Spotted fever group rickettsiae in ticks from the Masai Mara region of Kenya

We have identified for the first time Rickettsia africae, and the ticks that harbored them, in Kenya. A total of 5,325 ticks were collected from vegetation, livestock, and wild animals during two field trips to southwestern Kenya. Most were immature forms (85.2%) belonging to the genera Amblyomma or Rhipicephalus. The adults also included representatives from the genus Boophilus. Ticks were assessed for rickettsial DNA by a polymerase chain reaction (PCR) using primers for the spotted fever group (SFG)-specific rickettsial outer membrane protein A (rompA) gene, and positive amplicons were sequenced. While none of the immature ticks tested positive by PCR, 15.8% of the adult Amblyomma variegatum and less than 1% of the Rhipicephalus spp. were SFG positive. Sequences of amplified products were identified as R. africae. These findings extend the known range of R. africae.     

Spotted fever group rickettsiae from ticks captured in Sudan

Ticks were collected from ruminants in various areas of Sudan in 1998 and 2000. Primer pairs of rickettsial citrate synthase gene (gltA) and a spotted fever group (SFG) rickettsial 190-kDa surface antigen gene (rompA), respectively, were used for identification. Polymerase chain reaction (PCR)-positive products were used for DNA sequencing. The gltA gene was detected in 55% of the ticks examined (57/104). Among the 57 ticks studied, 19 were positive for the rompA gene. Thus, 18% of the ticks examined were found to be infected with SFG rickettsiae. The nucleotide sequences of the amplified rompA gene fragment of Hyalomma spp. and Amblyomma spp. were similar to those of Rickettsia aeschlimannii and Rickettsia africae, respectively. In this study, we succeeded in detecting the SFG rickettsiae gene in ticks, and established that there were at least two species of SFG rickettsiae in field ticks in Sudan.     

四川石渠县牦牛源蜱感染斑点热群立克次体分子流行病学调查

目的 了解四川省石渠县牦牛体表寄生蜱的种类及其斑点热群立克次体的感染情况。方法 采集牦牛体表的蜱,经形态学初步鉴定后,提取蜱总DNA,PCR扩增蜱16S rRNA基因及斑点热群立克次体ompA、ompB基因,并对扩得阳性产物进行测序和构建进化树分析,从而确定蜱及其携带斑点热群立克次体的种类。结果 在石渠县4个乡共采集到蜱818只,其中西藏革蜱占78.97%(646/818)、青海血蜱占21.03%(172/818)。在818只蜱中有408只扩得斑点热群立克次体ompA、ompB基因,总阳性率为49.8%。经比对分析, ompA基因总共得到4条序列(uncultured Rickettsia sp.shiqu1、R.raoultii.shiqu2、R.raoultii.shiqu3和R.raoultii.shiqu4),ompB基因也得到4条序列(uncultured Rickettsia sp.shiqu1、R.raoultii.shiqu2、R.raoultii.shiqu3和R.raoultii.shiqu4)。经遗传进化分析显示ompA基因的uncultured Rickettsia sp.shiqu1与我国青海未定种的Uncultured Rickettsia sp.(MG228270)亲缘关系最近;ompB基因的uncultured Rickettsia sp.shiqu1与韩国未定种的Rickettsia sp.(KC888953)和Candidatus Rickettsia longicornii(MG906675)亲缘关系最近;ompA和ompB基因的R.raoultii.shiqu2-4与对人有致病性的劳氏立克次体(R.raoultii)的亲缘关系最近。结论 首次在牦牛体表寄生的西藏革蜱中检出劳氏立克次体。石渠县存在西藏革蜱和青海血蜱,蜱传劳氏立克次体感染率较高并且具有感染人的风险。     

Rickettsia parkeri: a Rickettsial pathogen transmitted by ticks in endemic areas for spotted fever rickettsiosis in southern Uruguay

At first Rickettsia conorii was implicated as the causative agent of spotted fever in Uruguay diagnosed by serological assays. Later Rickettsia parkeri was detected in human-biting Amblyomma triste ticks using molecular tests. The natural vector of R. conorii, Rhipicephalus sanguineus, has not been studied for the presence of rickettsial organisms in Uruguay. To address this question, 180 R. sanguineus from dogs and 245 A. triste from vegetation (flagging) collected in three endemic localities were screened for spotted fever group (SFG) rickettsiosis in southern Uruguay. Tick extracted DNA pools were subjected to PCR using primers which amplify a fragment of the rickettsial gltA gene. Positive tick DNA pools with these primers were subjected to a second PCR round with primers targeting a fragment of the ompA gene, which is only present in SFG rickettsiae. No rickettsial DNA was detected in R. sanguineus. However, DNA pools of A. triste were found to be positive for a rickettsial organism in two of the three localities, with prevalences of 11.8% to 37.5% positive pools. DNA sequences generated from these PCR-positive ticks corresponded to R. parkeri. These findings, joint with the aggressiveness shown by A. triste towards humans, support previous data on the involvement of A. triste as vector of human infections caused by R. parkeri in Uruguay.     

Seroepidemiology of Rickettsia africae infection in Norwegian travellers to rural Africa

Rickettsia africae is the causative agent of African tick bite fever (ATBF), an acute febrile illness frequently accompanied by inoculation eschars, regional lymphadenitis, myalgia and severe headache. Recently, ATBF has been recognized as an emerging health problem for international travellers to rural sub-Saharan Africa. To estimate the incidence, risk factors for and proportion of symptomatic cases of travel-associated R. africae infection, we performed a seroepidemiological study of 152 first-time Norwegian travellers to rural areas in sub-Equatorial Africa. Seropositivity was based on the detection of specific antibodies to R. africae in microimmunofluorescence and/or Western blotting assays. Thirteen (8.6%) travellers were seropositive to R. africae. Eight (62%) seropositive travellers reported symptoms consistent with ATBF; of these, 2 had received antirickettsial therapy. Using multiple logistic regression, the following factors were found to be significantly associated with seropositivity: hunting as the purpose of travel [odds ratio (OR) 10.1; 95% confidence interval (CI) 1.5-69; p=0.019] and stay in rural areas of > 7 d (OR 6.0; 95% CI 1.5-24; p=0.012). This first seroepidemiological study on travel-associated R. africae infection suggests that the infection may be common in international travellers to rural sub-Saharan Africa but that most cases are asymptomatic or clinically mild and self-limited.     

Prevalence of tick-borne zoonotic bacteria in questing adult ticks from northern Spain

A total of 691 questing adult ixodid ticks of the genera Ixodes, Haemaphysalis, Dermacentor, and Rhipicephalus were tested by polymerase chain reaction (PCR) and reverse line blot (RLB) for the presence of Anaplasma phagocytophilum, Coxiella burnetii, Borrelia spp., and spotted fever group (SFG) rickettsiae. Ticks were collected by blanket dragging during 2 sampling years (2003-2005) in 10 recreational areas in the Basque Country (Northern Spain). Adult ticks were collected every month of the year and eight different species were identified among which Ixodes ricinus was the most abundant and widespread. Three pathogens for humans, Borrelia burgdorferi, A. phagocytophilum, and C. burnetii, as well as rickettsiae of unknown pathogenicity were detected. The latter were identified as Rickettsia sp. RpA4/DnS14 by sequencing of the citrate synthase (gltA) gene. The infection rates varied from 0.1%-6.9%. DNA of A. phagocytophilum was detected mainly in I. ricinus, but also in Haemaphysalis punctata, H. concinna, and Rhipicephalus bursa. Coxiella burnetii was detected in only one specimen of H. punctata, and Borrelia spp. in eight ticks. Furthermore, PCR-RLB analysis specific for B. burgdorferi sensu lato detected one H. punctata with positive hybridization with the B. burgdorferi sensu stricto probe, and two I. ricinus positive for B. afzelii and B. garinii. SFG rickettsiae were the pathogens most frequently found, present in 48 of 97 D. reticulatus analyzed. Mixed infections were not found in any of the analyzed ticks. These results are compared and discussed with data obtained in previous studies carried out in the same and other regions.     

A survey for spotted fever group rickettsiae and ehrlichiae in Amblyomma variegatum from St. Kitts and Nevis

Eighty-nine Amblyomma variegatum ticks were collected from the islands of St. Kitts and Nevis in the Caribbean and preserved in 70% ethanol or local rum. After being washed in sterile water, their DNA was extracted and analyzed by a polymerase chain reaction (PCR) for DNA of spotted fever group rickettsiae and ehrlichiae. None of the tested ticks was positive in a PCR assay using the primers 16S EHRD and 16S EHRR for the 16S rRNA gene of Ehrlichia spp.. Forty-one percent of the A. variegatum (36 of 89 of which 34 [47%] of 72 were adult males, 2 (13%) of 16 were adult females, and 0 (0%) of 1 were nymphs) were positive in a PCR assay using the primer pair 190-70 and 190-701 for the outer membrane protein A (ompA) gene of spotted fever group rickettsiae. All PCR amplification products obtained had 100% sequence homology with Rickettsia africae, the agent of African tick-bite fever.     

A focus of dogs and Rickettsia massiliae-infected Rhipicephalus sanguineus in California

A recurrent focus of Rhipicephalus sanguineus infestation was investigated in a suburban area of southern California after reports of suspected Rocky Mountain spotted fever in two dogs on the same property. Abundant quantities of Rh. sanguineus were collected on the property and repeatedly from each dog, and Rickettsia massiliae DNA was detected by polymerase chain reaction (PCR). Whole blood and serum samples from four dogs were tested by using PCR and microimmunofluorescent assay for antibodies against spotted fever group rickettsiae. Serum samples from all four dogs contained antibodies reactive with R. massiliae, R. rhipicephali, R. rickettsii, and 364D Rickettsia but no rickettsial DNA was detected by PCR of blood samples. Serum cross-absorption and Western blot assays implicated R. massiliae as the most likely spotted fever group rickettsiae responsible for seropositivity. To our knowledge, this is the first detection of R. massiliae in ticks in California.     

Detection of Rickettsia africae in patients and ticks along the coastal region of Cameroon

Rickettsia africae was identified in seven (6%) of 118 patients with acute fevers of unknown etiology proven not to be malaria or typhoid fever from clinics along the coastal region of Cameroon by polymerase chain reaction (PCR) amplification and sequencing of the citrate synthase (gltA) and outer membrane protein A (ompA) genes of Rickettsia. The majority (71%) of the patients were female. Clinical manifestations included fever (100%), headache (71%), myalgia (71%), arthralgia (43%), pulmonary involvement (29%), and diffuse rash (14%). Moreover, R. africae was detected by PCR amplification and sequence analysis of the gltA and ompA genes in 62 (75%) of 83 adult Amblyomma variegatum ticks collected from cattle in the same region. These results confirm the presence of a previously unrecognized infectious disease in the indigenous Cameroonian population, as well as extend the established range of R. africae.     

Risk factors for African tick-bite fever in rural central Africa

African tick-bite fever is an emerging infectious disease caused by the spotted fever group Rickettsia, Rickettsia africae, and is transmitted by ticks of the genus Amblyomma. To determine the seroprevalence of exposure to R. africae and risk factors associated with infection, we conducted a cross-sectional study of persons in seven rural villages in distinct ecological habitats of Cameroon. We examined 903 plasma samples by using an indirect immunofluorescence assay for antibodies to R. africae and analyzed demographic and occupational data collected from questionnaires. Of the 903 persons tested, 243 (26.9%) had IgG/IgM/IgA reactive with R. africae. Persons from four of the seven village sites were significantly more likely to be seropositive (P < 0.05), and lowland forest sites tended to have higher seroprevalences. These results suggest that African tick-bite fever is common in adults in rural areas of Cameroon and that ecological factors may play a role in the acquisition of R. africae infection.     

新疆克拉玛依地区蜱媒病原体的调查

目的 调查新疆克拉玛依地区蜱种类及其携带病原体。方法 于克拉玛依市不同区采集游离蜱和寄生蜱,鉴定蜱种类,利用荧光定量PCR或套氏PCR/半套氏PCR检测发热伴血小板减少综合征布尼亚病毒、斑点热群立克次体、克里米亚-刚果出血热病毒、蜱传脑炎病毒、巴贝斯虫/泰勒虫、贝氏立克次体、查菲埃立克次体、恙虫病东方体和伯氏疏螺旋体等9种病原体。结果 共采集蜱683只,分3属3种,其中亚东璃眼蜱数量(n=362)最多,其次为银盾革蜱(n=311)、图兰扇头蜱(n=10)。银盾革蜱中斑点热群立克次体的阳性率为3.2%(10/311),巴贝斯虫的阳性率为1.0%(3/311);亚东璃眼蜱中巴贝斯虫的阳性率为0.8%(3/362),泰勒虫阳性率为0.3%(1/362);其他病原体检测结果均呈阴性。PCR扩增阳性基因序列分析显示银盾革蜱携斑点热群立克次体阳性与劳氏立克次体最相关,银盾革蜱和亚东璃眼蜱携带驽巴贝斯虫。结论 新疆克拉玛依地区优势蜱为亚东璃眼蜱和银盾革蜱,银盾革蜱携带劳氏立克次体,银盾革蜱和亚东璃眼蜱携带驽巴贝斯虫,提示该地区存在斑点热、巴贝斯虫病的自然疫源地,因此有必要加强对这些蜱携疾病的防控。     

Detection of Rickettsia parkeri and Candidatus Rickettsia andeanae in Amblyomma maculatum Gulf Coast ticks collected from humans in the United States

Rickettsia parkeri, a spotted fever group (SFG) rickettsia recently found to be pathogenic to humans, causes an eschar-associated febrile illness. The R. parkeri rickettsiosis, Tidewater spotted fever, has been misdiagnosed as Rocky Mountain spotted fever due to serologic cross reactivity and the lack of specific diagnostic methods. Candidatus Rickettsia andeanae, also a SFG rickettsia, is a recently described agent of unknown pathogenicity originally identified in ticks collected from domestic animals during a fever outbreak investigation in northern Peru. Among 37 Amblyomma maculatum (collected from humans (n=35) and questing (n=2)) obtained from the southern United States during 2000-2009, nine and four A. maculatum nucleic acid preparations were found positive for R. parkeri and Candidatus R. andeanae, respectively, by newly developed genus- and species-specific quantitative real-time polymerase chain reaction assays. In addition Rickettsia felis was found in two A. maculatum nucleic acid preparations.     

Anaplasma phagocytophilum infection in domestic animals in ten provinces/cities of China

A nationwide epidemiologic investigation of domestic animal infections has been conducted in nine provinces and one city during 2007-2010. Serum samples from a total of 707 goats, 433 cattle, and 219 dogs were collected for detecting Anaplasma phagocytophilum IgG antibody by immunofluorescence assays and the average seroprevalences were 10.05% for dogs, 3.82% for goats, and 0.69% for cattle, respectively. A total of 472 goats, 201 cattle, 102 dog blood clots, and 1,580 ticks were collected for polymerase chain reaction (PCR) amplifying A. phagocytophilum 16S rRNA genes and the PCR-positive rates were 26.69% for goats, 23.38% for cattle, and 10.89% for dogs. Six species were identified and the average PCR-positive rates were 58.3% for Dermacentor silvarum, 43.9% for Haemaphysalis longicornis, 12.5% for Ixodes persulcatus, 7.5% (3 of 40) for Boophilus microplus, and 5.2% for Rhipicephalus sanguineus, respectively. The evidence in the study indicated the zoonotic Rickettsia is highly prevalent in China.     

African tick-bite fever imported into Norway: presentation of 8 cases.

We report on 8 Norwegian travellers to Southern Africa with African tick-bite fever (ATBF), a recently described spotted fever group rickettsiosis. All patients had acute flu-like symptoms and developed I or multiple inoculation eschars. The patients were treated with either doxycycline or ciprofloxacin, and all recovered. The diagnosis of ATBF was confirmed by the detection of specific IgM antibodies to Rickettsia africae by microimmunofluoroscence in convalescent-phase serum samples.     

Sub-acute neuropathy in patients with African tick bite fever

African tick bite fever (ATBF) caused by Rickettsia africae is an emerging health problem in travellers to sub-Saharan Africa. We here present 6 patients with evidence of long-lasting sub-acute neuropathy following ATBF contracted during safari trips to southern Africa. Three patients developed radiating pain, paresthaesia and/or motor weakness of extremities, 2 had hemi-facial pain and paresthaesia, and 1 developed unilateral sensorineural hearing loss. When evaluated 3-26 months after symptom onset, cerebrospinal fluid samples from 5 patients were negative for R. africae PCR and serology, but revealed elevated protein content in 3 and mild pleocytosis in 1 case. Despite extensive investigations, no plausible alternative causes of neuropathy could be identified. Treatment with doxycycline in 2 patients had no clinical effect. Given the current increase of international safari tourism to sub-Saharan Africa, more cases of sub-acute neuropathy following ATBF may well be encountered in Europe and elsewhere in the y to come.     

Ticks and associated pathogens collected from domestic animals in the Netherlands

Following an outbreak of autochthonous canine babesiosis in the Netherlands, a request made to veterinarians and the public to collect ticks from companion animals resulted in 4298 ticks submitted between July 2005 and October 2006 to our center. Ticks were identified as Ixodes ricinus adults (2907/4298, 67.6%), Ixodes sp. nymphs (529/4298, 12.3%) and Ixodes sp. larvae (385/4298, 9.0%), I. hexagonus adults (328/4298, 7.6%), Dermacentor reticulatus (72/4298, 1.7%), and several other exotic tick species such as Amblyomma flavomaculatum (formerly Aponomma flavomaculatum), Hyalomma marginatum rufipes, Rhipicephalus sanguineus, and R. turanicus (55/4298, 1.3%). Eight localities were surveyed for the presence of local D. reticulatus, a tick not indigenous to the Netherlands, based on multiple submissions of D. reticulatus ticks from these areas. D. reticulatus was collected from the vegetation in six of these localities, confirming the presence of populations of this tick in the Netherlands. Adult I. ricinus (n=251), I. hexagonus (n=237), and D. reticulatus (n=344) ticks were selected at random and subsequently screened by polymerase chain reaction (PCR) and reverse line blot (RLB) hybridization for the presence of Borrelia, Babesia, Theileria, Anaplasma, Ehrlichia, and Rickettsia species. I. ricinus ticks were infected with Rickettsia helvetica (24.7%), spirochetes belonging to the Borrelia burgdorferi sensu lato group (7.2%), the Ehrlichia-like "Schotii" variant (2.4%), Anaplasma phagocytophilum (1.6%), Babesia sp. (EU1) (1.2%), Babesia divergens (0.4%), and Babesia microti (0.4%). A. phagocytophilum (5.9%) and R. helvetica (0.8%) were also detected in adult I. hexagonus ticks. Spotted fever group Rickettsiae, previously reported as Rickettsia sp. DnS14/RpA4 (14.0%), and Borrelia burgdorferi sensu lato (0.3%) were detected in the D. reticulatus ticks, which appeared to be free from B. canis infection. We concluded that a much broader spectrum of ticks and tick-borne pathogens is present in the Netherlands than previously thought, including several potential zoonotic pathogens.     

Detection of Borrelia lusitaniae, Rickettsia sp. IRS3, Rickettsia monacensis, and Anaplasma phagocytophilum in Ixodes ricinus collected in Madeira Island, Portugal

A total of 300 Ixodes ricinus ticks were tested by polymerase chain reaction (PCR) for the presence of Borrelia spp., Rickettsia spp., and Anaplasma phagocytophilum. Sequence analysis demonstrated 8 (2.7%) ticks infected with B. lusitaniae, 60 (20%) with Rickettsia spp., and 1 (0.3%) with A. phagocytophilum. Seven (2.3%) ticks were coinfected with B. lusitaniae and Rickettsia spp., 2 (0.6%) with R. monacensis, and 5 (1.7%) with Rickettsia sp. IRS3. The results of this study suggest simultaneous transmission of multiple tick-borne agents on Madeira Island, Portugal.     

滇西横断山区家畜体表蜱类调查及鉴定

目的 调查滇西横断山区家畜体表蜱的种类。方法 采集家畜体表寄生的蜱虫,经形态学鉴定后,用PCR法扩增蜱虫样本的16S rRNA、12S rRNA、COI、ITS2的基因片断,测序后进行序列分析。结果 共采集成虫蜱样本1 874只,经形态学鉴定为1科、1属(扇头蜱属Rhipicephalus)、4种,其中微小扇头蜱(R. microplus)1 637只(占87.35%)),镰形扇头蜱(R. haemaphysaloides)218只(11.63%),短小扇头蜱(R. pumilio)11只(0.59%),血红扇头蜱(R. sanguineus)8只(0.43%)。样品分子鉴定结果与形态学鉴定结果一致。系统发育树分析显示,微小扇头蜱Y2 16S rRNA、12S rRNA、COI、ITS2的基因序列分别与来自印度(EU918188)、贵州(KC503259)、马来西亚(KM246873)、贵州(KC503274)的微小扇头蜱在同一分支上,而与以往云南发现的微小扇头蜱不在同一分支;镰形扇头蜱Y5的 16S rRNA、12S rRNA和COI基因序列分别与来自泰国(KC170743)、台湾(DQ003005)和湖南(KM083593)的镰形扇头蜱在同一分支上;短小扇头蜱Y6和Y01 的ITS2基因序列与来自澳大利亚的短小扇头蜱(AF271282)在同一分支上。结论 滇西横断山区家畜体表蜱以微小扇头蜱为优势种,短小扇头蜱为云南境内首次发现。     

The occurrence of spotted fever rickettsioses and other tick-borne infections in forest workers in Poland

The presence of antibodies to Rickettsia conorii, R. helvetica, R. felis, R. slovaca, R. sibirica, and R. massiliae in sera of 129 forest workers from northeastern and southern Poland was assayed by indirect immunofluorescence. Previous environmental studies revealed presence of spotted fever group (SFG) rickettsiae in ticks collected from these areas. Additionally, the workers were examinated for the presence of antibodies specific to other tick-borne bacteria: Anaplasma phagocytophilum, Bartonella spp., and B. burgdorferi. The results of the studies have shown the presence of specific SFG rickettsiae antibodies in 14.7% of tested forest workers, among them 78.9% had species-specific antibodies to R. massiliae. Contrary to previous detection R. helvetica and R. slovaca in ticks collected in the environment of the examined area, no species-specific antibodies to these species were detected in studied workers. Antibodies to B. burgdorferi (44%) were found in forest workers more often than antibodies to other tested pathogens. B. burgdorferi was also the main component of coinfections. The most frequent confirmed serologically coinfections were simultaneous infections with B. burgdorferi and Bartonella spp. found in 10% of tested individuals. So far, SFG rickettsiae infections have not been diagnosed in Poland; however, the presence of the bacteria in ticks and presence of specific antibodies in humans exposed to arthropods show the need for monitoring the situation. The list of tick-borne pathogens is increasing, but knowledge about the possibility of humans acquiring multipathogens infections after tick bite still needs evaluation.     

Tick-borne bacteria in free-living jaguars (Panthera onca) in Pantanal, Brazil

Tick-borne bacteria were investigated in 10 free-living jaguars and their ticks in the Pantanal biome, Brazil. Jaguar sera were tested by indirect fluorescent antibody assays using Rickettsia rickettsii, Rickettsia parkeri, Rickettsia amblyommii, Rickettsia rhipicephali, Rickettsia felis, Rickettsia bellii, Ehrlichia canis, and Coxiella burnetii as crude antigens. All 10 jaguar sera reacted (titer ≥ 64) to at least one Rickettsia species; 4 and 3 sera reacted with E. canis and C. burnetii, respectively. One jaguar presented antibody titer to R. parkeri at least fourfold higher than those to any of the other five Rickettsia antigens, suggesting that this animal was infected by R. parkeri. Ticks collected from jaguars included the species Amblyomma cajennense, Amblyomma triste, and Rhipicephalus (Boophilus) microplus. No Rickettsia DNA was detected in jaguar blood samples, but an A. triste specimen collected on a jaguar was shown by PCR to be infected by R. parkeri. The blood of two jaguars and samples of A. triste, A. cajennense, and Amblyomma sp. yielded Ehrlichia DNA by PCR targeting the ehrlichial genes 16S rRNA and dsb. Partial DNA sequences obtained from PCR products resulted in a new ehrlichial strain, here designated as Ehrlichia sp. strain Jaguar. A partial DNA sequence of the 16S rRNA gene of this novel strain showed to be closest (99.0%) to uncultured strains of Ehrlichia sp. from Japan and Russia and 98.7% identical to different strains of Ehrlichia ruminantium. The ehrlichial dsb partial sequence of strain jaguar showed to be at most 80.7% identical to any Ehrlichia species or genotype available in GenBank. Through phylogenetic analysis, Ehrlichia sp. strain jaguar grouped in a cluster, albeit distantly, with different genotypes of E. ruminantium. Results highlight risks for human and animal health, considering that cattle ranching and ecotourism are major economic activities in the Pantanal region of Brazil.