A leukotriene-dependent spleen-liver axis drives TNF production in systemic inflammation.

Production of the proinflammatory cytokine tumor necrosis factor (TNF) must be precisely regulated for effective host immunity without the induction of collateral tissue damage. Here, we showed that TNF production was driven by a spleen-liver axis in a rat model of systemic inflammation induced by bacterial lipopolysaccharide (LPS). Analysis of cytokine expression and secretion in combination with splenectomy and hepatectomy revealed that the spleen generated not only TNF but also factors that enhanced TNF production by the liver, the latter of which accounted for nearly half of the TNF secreted into the circulation. Using mass spectrometry-based lipidomics, we identified leukotriene B4 (LTB4) as a candidate blood-borne messenger in this spleen-liver axis. LTB4 was essential for spleen-liver communication in vivo, as well as for humoral signaling between splenic macrophages and Kupffer cells in vitro. LPS stimulated the splenic macrophages to secrete LTB4, which primed Kupffer cells to secrete more TNF in response to LPS in a manner dependent on LTB4 receptors. These findings provide a framework to understand how systemic inflammation can be regulated at the level of interorgan communication.

Pigment production by Fusarium solani BRM054066 and determination of antioxidant and anti-inflammatory properties.

The fungal kingdom has been widely studied as a source of bioactive compounds of interest to the pharmaceutical and food industry. This paper studies the production of natural red pigments by Fusarium solani BRM054066 in the submerged fermentation system, using Doehlert experimental design to determine optimal cultivation conditions. The chemical composition of the red pigment was determined by Nuclear Magnetic Resonance spectroscopy (NMR) and Ultra-Performance Liquid Chromatography coupled to Mass Spectrometry (UPLC-MS). Antioxidant activity was assessed by the ability to sequester of free radical DPPH. In the analysis of anti-inflammatory activity, murine peritoneal macrophages activated by LPS were used, and the gene expression of TNF-α, IL-1ß, IL-6, IL-10 and IL-17 was determined using qPCR. As a result, it was found that agitation at 200 rpm and glucose concentration ≥ 20 g/L promote the best results in the production of red pigment. The chemical compounds identified were two naphthoquinones, fusarubin and dihydrofusarubin, and an anthraquinone, a bostrycoidin, being fusarubin the majority compound. The red pigment showed antioxidant activity by scavenge 50% of the DPPH radical, in a concentration of 24 µg/mL. The pigment also showed an effective anti-inflammatory capacity by reducing the overexpression of the pro-inflammatory cytokines TNF-α, IL-1ß and IL-6 and promoting the production of anti-inflammatory IL-10 and IL-17, in murine macrophages activated by LPS (p < 0.05). According to the results, the fungus F. solani BRM054066, under optimized conditions of cultivation, proved to be a promising source of biologically active natural pigments with wide industrial applicability.

Molecular characteristics of methicillin-resistant Staphylococcus aureus isolates from hospital and community environments in northeastern Brazil

ABSTRACT This study characterized 30 MRSA isolates from intensive care unit (ICU) environment and equipment surfaces and healthy children. The SCCmec types I, IVa and V were detected in HA-MRSA isolates while CA-MRSA showed the SCCmec type IVa and V. Most isolates were classified as agr group II. All isolates presented the sei gene, and only HA-MRSA were positive for etb e tst genes. Three genotypes were related to Pediatric (ST5/SCCmecIV) and Berlin (ST45/SCCmecIV) clones. The present study showed molecular similarity between CA- and HA-MRSA isolates in hospital and community settings in a Brazilian region.

DMH-CBD, a cannabidiol analog with reduced cytotoxicity, inhibits TNF production by targeting NF-kB activity dependent on A2A receptor.

Cannabidiol (CBD) is a natural compound with psychoactive therapeutic properties well described. Conversely, the immunological effects of CBD are still poorly explored. In this study, the potential anti-inflammatory effects and underlying mechanisms of CBD and its analog Dimethyl-Heptyl-Cannabidiol (DMH-CBD) were investigated using RAW 264.7 macrophages. CBD and DMH-CBD suppressed LPS-induced TNF production and NF-kB activity in a concentration-dependent manner. Both compounds reduced the NF-kB activity in a µM concentration range: CBD (IC50 = 15 µM) and DMH-CBD (IC50 = 38 µM). However, the concentrations of CBD that mediated NF-kB inhibition were similar to those that cause cytotoxicity (LC50 = 58 µM). Differently, DMH-CBD inhibited the NF-kB activation without cytotoxic effects at the same concentrations, although it provokes cytotoxicity at long-term exposure. The inhibitory action of the DMH-CBD on NF-kB activity was not related to the reduction in IkBα degradation or either p65 (NF-kB) translocation to the nucleus, although it decreased p38 MAP kinase phosphorylation. Additionally, 8-(3-Chlorostyryl) caffeine (CSC), an A2A antagonist, reversed the effect of DMH-CBD on NF-kB activity in a concentration-dependent manner. Collectively, our results demonstrated that CBD reduces NF-kB activity at concentrations intimately associated with those that cause cell death, whereas DMH-CBD decreases NF-kB activity at non-toxic concentrations in an A2A receptor dependent-manner.

Ureaplasma diversum and Its Membrane-Associated Lipoproteins Activate Inflammatory Genes Through the NF-κB Pathway via Toll-Like Receptor 4.

Objectives:Ureaplasma diversum is a pathogen of cows that may cause intense inflammatory responses in the reproductive tract and interfere with bovine reproduction. The aims of this study were to evaluate the immune response of bovine blastocysts and macrophages to U. diversum infection and to evaluate the invasion capacity of this microorganism in bovine blastocysts. Methods: Viable and heat-inactivated U. diversum strains ATCC 49782 and CI-GOTA and their extracted membrane lipoproteins were inoculated in macrophages in the presence or absence of signaling blockers of Toll-Like Receptor (TLR) 4, TLR2/4, and Nuclear Factor KB (NF-κB). In addition, the same viable U. diversum strains were used to infect bovine blastocysts. RNA was extracted from infected and lipoprotein-exposed macrophages and infected blastocysts and assayed by qPCR to evaluate the expression of Interleukin 1 beta (IL-1ß), Tumor Necrosis Factor Alpha (TNF-α), TLR2 and TLR4 genes. U. diversum internalization in blastocysts was followed by confocal microscopy. Results: Both Ureaplasma strains and different concentrations of extracted lipoproteins induced a higher gene expression of IL-1ß, TNF-α, TLR2, and TLR4 in macrophages (p < 0.05) when compared to non-infected cells. The used blockers inhibited the expression of IL-1ß and TNF-α in all treatments. Moreover, U. diversum was able to internalize within blastocysts and induce a higher gene expression of IL-1b and TNF- α when compared to non-infected blastocysts (p < 0.05). Conclusion: The obtained results strongly suggest that U. diversum and its lipoproteins interact with TLR4 in a signaling pathway acting via NF-kB signaling to stimulate the inflammatory response. This is the first study to evaluate the in vitro immunological response of macrophages and bovine blastocysts against U. diversum. These results may contribute to a better understanding of the immunomodulatory activity and pathogenicity of this infectious agent.

Mycoplasma genitalium can modulate the local immune response in patients with endometriosis.

OBJECTIVE: To detect Mollicutes in women with endometriosis and healthy peritoneal tissues and evaluate the participation of these bacteria in the immune response during endometriosis. DESIGN: Cross-sectional study. SETTING: University hospitals. PATIENT (S): Women with endometriosis (n = 73) and without endometriosis (n = 31). INTERVENTION(S): Endocervical swabs, peritoneal fluid, and biopsied lesions of endometriosis of women with endometriosis (study group) and healthy peritoneal tissues (control group) were collected during surgery. Clinical characteristics were registered before surgery. MAIN OUTCOME MEASURE(S): We determined the infectious agents with the use of quantitative polymerase chain reaction (PCR). The cytokine secretion profile was determined with the use of Luminex. The expression of immune response related genes was determined with the use of a PCR array kit. RESULT(S): All target microorganisms were detected at least once in the swab samples analyzed. It was possible to observe higher diversity of microorganisms in the samples of swab and peritoneal fluid in the study group compared with the control. Ureaplasma parvum was associated with the severity of the symptom dyspareunia. Mycoplasma genitalium was associated with higher production of interferon-γ and interleukin-1ß. Genes of inflammatory response activation and antigen presentation were up-regulated in biopsied tissue of women with endometriosis. In women with endometriosis, peritoneal fluid cells showed a down-regulation of genes associated with the inflammatory response. This down-regulation profile was higher in presence of M. genitalium. CONCLUSION(S): Mycoplasma genitalium may play a key role in the immune tolerance process and, especially, the aggravation of this profile. More studies are needed to understand this immune tolerance profile of bacterial infections.

Detection of the Cytotoxic Penitrems A-F in Cheese from the European Single Market by HPLC-MS/MS.

Penitrems are fungal indole diterpene-derived tremorgenic secondary metabolites, which are mainly produced by Penicillium spp. Several cases of intoxications with penitrems and subsequent occurrences of penitrem A in foodstuff underline the need for reliable quantitation methods for the detection of these mycotoxins in food. In this study, a simple and fast high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the quantitative analysis of penitrems A-F in cheese was developed. Therefore, penitrems A-F were isolated from Penicillium crustosum as analytical reference standards. The analysis of 60 cheese samples from the European single market (EU) revealed the occurrence of penitrem A in 10% of the analyzed samples with an average concentration of 28.4 µg/kg and a maximum concentration of 429 µg/kg. In addition to penitrem A, other members of the group of penitrems, namely, penitrems B, C, D, E, and F, were for the first time quantitatively detected in food samples, although in lower concentrations and with lower incidence in comparison to penitrem A. Moreover, we report cytotoxic effects of all penitrems on two cell lines (HepG2 and CCF-STTG1). This clearly underlines their relevance and the importance to analyze food samples in order to get insights into the human exposure toward these mycotoxins.

Bacterial pathogens of the lower respiratory tract of calves from Brazilian rural settlement herds and their association with clinical signs of bovine respiratory disease / Patógenos bacterianos do trato respiratório inferior de bezerros criados em rebanhos de assentamentos brasileiros e sua associação com os sinais clínicos da doença respiratória dos bovinos

Bovine respiratory disease (BRD) is considered the major cause of economic losses in dairy and beef cattle production. The study aimed to detect the most important bacteria related to respiratory disease in tracheobronchial fluid samples of healthy and dairy calves with clinical signs of BRD in Brazilian rural settlements. Hundred and forty-one mongrel dairy calves were randomly selected from 42 family farm dairy herds from Brazilian settlements. Physical examination was performed and calves were classified as healthy (n=100) and BRD (n=41). Tracheobronchial fluid samples were collected. Isolation and molecular detection of Mycoplasma dispar, M. bovis and M. mycoides subsp. mycoides SC besides isolation of other aerobic bacteria were performed. Abnormal lung sounds (crackle/snoring/whistle), mucopurulent/purulent nasal discharge, body temperature >39.5°C and respiratory rate >40 breaths/min were higher in BRD calves compared to healthy calves (P<0.05). Bacillus sp., Staphylococcus intermedius and non-fermentative Gram-negative were the most prevalent bacteria isolated. Non-identified species from Enterobacteriaceae family was higher in BRD calves compared to healthy calves (P<0.05). Mollicutes were isolated in 7.4% of samples and only M. dispar was detected. Mollicutes was associated with purulent/mucopurulent nasal discharge (P=0.017). Pantoea agglomerans was associated to tachypnea (P=0.020), and Streptococcus spp. was associated with hyperthermia. Statistical tendencies were observed to M. dispar and tachypnea (P=0.066), and P. agglomerans and tachycardia (P=0.066). The obtained results describe the microorganisms found in tracheobronchial fluid of calves with BRD in some herds of Brazilian family farming and their relation to clinical signs of BRD.(AU)

A doença respiratória dos bovinos (DRB) é considerada a principal causa de perdas econômicas nas produções de leite e carne. O objetivo deste estudo foi detectar as mais importantes bactérias relacionadas a doença respiratória presentes em amostras de lavado traqueobrônquico de bezerros sadios e com sinais clínicos da DRB de assentamentos brasileiros. Cento e quarenta e um bezerros leiteiros sem raça definida foram randomicamente selecionados de 42 rebanhos leiteiros de assentamentos brasileiros. Exame físico foi realizado e os animais foram classificados em sadios (n=100) e com DRB (n=41). Amostras de lavado traqueobrônquico foram coletadas. Foram realizados o isolamento e a detecção molecular de Mycoplasma dispar, M. bovis e M. mycoides subsp. mycoides SC além de isolamento de outras bactérias aeróbias. Ruídos pulmonares anormais (crepitação/ ronco/sibilo), secreção nasal mucopurulenta/purulenta, temperatura corporal >39.5°C e frequência respiratória >40 movimentos respiratórios/min foram observados com maior frequência em bezerros com DRB comparado aos animais sadios (P<0.05). Bacillus sp, Staphylococcus intermedius e bactérias Gram-negativas não fermentadoras foram as bactérias mais prevalentes. Bactérias da família Enterobacteriaceae cuja espécie não fora identificada foram mais frequentes em bezerros com DRB comparado aos bezerros sadios (P<0.05). Mollicutes foram isolados em 7,4% das amostras e somente M. dispar foi detectado. Mollicutes foi associado à secreção nasal purulenta/mucopurulenta (P=0.017). Pantoea agglomerans foi associada a taquipneia (P=0.020), e Streptococcus spp. Foi associado a hipertermia. Tendência estatística foi observada para M. dispar e taquipneia (P=0.066), e P. agglomerans e taquicardia (P=0.066). Os resultados obtidos descrevem os micro-organismos encontrados no lavado traqueobrônquico de bezerros com DRB em rebanhos de agricultura familiar brasileira e sua relação com as manifestações clínicas da DRB.(AU)

Community-acquired methicillin-resistant Staphylococcus aureus carrying SCCmec type IV and V isolated from healthy children attending public daycares in northeastern Brazil

Abstract Nasal colonization with methicillin-resistant Staphylococcus aureus (MRSA) have increasingly been reported in healthy communities. This study aimed to assess the rate of S. aureus in general and MRSA in particular from nasal secretion of children in daycare centers in Vitória da Conquista, Brazil. The isolates were identified based on morphology, biochemical tests and by PCR. Detection of virulence genes, biofilm production, and susceptibility test by disk diffusion agar were performed. MRSA isolates were characterized by spa, SCCmec, and multilocus sequence typing (MLST). S. aureus were recovered from 70 (47.3%) of 148 children. Among the 11 MRSA strains (15.7%), two SCCmec types (IV and V) were detected. MLST identified four STs related to three clonal complexes (CC): 5, 45, and 398. Four spa types were found circulating in this setting. Resistance of S. aureus isolates to ampicillin, erythromycin, ciprofloxacin, clindamycin, and tetracycline was 80%, 32.8%, 7.1%, 7.1% and 4.3%, respectively. One isolate presented intermediate resistance to vancomycin detected by Etest methodology. All strains were biofilm producers. The virulence genes seb, sec, spa, and pvl were detected in some isolates. This study revealed a high rate of children carrying MRSA among healthy attendees in daycare centers in Vitória da Conquista, Brazil.

Molecular characterization of ureaplasmas isolated from reproductive tract of goats and sheep from Brazil

Abstract Ovine/caprine ureaplasmas have not yet been assigned a species designation, but they have been classified into nine serotypes. Herein ureaplasmas were searched for in 120 samples of vulvo vaginal mucous from sheep and 98 samples from goats at 17 farms. In addition, semen samples were collected from 11 sheep and 23 goats. The recovered ureaplasma were from sheep and goats from animals without any reproductive disorder symptoms, but not all animals presented positive cultures. In sheep, 17 (68%) cultures of vulvovaginal mucous were positive for ureaplasma and 11 (27%) samples of semen presented positive cultures in animals with clinical signs of orchitis, balanoposthitis or low sperm motility. In goats four ureaplasma isolates were obtained from vulvovaginal mucus, but the semen samples were all negative. The isolates were submitted to Pulsed-field gel electrophoresis methodology and their 16S rRNA genes were sequenced. Fifty percent of ureaplasma recovered from sheep allowed for PFGE typing. Eleven isolates showed eight profiles genetically close to the bovine ureaplasmas. The 16S rRNA gene sequencing showed differences or similarities of isolates from sheep and goats, and the reference strains of bovine and human ureaplasma. Four clinical isolates from sheep were grouped separately. The studied ureaplasma isolates showed to be a diverse group of mollicutes.

Anti-Inflammatory Activity of the Essential Oil Citral in Experimental Infection with Staphylococcus aureus in a Model Air Pouch.

This study proposes to implement an alternative and effective strategy for local treatment of disease provoked by S. aureus. For the analysis of possible anti-inflammatory activity of essential oil, after establishing an air pouch model, 48 male mice of Balb/c were treated, infected, and euthanized at 4 and 8 h. Thus, the total and differential white blood cells were counted in the animal's blood, and cytokines IL-1ß, IL-6, and TNF-α were titrated using ELISA in the air pouch lavage. Moreover, TNF-α, IL-1ß, and IL-6 gene expression was analyzed through an RT-qPCR array, and S. aureus was quantified using qPCR. Our results, p < 0.05, showed that EOC reduced the quantity of microorganisms. The group of mice treated with essential oil citral showed a significant decrease in TNF-α levels in tests demonstrating anti-inflammatory activity. There is no data about the mutual influence of the air pouch model, essential oil citral, and S. aureus. Thus, considering the interaction of these variables and the anti-inflammatory activity of the essential oil citral, we demonstrated, by alternative local treatment, a new antimicrobial agent that is not an antibiotic.

Metabolic profile and safety of piperlongumine.

Piperlongumine (PPL), a natural plant product, has been extensively studied in cancer treatment going up on clinical trials. Since the first report related to its use on cancer research (in 2011) around 80 papers have been published in less than 10 years, but a gap still remaining. There are no metabolism studies of PPL in human organism. For the lack of a better view, here, the CYP450 in vitro oxidation of PPL was described for the first time. In addition, the enzymatic kinetic data, the predicted in vivo parameters, the produced metabolites, the phenotyping study and possible piperlongumine-drug interactions in vivo is presented.

Intra-uterine experimental infection by Ureaplasma diversum induces TNF-α mediated womb inflammation in mice.

Ureaplasma diversum is an opportunistic pathogen associated with uterine inflammation, impaired embryo implantation, infertility, abortions, premature birth of calves and neonatal pneumonia in cattle. It has been suggested that the intra-uterine infection by Ureaplasma diversum can cause vascular changes that hinder the success of pregnancy. Thus, the aim of this study was to evaluate the changes of intrauterine site of A/J mice in estrus or proestrus phase inoculated with Ureaplasma diversum. The infection was monitored at 24, 48 and 72 hours by the PCR methodology to detect the Ureaplasma in the inoculation site and the profile of circulating blood cells. Morphological changes, intensity of inflammation and the production of cytokines were compared. The infected mice showed local inflammation through the production of IFN-γ and TNF-α. Ureaplasma diversum infections in the reproductive tract of studied mice seemed to be associated with the production of pro-inflammatory cytokines in uterine parenchyma. The levels of TNF-α of infected mice were dependent on the bacterial load of inoculated Ureaplasma. Uterine experimental infections by Ureaplasma diversum have not been mentioned yet and herein we presented the first report of an intrauterine infection model in mice.

Leishmanicidal evaluation of tetrahydroprotoberberine and spirocyclic erythrina-alkaloids.

Leishmaniasis is one of the World's most problematic diseases in developing countries. Traditional medicines to treat leishmaniasis have serious side effects, as well as significant parasite resistance problems. In this work, two alkaloids 1 and 2 were obtained from Corydalis govaniana Wall and seven alkaloids 3-9, were obtained from Erythrina verna. The structures of the compounds were confirmed by mass spectrometry and 1D- and 2D-NMR spectroscopy. The leishmanicidal activity of compounds 1-9 against Leishmania amazonensis was tested on promastigote forms and cytotoxicity against J774 (macrophage cell line) was assessed in vitro. Compound 1 showed potent activity (IC50 = 0.18 µg/mL), compared with the standard amphotericin B (IC50 = 0.20 µg/mL). The spirocyclic erythrina-alkaloids showed lower leishmanicidal activity than dibenzoquinolizine type alkaloids.

Isolation, pathogenicity and disinfection of Staphylococcus aureus carried by insects in two public hospitals of Vitória da Conquista, Bahia, Brazil

Currently, hospital infection is a serious public health problem, and several factors may influence the occurrence of these infections, including the presence of insects, which are carriers of multidrug-resistant bacterial species. The aim of this study was to isolate staphylococci carried by insects in two public hospitals of Vitoria da Conquista, Bahia and to identify the resistance profile, pathogenicity and efficacy of disinfection of the premises. A total of 91 insects were collected in 21 strategic points of these hospitals, and 32 isolated strains ofStaphylococcus aureus were isolated. Based on antibiogram and Minimum Inhibitory Concentration results, 95% of these strains were susceptible to oxacillin. These strains were also evaluated for the presence of resistance genes encoding resistance to oxacillin/methicillin by polymerase chain reaction, but the sample was negative for this gene. Pathogenicity tests were performed in vitro biofilm formation induced by glucose, where it was found that eight (27.58%) strains were classified as biofilm producers and 21 (72.4%) as stronger producers. In addition, we performed PCR for their virulence genes: Sea (enterotoxin A), SEB (B), Sec (C), PVL (Panton-Valentine Leukocidin), ClfA (clumping factor A) and Spa (protein A). Of these, Sea, Spa PVL were positive in 7 (21.8%), 2 (6.3%) and 1 (3.1%) samples, respectively. The analysis of cytokine induction in the inflammatory response of J774 macrophages by isolates from the two hospitals did not show statistical difference at the levels of IL-6, TNF-α, IL-1 and IL-10 production. In addition, we verified the antimicrobial activity of disinfecting agents on these strains, quaternary ammonium, 0.5% sodium hypochlorite, 1% sodium hypochlorite, 2% sodium hypochlorite, 2% glutaraldehyde, Lysoform®, 70% alcohol solution of chlorhexidine digluconate, 2% peracetic acid, and 100% vinegar. Resistance was seen in only for the following two disinfectants: 70% alcohol in 31 (96.8%) samples tested and vinegar in 30 (93.8%) samples. The study demonstrated the presence of resistant and pathogenic organisms conveyed by insects, thus suggesting improvement in efforts to control these vectors.

Brazilian red propolis: unreported substances, antioxidant and antimicrobial activities.

BACKGROUND: Chloroform, ethyl acetate and methanol extracts of a sample of red propolis from the state of Alagoas (northeast Brazil) were analyzed by gas chromatography-mass spectrometry and high-performance liquid chromatography-diode array detection-electrospray ionization-mass spectrometry. Antimicrobial and antioxidant activities were also obtained. RESULTS: The propolis sample contained low content of narigenin-8-C-hexoside, this being the first report of a C-glycoside in propolis. The main constituent found was characterized as 3,4,2',3'-tetrahydroxychalcone. Other important constituents were the chalcone isoliquiritigenin, the isoflavans (3S)-vestitol, (3S)-7-O-methylvestitol, the pterocarpan medicarpin, the phenylpropenes trans-anethol, methyl eugenol, elimicin, methoxyeugenol and cis-asarone, and the triterpenic alcohols lupeol and α- and ß- amyrins. The methanol extract exhibited high antioxidant activities by 2,2-diphenyl-1-picrylhydrazyl and ß-carotene/linoleic acid assay methods, and antimicrobial activity toward Gram-positive and Gram-negative bacteria. CONCLUSION: Structures are suggested for new substances never before seen in any kind of propolis. This is the first report of 3,4,2',3'-tetrahydroxychalcone and a flavone C-glycoside in a propolis sample.

Survey of feline leukemia virus and feline coronaviruses in captive neotropical wild felids from Southern Brazil.

A total of 57 captive neotropical felids (one Leopardus geoffroyi, 14 Leopardus pardalis, 17 Leopardus wiedii, 22 Leopardus tigrinus, and three Puma yagouaroundi) from the Itaipu Binacional Wildlife Research Center (Refúgio Bela Vista, Southern Brazil) were anesthetized for blood collection. Feces samples were available for 44 animals, including one L. geoffroyi, eight L. pardalis, 14 L. wiedii, 20 L. tigrinus, and one P. yagouaroundi. Total DNA and RNA were extracted from blood and feces, respectively, using commercial kits. Blood DNA samples were evaluated by polymerase chain reaction (PCR) for feline leukemia virus (FeLV) proviral DNA, whereas reverse transcriptase-PCR was run on fecal samples for detection of coronavirus RNA. None of the samples were positive for coronaviruses. A male L. pardalis and a female L. tigrinus were positive for FeLV proviral DNA, and identities of PCR products were confirmed by sequencing. This is the first evidence of FeLV proviral DNA in these species in Southern Brazil.