where the organism proliferates in ER-derived replicative organelles (rBCVs), which are lysosomal-associated membrane protein 1 (LAMP1)-negative and calreticulin-positive. Later on (i.e. at 48 hpi and 72 hpi), the BCV becomes LAMP-1-positive and calreticulin-negative, which is suggestive of autophagic vacuoles (aBCV) (Starr et al., 2012). NO production, catalyzed by iNOS, constitutes one of the main bactericidal mechanisms of macrophages (Macmicking et al., 1997; Wang et al., 2001). Although NO apparently plays only a partial role in controlling intracellular growth of Brucella sp. in macrophages (Jiang et al., 1993; Sun et al., 2002), here we demonstrated that B. abortus-infected bovine macrophages induce only a mild production of NO. Similar results have been previously reported with LPS or alive Brucella (López-Urrutia et al., 2000; Wang et al., 2001), B. suis and B. melitensis (Bagües et al., 2004) in murine macrophage cell line J774.A1, in which there is also induction of iNOS and production of NO. Our results suggest that the ability of Nelore macrophages to control more efficiently the replication of B. abortus may be related to a higher production of NO. However, a lower NO production in bovine macrophage challenged with B. abortus compared to the Salmonella Typhimurium, suggests that other mechanisms may play a role as bactericidal mechanisms (Jiang et al., 1993; Ko et al., 2002; Sun et al., 2002). Innate immunity not only acts as the first line of defense, but it also induces and modulates the adaptive immune response (Adams and Schutta, 2010). Interestingly, transcription levels of IL-12 and TNF-␣ were higher in infected Nelore macrophages when compared to infected Holstein macrophages. Conversely, during the course of infection with B. abortus the more permissive Holstein macrophages had higher levels of IL-4 and IL-10 mRNA than macrophages from Nelore. These results are consistent with the role of IL-12 and TNF-␣ that play an important role in resistance against infection with intracellular pathogens, including Brucella (Zhan et al., 1996). B. abortus-infected macrophages have upregulation of proinflammatory cytokines including TNF-␣, IL-1␤, and modulatory molecules associated with CD4+ T cells subtype 1 (Th1) response such as IL-12 and major histocompatibility complex class II (MHC II) during early stages of infection (Eskra et al., 2003). This response favors a control of Brucella intracellular replication (Ko and Splitter, 2003). Activation of Th1 lymphocytes response is due to stimulation by macrophage-secreted IL-12 during B. abortus infection in vitro and in vivo (Jones and Winter, 1992; Jiang and Baldwin, 1993). Interferon ␥ (IFN-␥) is one of the most important cytokine synthesized by Th1 lymphocytes, and it is widely described as major activator of macrophage antibacterial functions (Jiang and Baldwin, 1993; Splitter et al., 1996; Oliveira et al., 2002). Furthermore, IL-12 is involved in adaptive immune response by direct stimulation of cytotoxic activity by CD8+ T lymphocytes (Zhan et al., 1996). Conversely, cytokines that drive a Th2 response such as IL-4 and IL-10 tend to favor intracellular survival and

replication of B. abortus in macrophages (Fernandes and Baldwin, 1995), and the establishment of persistent infection in mice (Golding et al., 2001). IL-10 is a cytokine with pleiotropic effects in immunoregulation and inflammation. It downregulates expression of Th1 cytokines, MHC class II, and macrophage co-stimulatory molecules (Fiorentino et al., 1991; Gazzinelli et al., 1992; Fernandes and Baldwin, 1995). It is noteworthy that induction of transcription in response to B. abortus infection is limited, and typically within a 10-fold change range as observed in this study. B. abortus is known to be a stealth pathogen that can modulate or inhibit the innate host response (Barqueiro-Calvo et al., 2007; Carvalho Neta et al., 2008; Paixão et al., 2010). Iron deprivation is a mechanism that prevents growth of intracellular pathogens including Mycobacterium and Brucella. Nramp1 (currently named Solute Carrier Family 11 Member 1 – Slc11a1) is a transmembrane protein that is tought to transport iron from the phagolysosome into the cytosol (Gruenheid et al., 1995). Previous studies have demonstrated a link between Nramp1 and control of replication of intracellular pathogens (Adams and Templeton, 1998; Blackwell and Searle, 1999; Barthel et al., 2001), but the significance of certain bovine Slc11a1 polymorphisms in in controlling B. abortus infection remains controversial (Paixão et al., 2007, 2012; Martínez et al., 2010). There were no significant differences in Nramp1 mRNA levels between macrophages from Nelore or Holstein, and therefore the increased resistance of Nelore macrophages cannot be attributed to Nramp1 transcription levels. In addition, Holstein macrophage is more permissive to B. abortus replication at 48 hpi independently of iron availability or the ability of the bacteria to uptake iron within the phagolysosome. Iron is the most abundant metal ion in biological systems and it is required for several metabolic processes (Payne, 1993). Thus, it was postulated that iron acquisition is a prerequisite for pathogenesis of intracellular bacteria (Ratledge and Dover, 2000). Under iron limiting conditions, Gram-negative bacteria release iron chelating agents with low molecular weight and high affinity called siderophores (Neilands, 1992). B. abortus has two known siderophores, the 2,3-dihydroxybenzoic acid (2,3-DHBA) (encoded by the dhbCEBA operon) and brucebactin (Bellaire et al., 2003a,b; ˜ et al., 1992; Martínez et al., 2006; Parent et al., Lopez-Goni 2002). Our results demonstrated that B. abortus dhbC was attenuated in bovine macrophages from both breeds and this phenotype was exacerbated under intralysosomal iron depletion. Bellaire et al. (1999) demonstrated that the siderophore 2,3-DHBA is not required for virulence in murine macrophages or for the establishment of infection in BALB/c mice. However, other study from the same group has shown that this siderophore synthesis is essential for maintaining the virulence of B. abortus in vivo in ruminants (Bellaire et al., 2003a,b). In conclusion, macrophages from Nelore cattle are more efficient for controlling intracellular replication of B. abortus in vitro. There macrophages exhibit greater transcription of proinflamatory cytokines and generate higher levels of NO during B. abortus infection when compared to macrophages from Holstein. These results suggest that Nelore may have a higher degree of resistance against brucellosis when compared to Holstein.

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Conflict of interest statement The authors confirm that they have no conflicts of interest in this work. Acknowledgements We thank Dr. Sergio Oliveira Costa for technical support in NO assays. We thank the Center of Microscopy at the Universidade Federal de Minas Gerais, particularly Dr. Elizabeth Ribeiro da Silva for providing equipment and technical support in experiments involving electron microscopy. Work in RLS lab is supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil) and FAPEMIG (Fundac¸ão de Amparo a Pesquisa do Estado de Minas Gerais, Brazil). AAM and RLS have fellowships from CNPq (Brazil). References Adams, L.G., Schutta, J.C., 2010. Natural resistance against brucellosis: a review. Open. Vet. Sci. J. 4, 61–71. Adams, L.G., Templeton, J.W., 1998. Genetic resistance to bacterial diseases of animals. Rev. Sci. Tech. Off. Int. Epiz. 17, 200–219. Bagües, M.P.J., Gross, A.T., Dornand, J., 2004. Different responses of macrophages to smooth and rough Brucella spp.: relationship to virulence. Infect. Immun. 72, 2429–2433. Barqueiro-Calvo, E., Chaves-Olarte, E., Weiss, D.S., Guzman-Verri, C., Chacon-Diaz, C., Rucavado, A., Moriyon, I., Moreno, E., 2007. Brucella abortus uses a stealthy strategy to avoid activation of the innate immune system during the onset of infection. PLoS One 7, e631. Barthel, R., Feng, J., Piedrahita, J.A., Mcmurray, J.A., Templeton, D.N., Adams, L.G., 2001. Stable transfection of bovine Nramp1 gene into murine RAW264.7 cells: effect on Brucella abortus survival. Infect. Immun. 69, 3110–3119. Bellaire, B.H., Elzer, P.H., Baldwin, C.L., Roop II, R.M., 1999. The siderophore 2,3-dihydroxybenzoic acid is not required for virulence of Brucella abortus in BALB/c mice. Infect. Immun. 67, 2615–2618. Bellaire, B.H., Elzer, P.H., Baldwin, C.L., Roop II, R.M., 2003a. Production of the siderophore 2,3-dihydroxybenzoic acid is required for wild-type growth of Brucella abortus in the presence of erythritol under low-iron conditions in vitro. Infect. Immun. 71, 2927–2932. Bellaire, B.H., Elzer, P.H., Hagius, S., Walker, J., Baldwin, C.L., Roop II, R.M., 2003b. Genetic organization and iron-responsive regulation of the Brucella abortus 2,3-dihydroxybenzoic acid biosynthesis operon, a cluster of genes required for wild type virulence in pregnant cattle. Infect. Immun. 71, 1794–1803. Blackwell, J.M., Searle, S., 1999. Genetic regulation of macrophage activation: understanding the function of Nramp1 (=Ity/Lsh/Bcg). Immunol. Lett. 65, 73–80. Bock, R.E., Kingston, T.G., De Vos, A.J., 1999. Effect of breed of cattle on transmission rate and innate resistance to infection with Babesia bovis. Babesia bigemina transmitted by Boophilus microplus. Aust. Vet. J. 77, 461–464. Campbell, G.A., Adams, L.G., 1992. The long-term culture of bovine monocyte-derived macrophages and their use in the study of intracellular proliferation of Brucella abortus. Vet. Immunol. Immunopathol. 34, 291–305. Campbell, G.A., Adams, L.G., Sowa, B.A., 1994. Mechanisms of binding of Brucella abortus to mononuclear phagocytes from cows naturally resistant or susceptible to brucellosis. Vet. Immunol. Immunopathol. 41, 295–306. Carvalho Neta, A.V., Mol, J.P.S., Xavier, M.N., Paixão, T.A., Lage, A.P., Santos, R.L., 2010. Pathogenesis of bovine brucellosis. Vet. J. 184, 146–155. Carvalho Neta, A.V., Steynen, A.P.R., Paixão, T.A., Miranda, K.L., Silva, F.L., Roux, C.M., Tsolis, R.M., Everts, R.E., Lewin, H.A., Adams, L.G., Carvalho, O.A.F., Lage, A.P., Santos, R.L., 2008. Modulation of bovine trophoblastic innate immune response by Brucella abortus. Infect. Immun. 76, 1897–1907. Eaglesome, M.D., Garcia, M.M., 1992. Microbial agents associated with bovine genital tract infection and semen. Part I. Brucella abortus, Leptospira, Campylobacter fetus and Tritrichomonas foetus. Vet. Bull. 62, 743–745.

301

Emminger, A.C., Schalm, O.W., 1943. The effect of Brucella abortus on the bovine udder and its secretion. Am. J. Vet. Res. 4, 100–109. Enright, F.M., Walker, J.V., Jeffers, G., Deyoe, B.L., 1984. Cellular and humoral responses of Brucella abortus-infected bovine fetuses. Am. J. Vet. Res. 45, 424–430. Eskra, L., Mathison, A., Splitter, G., 2003. Microarray analysis of mRNA levels from RAW264.7 macrophages infected with Brucella abortus. Infect. Immun. 71, 1125–1133. Fernandes, D.M., Baldwin, C.L., 1995. Interleukin-10 downregulates protective immunity to Brucella abortus. Infect. Immun. 63, 1130–1133. Fiorentino, D.F.A., Zlotnik, P., Viera, T.R., Mosmann, M., Howard, K., Moore, W., O’Garra, A., 1991. IL-10 acts on the antigen presenting cell to inhibit cytokine production by Th1 cells. J. Immunol. 146, 3444–3451. Forbes, J.R., Gros, P., 2003. Iron, manganese, and cobalt transport by Nramp1 (Slc11a1) and Nramp2 (Slc11a2) expressed at the plasma membrane. Blood 102, 1884–1892. Gazzinelli, R.T., Oswald, I.P., James, S.L., Sher, A., 1992. IL-10 inhibits parasite killing and nitrogen oxide dependent production by IFN-gactivated macrophages. J. Immunol. 148, 1792–1796. Golding, B., Scott, D.E., Scharf, O., Huang, L.Y., Zaitseva, M., Laphan, C., Eller, N., Golding, H., 2001. Immunity and protection against Brucella abortus. Microb. Infect. 3, 43–48. Gruenheid, S., Canonne-Hergaux, F., Gauthier, S., Hackam, D.J., Grinstein, S., Gros, P., 1995. The iron transport protein Nramp2 is an integral membrane glycoprotein that colocalizes with transferrin in recycling endosomes. J. Exp. Med. 189, 831–841. Harmon, B.G., Adams, L.G., Templeton, J.W., Smith III, R., 1989. Macrophage function in mammary glands of Brucella abortus-infected cows and cows resistants infection after inoculation of Brucella abortus. Am. J. Vet. Res. 50, 459–465. Heller, M.C., Watson, J.L., Blanchard, M.T., Jackson, K.A., Stott, J.L., Tsolis, R.M., 2012. Characterization of Brucella abortus infection of bovine monocyte-derived dendritic cells. Vet. Immunol. Immunopathol. 149, 255–261. Jiang, X., Baldwin, C.L., 1993. Effects of cytokines on intracellular growth of Brucella abortus. Infect. Immun. 61, 124–134. Jiang, X., Leonard, B., Benson, R., Baldwin, C.L., 1993. Macrophage control of Brucella abortus: role of reactive oxygen intermediates and nitric oxide. Cell. Immunol. 151, 309–319. Jones, S.M., Winter, A.J., 1992. Survival of virulent and attenuated strains of Brucella abortus in normal and gamma interferon-activated murine peritoneal macrophages. Infect. Immun. 60, 3011–3014. Ko, J., Gendron-Fitzpatrick, A., Ficht, T.A., Splitter, G.A., 2002. Virulence criteria for Brucella abortus strains as determined by interferon regulatory factor-1 deficient mice. Infect. Immun. 70, 7004–7012. Ko, J., Splitter, G.A., 2003. Molecular host–pathogen interaction in brucellosis: current understanding and future approaches to vaccine development for mice and humans. Clin. Microbiol. Rev. 6, 65–78. Kudi, A.C., Kalla, D.J.U., Kudi, M.C., Kapio, G.I., 1997. Brucellosis in camels. J. Arid. Environ. 37, 413–417. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C (T)) method. Methods 25, 402–408. ˜ I., Moriyon, I., Neilands, J.B., 1992. Identification of 2,3Lopez-Goni, dihydroxybenzoic acid as a Brucella abortus siderophore. Infect. Immun. 60, 4496–4503. López-Urrutia, L., Alonso, A., Nieto, M.L., Bayon, Y., Orduna, A., 2000. Lipopolysaccharides of Brucella abortus and Brucella melitensis induce nitric oxide synthesis in rat peritoneal macrophages. Infect. Immun. 68, 1740–1745. Macmicking, J., Xie, Q.W., Nathan, C., 1997. Nitric oxide macrophage functions. Annu. Rev. Immunol. 15, 323–350. ˜ J., 2010. Martínez, R., Dunner, S., Toro, R., Tobón, J., Gallego, J., Canón, Effect of polymorphisms in the Slc11a1 coding region on resistance to brucellosis by macrophages in vitro and after challenge in two Bos breeds (Blanco Orejinegro and Zebu). Genet. Mol. Biol. 33, 463–470. Martínez, M., Ugalde, R.A., Almirón, M., 2006. Irr regulates brucebactin and 2,3-dihydroxybenzoic acid biosynthesis, and is implicated in the oxidative stress resistance and intracellular survival of Brucella abortus. Microbiology 152, 2591–2598. Neilands, J.B., 1992. Identification of 2,3-dihydroxybenzoic acid as a Brucella abortus siderophore. Infect. Immun. 60, 4496–4503. Oliveira, S.C., Soeurt, N., Splitter, G.A., 2002. Molecular and cellular interactions between Brucella abortus antigens and host immune responses. Vet. Microbiol. 90, 417–424.

302

A.A. Macedo et al. / Veterinary Immunology and Immunopathology 151 (2013) 294–302

Paixão, T.A., Poester, F.P., Carvalho Neta, A.V., Borges, A.M., Lage, A.P., Santos, R.L., 2007. Nramp1 3 UTR polymorphisms are not associated with natural resistance to Brucella abortus in cattle. Infect. Immun. 75, 2493–2499. Paixão, T.A., Martinez, R., Santos, R.L., 2012. Polymorphisms of the coding region of Slc11a1 (Nramp1) gene associated to natural resistance against bovine brucellosis. Arq. Bras. Med. Vet. Zootec. 64, 1081–1084. Paixão, T.A., Roux, C.M., Hartigh, A.B.D., Sankaran, S., Dandekar, S., Santos, R.L., Tsolis, R.M., 2010. Establishment of systemic Brucella melitensis infection through the digestive tract requires urease, the type IV secretion system, and lipopolysaccharide O-antigen. Infect. Immun. 77, 4197–4208. Parent, M.A., Bellaire, B.H., Murphy, E.A., Roop II, R.M., Elzer, P.H., Baldwin, C.L., 2002. Brucella abortus siderophore 2,3-dihydroxybenzoic acid (DHBA) facilitates intracellular survival of the bacteria. Microb. Pathog. 32, 239–248. Parker, R.J., Shepherd, R.K., Trueman, K.F., Jones, G.W., Kent, A.S., Polkinghorne, I.G., 1985. Susceptibility of Bos indicus and Bos taurus to Anaplasma marginale and Babesia bigemina infections. Vet. Parasitol. 17, 205–213. Payne, S.M., 1993. Iron acquisition in microbial pathogenesis. Trends Microbiol. 1, 66–69. ˜ M.T., Miller, J.E., Wyatt, W., Kearney, M.T., 2000. Differences in susPena, ceptibility to gastrointestinal nematode infection between Angus and Brangus cattle in south Louisiana. Vet. Parasitol. 8, 51–61. Persson, H.L., Yu, Z., Tirosh, O., Eaton, J.W., Brunk, U.T., 2003. Prevention of oxidant-induced cell death by lysosomotropic iron chelators. Free Radic. Biol. Med. 34, 1295–1305. Pizarro-Cerdá, J., Moreno, E., Gorvel, J.P., 2000. Invasion and intracellular trafficking of Brucella abortus in nonphagocytic cells. Microb. Infect. 2, 829–835. Price, R.E., Templeton, J.W., Smith III, R., Adams, L.G., 1990. Ability of phagocytes from cattle naturally resistant or susceptible to brucellosis to control in vitro intracellular survival of Brucella abortus. Infect. Immun. 58, 879–886. Qureshi, T., Templeton, J.W., Adams, L.G., 1996. Intracellular survival of Brucella abortus, Mycobacterium bovis BCG. Salmonella Dublin and Salmonella typhimurium in macrophages from cattle genetically resistant to Brucella abortus. Vet. Immunol. Immunopathol. 50, 55–65. Ratledge, C., Dover, L.G., 2000. Iron metabolism in pathogenic bacteria. Annu. Rev. Microbiol. 54, 881–941. Rechav, Y., Kostrzewski, M.W., 1991. Relative resistance of six cattle breeds to the tick Boophilus decoloratus in South Africa. Onderstepoort J. Vet. Res. 58, 181–186. Rogers, R.J., Cook, D.R., Kettlerer, P.J., Baldock, F.C., Blachall, P.J., Stewart, S.W., 1989. An evaluation of three serological tests for antibody to Brucella suis in pigs. Aust. Vet. J. 66, 77–80. Rossetti, C.A., Galindo, C.L., Everts, R.E., Lewin, H.A., Garner, H.R., Adams, L.G., 2011. Comparative analysis of the early transcriptome of

Brucella abortus – infected monocyte-derived macrophages from cattle naturally resistant or susceptible to brucellosis. Res. Vet. Sci. 91, 40–51. Salcedo, S.P., Marchesini, M.I., Lelouard, H., Fugier, E., Jolly, G., Balor, S., Muller, A., Lapaque, N., Demaria, O., Alexopoulou, L., Comerci, D.J., Ugalde, R.A., Pierre, P., Gorvel, J.P., 2008. Brucella control of dendritic cell maturation is dependent on the TIR-containing protein Btp1. PLoS Pathog. 4, e21. Samartino, L.E., Enright, F.M., 1992. Interaction of bovine chorioallantoic membrane explants with three strains of Brucella abortus. Am. J. Vet. Res. 53, 359–363. Splitter, G., Oliveira, S.C., Carey, M., Miller, C., Ko, J., Covert, J., 1996. T lymphocyte mediated protection against facultative intracellular bacteria. Vet. Immunol. Immunopathol. 54, 309–319. Starr, T., Child, R., Wehrly, T.D., Hansen, B., Hwang, S., López-Otin, C., Virgin, H.W., Celli, J., 2012. Selective subversion of autophagy complexes facilitates completion of the Brucella intracellular cycle. Cell Host Microbe 11, 33–45. Sun, Y.H., Hartigh, A.B.D., Santos, R.L., Adams, L.G., Tsolis, R.M., 2002. VirB-mediated survival of Brucella abortus in mice and macrophages is independent of a functional inducible nitric oxide synthase or macrophage NADPH oxidase in macrophages. Infect. Immun. 70, 4826–4832. Tatum, F.M., Detilleux, P.G., Sacks, J.M., Halling, S.M., 1992. Construction of Cu-Zn superoxide dismutase deletion mutants of Brucella abortus: analysis of survival in vitro in epithelial and phagocytic cells and in vivo in mice. Infect. Immun. 60, 2863–2869. Templeton, J.W., Smith III, R., Adams, L.G., 1988. Natural disease resistance in domestic animals. J. Am. Vet. Med. Assoc. 192, 1306–1315. Utech, K.B.W., Wharton, R.H., Kerr, J.D., 1978. Resistance to Boophilus microplus (Canestrini) in different breeds of cattle. Aust. J. Agric. Res. 29, 885–895. Xavier, M.N., Paixão, T.A., Poester, F.P., Lage, A.P., Santos, R.L., 2009. Pathology, immunohistochemistry, and bacteriology of tissues and milk of cows and fetuses experimentally infected with Brucella abortus. J. Comp. Pathol. 140, 149–157. Wambura, P.N., Gwakisa, P.S., Silayo, R.S., Rugaimukamu, E.A., 1998. Breed-associated resistance to tick infestation in Bos indicus and their crosses with Bos taurus. Vet. Parasitol. 77, 63–70. Wang, M., Qureshi, N., Soeurt, N., Splitter, G., 2001. High levels of nitric oxide production decrease early but increase late survival of Brucella abortus in macrophages. Microb. Pathol. 31, 221–230. Young, E.J., 1983. Human brucellosis. Rev. Infect. Dis. 5, 821–842. Zhan, Y., Liu, Z., Cheers, C., 1996. Tumor necrosis factor alpha and interleukin 12 contribute to resistance to the intracellular bacterium Brucella abortus by different mechanisms. Infect. Immun. 64, 2782–2786.