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Background: Hereditary equine regional dermal asthenia (HERDA) is a genetic disease that alters collagen biosynthesis. Affected horses exhibit fragile, hyperextensible skin, especially over the dorsal region. Although ultraviolet (UV) radiation seems to contribute to the regional distribution of lesions and worsening of clinical signs, the molecular mechanisms involved are largely unknown., Objectives: To evaluate the effect of solar radiation on matrix metalloproteinase MMP1, MMP8 and MMP13 gene expression in the dorsal and ventral skin of HERDA-affected and HERDA-unaffected horses [wild-type (WT) horses]., Animals: Six HERDA-affected and six unaffected Quarter horses (WT) were paired according to age, sex and coat colour., Materials and Methods: Horses were submitted to 30 day sunlight restriction, followed by 15 day sunlight exposure. Dorsal and ventral skin biopsies were obtained at six sampling times over 45 days. The expression of MMP1, MMP8 and MMP13 genes was measured by quantitative PCR., Results: Although solar radiation modulated MMP1, MMP8 and MMP13 expression, the effects were more pronounced on MMP1. Sun exposure for three days significantly upregulated MMP1 in the dorsal region when compared to the ventral skin in both unaffected and HERDA-affected horses., Conclusions and Clinical Relevance: This study shows that solar irradiation leads to upregulation of skin collagenase genes particularly MMP1 in the dorsal, sun-exposed skin of horses. Furthermore, this was more marked in HERDA-affected horses. The increased activity of collagenases on the disorganised collagen present in HERDA affected horses would explain why UV radiation leads to deterioration of clinical signs in affected individuals., (© 2022 ESVD and ACVD.)

Brazil currently ranks second in absolute deaths by COVID-19, even though most of its population has completed the vaccination protocol. With the introduction of Omicron in late 2021, the number of COVID-19 cases soared once again in the country. We investigated in this work how lineages BA.1 and BA.2 entered and spread in the country by sequencing 2173 new SARS-CoV-2 genomes collected between October 2021 and April 2022 and analyzing them in addition to more than 18,000 publicly available sequences with phylodynamic methods. We registered that Omicron was present in Brazil as early as 16 November 2021 and by January 2022 was already more than 99% of samples. More importantly, we detected that Omicron has been mostly imported through the state of São Paulo, which in turn dispersed the lineages to other states and regions of Brazil. This knowledge can be used to implement more efficient non-pharmaceutical interventions against the introduction of new SARS-CoV variants focused on surveillance of airports and ground transportation.

Non-SARS-CoV-2 respiratory viral infections, such as influenza virus (FluV) and human respiratory syncytial virus (RSV), have contributed considerably to the burden of infectious diseases in the non-COVID-19 era. While the rates of co-infection in SARS-CoV-2-positive group (SCPG) patients have been determined, the burden of other respiratory viruses in the SARS-CoV-2-negative group (SCNG) remains unclear. Here, we conducted a cross-sectional study (São José do Rio Preto county, Brazil), and we collected our data using a meta-analysis to evaluate the pooled prevalence of FluV and RSV among SCNG patients. Out of the 901 patients suspected of COVID-19, our molecular results showed positivity of FluV and RSV in the SCNG was 2% (15/733) and 0.27% (2/733), respectively. Co-infection with SARS-CoV-2 and FluV, or RSV, was identified in 1.7% of the patients (3/168). Following our meta-analysis, 28 studies were selected ( n = 114,318 suspected COVID-19 patients), with a pooled prevalence of 4% (95% CI: 3-6) for FluV and 2% (95% CI: 1-3) for RSV among SCNG patients were observed. Interestingly, FluV positivity in the SCNG was four times higher (OR = 4, 95% CI: 3.6-5.4, p < 0.01) than in the SCPG. Similarly, RSV positivity was significantly associated with SCNG patients (OR = 2.9, 95% CI: 2-4, p < 0.01). For subgroup analysis, cold-like symptoms, including fever, cough, sore throat, headache, myalgia, diarrhea, and nausea/vomiting, were positively associated ( p < 0.05) with the SCPG. In conclusion, these results show that the pooled prevalence of FluV and RSV were significantly higher in the SCNG than in the SCPG during the early phase of the COVID-19 pandemic.

Zika virus (ZIKV), a mosquito-borne pathogen, is an emerging arbovirus associated with sporadic symptomatic cases of great medical concern, particularly among pregnant women and newborns affected with neurological disorders. Serological diagnosis of ZIKV infection is still an unmet challenge due to the co-circulation of the dengue virus, which shares extensive sequence conservation of structural proteins leading to the generation of cross-reactive antibodies. In this study, we aimed to obtain tools for the development of improved serological tests for the detection of ZIKV infection. Polyclonal sera (pAb) and a monoclonal antibody (mAb 2F2) against a recombinant form of the ZIKV nonstructural protein 1 (NS1) allowed the identification of linear peptide epitopes of the NS1 protein. Based on these findings, six chemically synthesized peptides were tested both in dot blot and ELISA assays using convalescent sera collected from ZIKV-infected patients. Two of these peptides specifically detected the presence of ZIKV antibodies and proved to be candidates for the detection of ZIKV-infected subjects. The availability of these tools opens perspectives for the development of NS1-based serological tests with enhanced sensitivity regarding other flaviviruses.

COVID-19 has posed a worldwide public health challenge affecting millions of people in different countries. Rapid and efficient detection of SARS-CoV-2 is essential for pandemic control. Reverse Transcription quantitative PCR (RT-qPCR) of nasopharyngeal swabs is the gold standard method for the virus detection, but the high demand for tests has substantially increased the costs and reduced the availability of reagents, including genetic material purification kits. Thus, the present study aimed to compare two bead-based RNA extraction methods (an in-house and a commercial kit) from nasopharyngeal swabs and RT-qPCR detection of SARS-CoV-2. Twenty-five positive and five negative nasopharyngeal swab samples were subjected to extraction of nucleic acids using both methods in an automated platform. Both protocols revealed a high correlation between Cycle Quantifications (Cqs) (r = 0.99, p < 0.0001). In addition, the in-house kit was 89.5 % cheaper when compared to the mean cost of commercial RNA extraction kits. The results show that the in-house protocol is an affordable and reliable option for RNA extraction for SARS-CoV-2 detection from nasopharyngeal swabs., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The emergence of several SARS-CoV-2 lineages presenting adaptive mutations is a matter of concern worldwide due to their potential ability to increase transmission and/or evade the immune response. While performing epidemiological and genomic surveillance of SARS-CoV-2 in samples from Porto Ferreira-São Paulo-Brazil, we identified sequences classified by pangolin as B.1.1.28 harboring Spike L452R mutation, in the RBD region. Phylogenetic analysis revealed that these sequences grouped into a monophyletic branch, with others from Brazil, mainly from the state of São Paulo. The sequences had a set of 15 clade defining amino acid mutations, of which six were in the Spike protein. A new lineage was proposed to Pango and it was accepted and designated P.4. In samples from the city of Porto Ferreira, P.4 lineage has been increasing in frequency since it was first detected in March 2021, corresponding to 34.7% of the samples sequenced in June, the second in prevalence after P.1. Also, it is circulating in 30 cities from the state of São Paulo, and it was also detected in one sample from the state of Sergipe and two from the state of Rio de Janeiro. Further studies are needed to understand whether P.4 should be considered a new threat., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bittar, Possebon, Ullmann, Geraldini, da Costa, de Almeida, da S. Sanches, Nascimento-Júnior, Cilli, Artico Banho, Campos, Ferreira, Sacchetto, da Silva, Parra, Moraes, da Costa, Vasconcelos, Spilki, Nogueira, Rahal and Araujo Jr.)

Equine infectious anemia virus (EIAV) is a persistent lentivirus that causes equine infectious anemia (EIA). In Brazil, EIAV is endemic in the Pantanal region, and euthanasia is not mandatory in this area. All of the complete genomic sequences from field viruses are from North America, Asia, and Europe, and only proviral genomic sequences are available. Sequences from Brazilian EIAV are currently available only for gag and LTR regions. Thus, the present study aimed for the first time to sequence the entire EIAV genomic RNA in naturally infected horses from an endemic area in Brazil. RNA in plasma from naturally infected horses was used for next-generation sequencing (NGS), and gaps were filled using Sanger sequencing methodology. Complete viral genomes of EIAV from two horses were obtained and annotated (Access Number: MN560970 and MN560971). Putative genes were analyzed and compared with previously described genes, showing conservation in gag and pol genes and high variations in LTR and env sequences. Amino acid changes were identified in the p26 protein, one of the most common targets used for diagnosis, and p26 molecular modelling showed surface amino acid alterations in some epitopes. Brazilian genome sequences presented 88.6% nucleotide identity with one another and 75.8 to 77.3% with main field strains, such as EIAV Liaoning, Wyoming, Ireland, and Italy isolates. Furthermore, phylogenetic analysis suggested that this Brazilian strain comprises a separate monophyletic group. These results may help to better characterize EIAV and to overcome the challenges of diagnosing and controlling EIA in endemic regions.

Ehrlichia minasensis is a tick-borne pathogen affecting cattle, cervids, and dogs, and it is closely related to the monocytotropic pathogen Ehrlichia canis Here, we announce the draft genome sequence of Ehrlichia minasensis strain Cuiabá, isolated from a naturally infected calf from Santo Antônio do Leverger, Mato Grosso, Brazil., (Copyright © 2019 Aguiar et al.)

Porcine circovirus 2 (PCV2) is an icosahedral, non-enveloped, and single-stranded circular DNA virus that belongs to the family Circoviridae, genus Circovirus, and is responsible for a complex of different diseases defined as porcine circovirus diseases (PCVDs). These diseases - including postweaning multisystemic wasting syndrome (PMWS), enteric disease, respiratory disease, porcine dermatitis and nephropathy syndrome (PDNS), and reproductive failure - are responsible for large economic losses in the pig industry. After serial passages in swine testicle (ST) cells of a wild-type virus isolated from an animal with PMWS, we identified three PCV2b viruses with capsid protein (known as Cap protein) cumulative mutations, including two novel mutants. The mutant viruses were introduced into new ST cell cultures for reisolation and showed, in comparison to the wild-type PCV2b, remarkable viral replication efficiency (> 10 11 DNA copies/ml) and cell death via necrosis, which were clearly related to the accretion of capsid protein mutations. The analysis of a Cap protein/capsid model showed that the mutated residues were located in solvent-accessible positions on the external PCV2b surface. Additionally, the mutated residues were found in linear epitopes and participated in pockets on the capsid surface, indicating that these residues could also be involved in antibody recognition. Taking into account the likely natural emergence of PCV2b variants, it is possible to consider that the results of this work increase knowledge of Circovirus biology and could help to prevent future serious cases of vaccine failure that could lead to heavy losses to the swine industry., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Bat rabies surveillance data and risk factors for rabies spillover without human cases have been evaluated in Curitiba, the ninth biggest city in Brazil, during a 6-year period (2010-2015). A retrospective analysis of bat complaints, bat species identification and rabies testing of bats, dogs and cats has been performed using methodologies of seasonal decomposition, spatial distribution and kernel density analysis., Results: Overall, a total of 1003 requests for bat removal have been attended to, and 806 bats were collected in 606 city locations. Bat species were identified among 13 genera of three families, with a higher frequency of Nyctinomops in the central-northern region and Molossidae scattered throughout city limits. Out of the bats captured alive, 419/806 (52.0%) healthy bats were released due to absence of human or animal contacts. The remaining 387/806 (48.0%) bats were sent for euthanasia and rabies testing, which resulted in 9/387 (2.32%) positives. Linear regression has shown an increase on sample numbers tested over time (regression: y = 2.02 + 0.17×; p < 0.001 and r 2  = 0.29), as well as significant seasonal variation, which increases in January and decreases in May, June and July. The Kernel density analysis showed the center-northern city area to be statistically important, and the southern region had no tested samples within the period. In addition, a total of 4769 random and suspicious samples were sent for rabies diagnosis including those from dogs, cats, bats and others from 2007 to 2015. While all 2676 dog brains tested negative, only 1/1136 (0.088%) cat brains tested positive for rabies., Conclusion: Only non-hematophagous bats were collected during the study, and the highest frequency of collections occurred in the center-northern region of the city. Rabies spillover from bats to cats may be more likely due to the registered exposure associated with cats' innate hunting habits, predisposing them to even closer contact with potentially infected bats. Although associated with a very low frequency of rabies, cats should always be included in rabies surveillance and vaccination programs.

Preeclampsia is a pregnancy disorder characterized by imbalance between pro- and anti-inflammatory cytokines associated with high plasma levels of uric acid and Interleukin-1 beta (IL-1β). The inflammasome is a protein complex that mediates innate immune responses via caspase-1 activation promoting secretion of IL-1β and IL-18 in their active forms, and also release of the high-mobility group box 1 protein (HMGB1). As the placenta seems to play an important role in the pathogenesis of PE, the present study investigated the expression of genes and proteins related to the inflammasome in placentas from pregnant women with severe preeclampsia. Placental tissue was collected from 20 normotensive pregnant women and 20 preeclamptic women, and inflammasome components, NLRP3 (NOD-like receptor family, pyrin domain-containing protein 3), caspase-1, IL-1β and IL-18, as well as tumor necrosis factor-alpha (TNF-α) and HMGB1 were evaluated by immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and also quantified by reverse transcription-qPCR (RT-qPCR). Compared with normotensive pregnant women, placenta from women with PE showed a significant increase in NLRP3, caspase-1, IL-1β, TNF-α and HMGB1 mRNA. Immunohistochemical staining of NLRP3, caspase-1, IL-1β and TNF-α in placental villi, as well as the levels of caspase-1, IL-1β, TNF-α and HMGB1 in placental homogenate were significantly higher in the preeclamptic group than in the normotensive group. However, mRNA expression of IL-18 and its protein concentrations were lower in placentas from preeclamptic women. The results suggest that placentas from pregnant women with preeclampsia show higher expression of NLRP3 inflammasome, which may be involved in the exaggerated inflammatory state in preeclampsia., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Aural plaques can be found on the inner surfaces of one or both ears of horses. Despite their low malignancy, these lesions can sometimes cause discomfort and sensitivity in horses, and a loss in commercial value due to their aesthetic effect. There has been a study describing the epidemiological features and the clinical prevalence of equine aural plaques in Brazil., Hypothesis/objectives: To determine the clinical prevalence and selected associated factors of aural plaques., Animals: In the study, 891 horses were assessed for aural plaques. The sample group had a median age of 5 years and comprised both sexes and various breeds from different regions of Brazil., Methods: Horses were evaluated by a general observation of the body and a detailed observation of both ears. Data on the management system, characteristics of the lesions, the presence of ticks and ear grooming were collected for 109 clinically affected horses. An assessment of the frequency distribution of the disease and its characteristics was performed. Association tests were conducted to establish the relationships between the variables studied., Results: In 85% (40 of 47) of farms assessed, at least one horse presented with aural plaques. In 14.8% (132 of 891) of the horses, lesions characteristic of aural plaque were detected. Significant associations between the prevalence of "coalescing" lesions and a "semi-intensive" management system and ear grooming were detected., Conclusions: The findings confirm the extensive distribution of this disease in Brazil and its association with several management factors., (© 2016 ESVD and ACVD.)

Background: Aural plaques are a dermatopathy associated with Equus caballus papillomavirus (EcPV). This disease affects horses of all ages, genders and breeds, and causes sensitivity of the ears., Hypothesis/objectives: The aim of this study was to evaluate the clinical efficacy of 5% imiquimod cream for the treatment of aural plaques and to compare the PCR detection of EcPV 3, 4, 5 and 6 before and after treatment., Animals: Eight horses diagnosed with aural plaques (14 ears) were used. Three mares with unilateral aural plaques were used as untreated controls., Methods: Imiquimod cream was applied every 48 h until complete resolution of the aural plaques was observed. Animals were evaluated clinically for 180 days after the end of treatment. PCR for detecting EcPV 3, 4, 5 and 6 was performed using aural plaque biopsies collected before and at 90 days after the end of treatment., Results: Clinical resolution was observed in 93% of the treated ears. Imiquimod treatment promoted the clearance of EcPV in 71.4% of the treated ears. Clinical remission of the aural plaques and changes in EcPV DNA positivity between the first and second biopsies were not observed in the control group. In 75% of horses, sedation was required in order to carry out pretreatment cleaning., Conclusions: The results of this study support the hypothesis that 5% imiquimod cream may be used as an effective treatment for aural plaques in horses., (© 2016 ESVD and ACVD.)

Sequence-independent methods for viral discovery have been widely used for whole genome sequencing of viruses. Different protocols for viral enrichment, library preparation and sequencing have increasingly been more available and at lower costs. However, no study to date has focused on optimization of viral sample preparation for commercial deep sequencing. Accordingly, the aim of the present study was to evaluate an In-House enzymatic protocol for double-stranded DNA (dsDNA) synthesis and also compare the use of a commercially available kit protocol (Nextera XT, Illumina Inc, San Diego, CA, USA) and its combination with a library quantitation kit (Kapa, Kapa Biosystems, Wilmington, MA, USA) for deep sequencing (Illumina Miseq). Two RNA viruses (canine distemper virus and dengue virus) and one ssDNA virus (porcine circovirus type 2) were tested with the optimized protocols. The tested method for dsDNA synthesis has shown satisfactory results and may be used in laboratory setting, particularly when enzymes are already available. Library preparation combining commercial kits (Nextera XT and Kapa) has yielded more reads and genome coverage, probably due to a lack of small fragment recovering at the normalization step of Nextera XT. In addition, libraries may be diluted or concentrated to provide increase on genome coverage with Kapa quantitation., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In this paper we report the occurrence of zoonotic species of Cryptosporidium (C. ubiquitum and C. parvum subtype IIaA15G2R1) and the host-specific Giardia duodenalis genotype E in a sheep farm from Brazil. Fecal specimens were collected from 100 animals and screened for the presence of G. duodenalis and Cryptosporidium spp. by enzyme immunoassay (EIA) and polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) and sequence analysis of selected samples was used to confirm the species and to characterize the isolates. Of 100 specimens, 34 were positive for Giardia (34%) and 25 were positive for Cryptosporidium (25%). The prevalence of Giardia and Cryptosporidium was higher in lambs than in ewes. Co-infection was observed in 14% of animals. All G. duodenalis isolates belonged to genotype E. The genetic characterization performed in 22 Cryptosporidium-positive samples revealed the presence of zoonotic C. ubiquitum and C. parvum in 76% (19/25) and 12% (3/25) of infected animals, respectively. The presence of zoonotic Cryptosporidium subtypes in lambs was of epidemiological and public health relevance.

Porcine circovirus type 2 (PCV2) is difficult to isolate. Currently, no published articles have used the shell vial technique to isolate PCV2. In addition, the action of d-glucosamine on swine testicle cells (ST) has not been evaluated properly. Thus, the aim of this study was to determine an optimal concentration of d-glucosamine and to test the shell vial technique for PCV2 propagation in ST cells. The optimal concentration of d-glucosamine was determined to be 100mM. Because PCV2 is noncytopathic, the traditional adsorption was compared to the shell vial technique for 15 passages by qPCR, and RT-qPCR for passages 12 through 15. The quantities of viral DNA (P=0.013) and ORF1-mRNA detected with the shell vial technique were two-fold higher than the obtained with traditional adsorption. The levels of ORF2-mRNA were similar for both methods; however, by passage 15, a six-fold increase in levels was observed with the shell vial technique. Therefore, the shell vial technique was more efficient for the cultivation of PCV2, and qPCR/RT-qPCR can be used to monitor viral replication. In addition, a high viral load (>2.7×10(10) DNA copies/ml) and high levels of viral mRNA expression indicated that the ST cells were persistently infected., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Feline immunodeficiency virus (FIV) infection has been the focus of several studies because this virus exhibits genetic and pathogenic characteristics that are similar to those of the human immunodeficiency virus (HIV). FIV causes acquired immunodeficiency syndrome (AIDS) in cats, nevertheless, a large fraction of infected cats remain asymptomatic throughout life despite of persistent chronic infection. This slow disease progression may be due to the presence of factors that are involved in the natural resistance to infection and the immune response that is mounted by the animals, as well as due to the adaptation of the virus to the host. Therefore, the study of virus-host interaction is essential to the understanding of the different patterns of disease course and the virus persistence in the host, and to help with the development of effective vaccines and perhaps the cure of FIV and HIV infections., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Aural plaques occur on the skin of the medial surface of the pinnae of horses. In this study the presence of Equus caballus papillomavirus (EcPV)-3 and -4 DNA was assessed in 45 such plaques using a 'touchdown' PCR. Papillomaviruses (PVs) were detected in 62.3% (28/45) of samples: EcPV-3 and -4 DNA in 8.89% (4/45) and 37.78% (17/45) of samples, respectively, with 15.56% (7/45) of samples exhibiting co-infection. Viral DNA was not detected in 37.78% (17/45) of samples, suggesting the possible existence of other equine PVs. Neither EcPV-3 nor -4 were detected in negative control skin. This study is the first to evaluate the prevalence of these two viruses in equine aural plaques., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Plasmatic concentrations of von Willebrand Factor (vWF) increase during pregnancy in humans and dogs; however the mechanism of such increase is still not well defined. The aims of this study were: (i) to evaluate changes in vWF concentration during pregnancy and during the subsequent oestrous cycle in bitches affected and unaffected by von Willebrand Disease (vWD); (ii) to correlate the vWF levels and cortisol levels in both groups. Seven vWD affected (GI) and nine unaffected (GII) bitches were used. The animals were assessed during pregnancy, parturition, lactation and non-gestational oestrous cycle in 11 moments (Pregnancy 1, Pregnancy 2, Parturition, Lactation 1, Lactation 2, Lactation 3, Anestrus, Proestrus, Oestrus, Diestrus 1, and Diestrus 2). The following tests were performed; measurement of von Willebrand factor antigen (vWF:Ag), albumin and cortisol. In both groups, vWF concentration remained stable during the non-gestational oestrous cycle, but increased during pregnancy, with the highest value observed at parturition. Increases of 70% and 124% in vWF were seen in GI and GII, respectively, compared to anestrus. No correlation was found between vWF and cortisol. Values of vWF:Ag changed during pregnancy, with a peak at parturition, both in vWD affected and unaffected animals. Values of vWF were not altered in the different phases of the oestrous cycle following pregnancy in both groups. Evaluation of vWF during pregnancy can cause false negative results for vWD, but assessment can be performed at any point in the oestrous cycle of non-pregnant bitches., (© 2012 Blackwell Verlag GmbH.)

Papillomaviruses (PVs) infect a wide range of animal species and show great genetic diversity. To date, excluding equine sarcoids, only three species of PVs were identified associated with lesions in horses: Equus caballus papillomavirus 1 (EcPV1-cutaneous), EcPV2 (genital) and EcPV3 (aural plaques). In this study, we identified a novel equine PV from aural plaques, which we designated EcPV4. Cutaneous samples from horses with lesions that were microscopically diagnosed as aural plaques were subjected to DNA extraction, amplification and sequencing. Rolling circle amplification and inverse PCR with specific primers confirmed the presence of an approximately 8 kb circular genome. The full-length EcPV4 L1 major capsid protein sequence has 1488 nucleotides (495 amino acids). EcPV4 had a sequence identity of only 53.3%, 60.2% and 51.7% when compared with the published sequences for EcPV1, EcPV2 and EcPV3, respectively. A Bayesian phylogenetic analysis indicated that EcPV4 clusters with EcPV2, but not with EcPV1 and EcPV3. Using the current PV classification system that is based on the nucleotide sequence of L1, we could not define the genus of the newly identified virus. Therefore, a structural analysis of the L1 protein was carried out to aid in this classification because EcPV4 cause lesion similar to the lesion caused by EcPV3. A comparison of the superficial loops demonstrated a distinct amino acid conservation pattern between EcPV4/EcPV2 and EcPV4/EcPV3. These results demonstrate the presence of a new equine PV species and that structural studies could be useful in the classification of PVs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Cytokines play an essential role during active tuberculosis disease and cytokine genes have been described in association with altered cytokine levels. Therefore, the aim of this study was to verify if IFNG, IL12B, TNF, IL17A, IL10, and TGFB1 gene polymorphisms influence the immune response of Brazilian patients with pulmonary tuberculosis (PTB) at different time points of antituberculosis treatment (T1, T2, and T3). Our results showed the following associations: IFNG +874 T allele and IFNG +2109 A allele with higher IFN- γ levels; IL12B +1188 C allele with higher IL-12 levels; TNF -308 A allele with higher TNF- α plasma levels in controls and mRNA levels in PTB patients at T1; IL17A A allele at rs7747909 with higher IL-17 levels; IL10 -819 T allele with higher IL-10 levels; and TGFB1 +29 CC genotype higher TGF- β plasma levels in PTB patients at T2. The present study suggests that IFNG +874T/A, IFNG +2109A/G, IL12B +1188A/C, IL10 -819C/T, and TGFB1 +21C/T are associated with differential cytokine levels in pulmonary tuberculosis patients and may play a role in the initiation and maintenance of acquired cellular immunity to tuberculosis and in the outcome of the active disease while on antituberculosis treatment.

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, a deep mycosis endemic in Latin America. Studies to elucidate the host-parasite relationship in this mycosis have demonstrated that non-activated phagocytes fail to kill the etiologic agent. Investigations of human monocytes have shown that the lack of fungicidal activity is partially associated with the capacity of a high-virulence strain to induce PGE(2) release by these cells. This eicosanoid inhibits production of TNF-α, the cytokine involved in cell activation for release of H(2)O(2), the fungicidal metabolite. Cell priming with IFN-γ was shown to partially reverse this inhibitory effect. In this study, we asked whether monocyte challenge with a low-virulence strain of this fungus would also result in PGE(2) release and consequently inhibition of antifungal activities. We also assessed whether PGE(2,) besides inhibiting production of TNF-α, a monocyte-activating cytokine, also affects IL-10. The latter, in contrast to TNF-α is a monocyte-suppressing cytokine. Finally, we evaluated whether priming cells with other cytokines, namely TNF-α and GM-CSF, could be more effective than IFN-γ in reversing the PGE(2) inhibitory effect. The results revealed that the less virulent P. brasiliensis strain also induces human monocytes to release PGE(2). However, the inhibitory effect of PGE(2) was less pronounced when cells were challenged with this strain than with the more virulent one. It was also demonstrated that PGE(2), while inhibits TNF-α production, tends to increase IL-10 levels. Priming with GM-CSF or TNF-α was more effective than IFN-γ in compensating for the inhibitory PGE(2) effect, since these cytokines induce cells to produce higher H(2)O(2) and TNF-α levels.

In this study we investigated the role of nitric oxide (NO) in monocyte fungicidal activity against Paracoccidioides brasiliensis. We found that cells primed with IFN-γ, TNF-α or GM-CSF and challenged with a high-(Pb18) or low-virulence (Pb265) strain of the fungus increase their fungicidal activity. Expression of iNOS mRNA was increased after priming cells with each cytokine, and tended to be inhibited by Pb18. Despite up-regulation of iNOS mRNA expression by Pb265, an equivalent increase in NO production was not detected, as metabolite levels were similar in all cultures. The results indicated that high expression of human monocyte iNOS mRNA induced by P. brasiliensis is not correlated with NO concentrations produced., (Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Background: Porcine circovirus type 2 (PCV2) has been associated with several disease complexes, including reproductive failure. The aim of this study was to identify the subtypes of PCV2 that are associated with reproductive failure in pigs from the State of São Paulo, Brazil and to investigate co-infections with other infectious organisms., Findings: Samples of 168 aborted foetuses or mummified foetuses from five farrow-to-finish swine farms known to be infected with PCV2 and located in the State of São Paulo were tested for PCV2 by polymerase chain reaction (PCR). Positive samples were additionally tested for porcine parvovirus (PPV), Leptospira spp. and Brucella spp. by PCR. PCV2 was detected in 18 of the samples (10.7%). PPV, Brucella spp. and Leptospira spp were found in 2, 10 and 0 cases, respectively. Eleven PCV2 strains were sequenced and determined to be either genotype 2a (n = 1) or 2b (n = 10)., Conclusions: The findings indicate that the frequency of PCV2 infections in aborted porcine foetuses from the State of São Paulo is rather low (10.7%) and that co-infection with other pathogens is common and may be involved in PCV2 associated reproductive failure. No repeatable, characteristic amino acid motifs for regions of the PCV2 capsid protein seemed to be associated with abortion in sows.

Background: In Brazil, coffee (Coffea arabica) husks are reused in several ways due to their abundance, including as stall bedding. However, field veterinarians have reported that horses become intoxicated after ingesting the coffee husks that are used as bedding. The objective of this study was to evaluate whether coffee husk consumption causes intoxication in horses., Results: Six horses fed coast cross hay ad libitum were given access to coffee husks and excitability, restlessness, involuntary muscle tremors, chewing movements and constant tremors of the lips and tongue, excessive sweating and increased respiration and heart rates were the most evident clinical signs. Caffeine levels were measured in the plasma and urine of these horses on two occasions: immediately before the coffee husks were made available to the animals (T0) and at the time of the clinical presentation of intoxication, 56 h after the animals started to consume the husks (T56). The concentrations of caffeine in the plasma (p < 0.001) and urine (p < 0.001) of these animals were significantly greater at T56 than at T0., Conclusions: It was concluded that consumption of coffee husks was toxic to horses due to the high levels of caffeine present in their composition. Therefore, coffee husks pose a risk when used as bedding or as feed for horses.

The intestinal protozoan parasite Giardia duodenalis (syn. Giardia intestinalis and Giardia lamblia) is a widespread enteric pathogen in human and domestic animals. This organism is one of the most common parasites in domestic dogs in Brazil. In this study, we determined the occurrence and genetic characterization of G. duodenalis isolated from dogs from south-central São Paulo state, Brazil. A total of 300 fecal samples were collected. Fecal specimens were screened for the presence of G. duodenalis using microscopy (zinc sulfate solution flotation technique) and polymerase chain reaction (PCR) targeting the small subunit ribosomal (SSU-rDNA) and glutamate dehydrogenase (GDH) genes. Genetic characterization was performed using restriction fragment length polymorphisms (RFLP) and sequencing analysis of the GDH gene. In addition, selected samples were further characterized by RFLP and sequencing of the β-giardin gene. The overall occurrence of G. duodenalis was 17.3% (52/300). The occurrence was higher in stray dogs (28%) than in household dogs (6.25%). Of the 36 PCR-positive samples that were selected for genotyping, only dog-specific genotype C (20 isolates), D (11 isolates) and mixed C + D (five isolates) isolates were detected in the study. This study provides current information on the infection rates of G. duodenalis genotypes in canine populations and describes for the first time the presence of mixed infections within host-specific C and D genotypes in dogs in Brazil. These genotypes were widespread and commonly found in domestic dogs living in urban and suburban environments of the studied area and confirmed the endemic status of Giardia in this region.

Monocytes and macrophages play a central role in innate and adaptive immune response against systemic fungal infections. Imbalances in suppressor or stimulatory cytokine secretion caused by these cells may influence disease development, microorganism death, and the nature of the adaptive immune response. This study analyzed the monocyte cytokine profiles of healthy individuals challenged with high and low virulent strains of P. brasiliensis and mRNA cytokine expression kinetics by reverse transcription polymerase chain reaction (RT-PCR). Peripheral blood monocytes from healthy volunteers were cultured in vitro with and without virulent (Pb18) or low virulence (Pb265) strains from P. brasiliensis viable yeast cells. Interleukin-1 beta (IL-1beta), IL-6, IL-8, IL-10, tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta (TGF-beta1) were measured in culture supernatants by enzyme immunoassay (ELISA), and mRNA cytokine expression was determined by RT-PCR at 0, 4, 8, 12, 18 and 48 hr. Both P. brasiliensis strains induced monocyte production of IL-1beta, IL-6, IL-10 and TNF-alpha. Pb18 induced higher levels of IL-1beta, IL-6, and IL-10 than Pb265. IL-8 and TGF-beta1 levels were not significantly different from those cultured without stimulus. The mRNA cytokine expression was similar to supernatant cytokines measured by ELISA. In vitro monocyte challenge with virulent P. brasiliensis strain induces earlier and higher levels of pro- and anti-inflammatory cytokines than low virulence strain.

Babesia spp. infections were investigated in Bos taurus x Bos indicus dairy cows and calves and in Boophilus microplus engorged female ticks and eggs. Blood samples and engorged female ticks were collected from 25 cows and 27 calves. Babesia spp. was detected in ticks by microscopic examination of hemolymph of engorged female and by squashes of egg samples. Cattle infection was investigated in blood thin smears and by DNA amplification methods (PCR and nested PCR), using specific primers for Babesia bovis and Babesia bigemina. Merozoites of B. bovis (3 animals) and B. bigemina (12 animals) were detected exclusively in blood smears of calves. DNA amplification methods revealed that the frequency of B. bigemina infection in calves (92.6%) and in cows (84%) and of B. bovis in calves (85.2%) and in cows (100%) did not differ significantly (P > 0.05). Babesia spp. infection was more frequent in female ticks and eggs collected from calves (P < 0.01) than from cows, especially in those which had patent parasitemia. Hatching rates of B. microplus larvae were assessed according to the origin of engorged females, parasitemia of the vertebrate host, frequency and intensity of infection in engorged female tick, and frequency of egg infection. Hatching rate was lower in samples collected from calves (P < 0.01) than from cows, and in those in which Babesia spp. was detected in egg samples (P < 0.01).

PCR and nested-PCR methods were used to assess the frequency of Babesia bovis and Babesia bigemina infection in Boophilus microplus engorged females and eggs and in cattle reared in an area with endemic babesiosis. Blood and the engorged female ticks were from 27 naturally infested calves and 25 crossbred cows. The frequency of both Babesia species was similar in calves and cows (P>0.05). Babesia bovis was detected in 23 (85.2%) calves and in 25 (100%) cows and B. bigemina was detected in 25 (92.6%) calves and in 21 (84%) cows. Mixed infections with the both Babesia species were identified in 42 animals, 21 in each age category. Of female ticks engorged on calves, 34.9% were negative and single species infection with B. bigemina (56.2%) was significantly more frequent (P<0.01) than with B. bovis (4.7%). Most of the females (60.8%) engorged on cows did not show Babesia spp. infection and the frequency of single B. bovis infection (17.6%) was similar (P>0.05) to the frequency of single B. bigemina infection (15.9%). Mixed Babesia infection was lower (P<0.01) than single species infection in female ticks engorged either in cows (5.7%) or in calves (4.3%). An egg sample from each female was analysed for the presence of Babesia species. Of the egg samples from female ticks infected with B. bovis, 26 (47.3%) were infected while from those from female ticks infected with B. bigemina 141 (76.6%) were infected (P<0.01). The results showed that although the frequency of both species of Babesia was similar in calves and cows, the infectivity of B. bigemina was higher to ticks fed on calves while to those ticks fed on cows the infectivity of both Babesia species was similar.