Effects of supplementing direct-fed microbials on health and growth of pre-weaning Gyr × Holstein dairy calves.

This study aimed to evaluate the effects of direct-fed microbials (DFM) on health and growth responses of pre-weaning Bos indicus × B. taurus (Gyr × Holstein) crossbred calves. Ninety newborn heifer calves [initial body weight (BW) 35 ± 4.0 kg] were used. At birth, calves were ranked by initial BW and parity of the dam and assigned to: 1) whole milk without DFM supplementation (CON; n = 30), 2) whole milk with the addition of 1.0 g/calf per day of a Bacillus-based DFM (BAC; n = 30), or 3) whole milk with the addition of 1.0 g/calf per day of BAC and 1.2 g/calf per day of Enterococcus faecium 669 (MIX; n = 30). Milk was fed individually during the study (77 d) and the BAC and MIX treatments were offered daily throughout the 77-d pre-weaning period. All calves were offered a starter supplement and corn silage starting on d 1 and 60 of age, respectively. Milk and starter supplement intake were evaluated daily, and BW was recorded on d 0 and at weaning (d 77). Diarrhea and pneumonia were assessed daily, and fecal samples were collected on d 0, 7, 14, 21, and at weaning (d 77) for assessment of the presence of bacterial and protozoal pathogens via qPCR. All data were analyzed using SAS (v. 9.4) with calf as the experimental unit and using single-df orthogonal contrasts (BAC + MIX vs. CON; BAC vs. MIX). Daily feeding of DFM, regardless of type, improved weaning BW. Odds ratio for occurrence of pneumonia was lower for DFM-supplemented calves, but occurrence of both did not differ between BAC and MIX calves. No Salmonella spp. or E. coli F41 were detected in any of the calves. The proportion of calves positive for E. coli F17 was greater for DFM calves on d 7 (92 and 96% vs. 81% for BAC, MIX, and CON, respectively), 21 (13 and 26% vs. 7% for BAC, MIX, and CON, respectively), and at weaning (48 and 35% vs. 22% for BAC, MIX, and CON, respectively). For C. difficile, more DFM calves were positive on d 7 (65 and 30% vs. 35% for BAC, MIX, and CON, respectively) and 14 (20 and 28% vs. 7% for BAC, MIX, and CON, respectively), but also greater for BAC vs. MIX on d 7. More CON calves were positive for C. perfringens on d 14 (14% vs. 3 and 8% for CON, BAC, and MIX, respectively) compared with DFM-fed calves. Incidence of calves positive for C. perfringens was greater in BAC vs. MIX on d 7 (50 vs. 18%), and greater for MIX vs. BAC at weaning (9 vs. 0%). For protozoa occurrence, a lower proportion of DFM calves were positive for Cryptosporidium spp. on d 7 (58 and 48% vs. 76% for BAC, MIX, and CON, respectively), but opposite results were observed on d 21 for Cryptosporidium spp. (3 and 11% vs. 0% for BAC, MIX, and CON, respectively) and Eimeria spp. on d 14 (7 and 8% vs. 0% for BAC, MIX, and CON, respectively) and 21 (50 and 59% vs. 38% for BAC, MIX, and CON, respectively). In summary, DFM feeding alleviated the occurrence of pneumonia, improved growth rates, while also modulating the prevalence of bacteria and protozoa in pre-weaning Gyr × Holstein calves.

Strategies of the control of an outbreak of leptospiral infection in dairy cattle in Northeastern Brazil.

The aim of the present study was to describe the strategies of the control of an outbreak of leptospiral infection in dairy cattle in Maranhão State, Northeastern Brazil. In the period from January to July 2015, 18 (17%) out of 106 cows presented abortion, six (5.7%) stillbirth, and 12 (11.3%) repeated estrus, totaling 24 animals with reproductive problems. The diagnosis of leptospirosis was based on serology (microscopic agglutination test-MAT), bacteriological culture, and polymerase chain reaction (PCR). Antibiotic therapy, vaccination protocols, and changes in management practices were suggested as control measures. Of all animals on the farm (n = 280), 136 (48.6%) were seropositive for at least one serovar of Leptospira sp. No pure leptospiral culture was obtained. Eight of the animals with reproductive problems yielded positive PCR results (vaginal fluid of seven animals and urine and vaginal fluid of one animal). Genetic sequencing of a vaginal fluid/urine PCR-positive sample revealed Leptospira borgpetersenii. One year after the adoption of control measures, no reproductive problems were observed. Thus, leptospirosis probably caused the reproductive failures in the herd, and the control and prevention measures implemented were efficient in controlling the disease.

Equine infectious anemia virus in naturally infected horses from the Brazilian Pantanal.

Equine infectious anemia (EIA) has a worldwide distribution, and is widespread in Brazil. The Brazilian Pantanal presents with high prevalence comprising equine performance and indirectly the livestock industry, since the horses are used for cattle management. Although EIA is routinely diagnosed by the agar gel immunodiffusion test (AGID), this serological assay has some limitations, so PCR-based detection methods have the potential to overcome these limitations and act as complementary tests to those currently used. Considering the limited number of equine infectious anemia virus (EIAV) sequences which are available in public databases and the great genome variability, studies of EIAV detection and characterization molecular remain important. In this study we detected EIAV proviral DNA from 23 peripheral blood mononuclear cell (PBMCs) samples of naturally infected horses from Brazilian Pantanal using a semi-nested-PCR (sn-PCR). The serological profile of the animals was also evaluated by AGID and ELISA for gp90 and p26. Furthermore, the EIAV PCR amplified DNA was sequenced and phylogenetically analyzed. Here we describe the first EIAV sequences of the 5' LTR of the tat gene in naturally infected horses from Brazil, which presented with 91% similarity to EIAV reference sequences. The Brazilian EIAV sequences also presented variable nucleotide similarities among themselves, ranging from 93,5% to 100%. Phylogenetic analysis showed that Brazilian EIAV sequences grouped in a separate clade relative to other reference sequences. Thus this molecular detection and characterization may provide information about EIAV circulation in Brazilian territories and improve phylogenetic inferences.

Molecular Method Confirms Canine Leishmania Infection Detected by Serological Methods in Non-Endemic Area of Brazil.

In Brazil, visceral leishmaniasis (VL) is expanding and becoming urbanized, especially in non-endemic areas such as the State of Rio Grande do Sul. Considering that infected dogs are the main reservoir for zoonotic VL, this study evaluated the prevalence of canine visceral leishmaniasis (CVL) in the metropolitan area of Porto Alegre, a new area of expansion of VL in Brazil. Serum and plasma from 405 asymptomatic dogs from the municipalities of Canoas (n=107), São Leopoldo (n=216), and Novo Hamburgo (n=82) were tested for CVL using immunochromatographic (DPP®) and ELISA EIE® assays (2 assays officially adopted by the Brazilian government for the diagnosis of CVL) and real-time PCR to confirm the results. There was no agreement among serological and real-time PCR results, indicating that the Leishmania infection in asymptomatic animals with low parasite load, confirmed by negative parasitological tests (smears and parasite culture), need to be evaluated by molecular methods. The prevalence of LVC in the metropolitan region of Porto Alegre, confirmed by real-time PCR was 4% (5.6% in Canoas and 4.6% in São Leopoldo). The use of molecular method is essential for accurate diagnosis of CVL, especially in asymptomatic dogs in non-endemic areas.

Sinuolinea niloticus n. sp., a myxozoan parasite that causes disease in Nile tilapia (Oreochromis niloticus).

Sinuolinea species are myxozoans of the order Bivalvulida, suborder Variisporina, and family Sinuolineidae, which can be parasites for freshwater and marine fish. The aim of this study was to describe the occurrence of Sinuolinea niloticus n. sp. infecting Nile tilapia (Oreochromis niloticus) from aquaculture and from river sources with morphological and molecular analyses. Between March 2010 and November 2012, 116 Nile tilapia were randomly sampled from aquaculture net fishing (n = 56) in Mira Estrela, São Paulo, and from the Capivari River (n = 60) in Botucatu, São Paulo. The fishes that were sampled were examined by necropsy, microscopic observation and molecular techniques for detection and identification of the myxozoan causing disease in tilapia. All of the tissues that were sampled for analysis showed the presence of the parasite. It was observed by microscopy that the myxozoan belongs to the Sinuolinea genus. This identification was performed based on morphological characteristics and histopathology findings, such as structures consistent with myxozoan in the interstices in all analysed tissues, coagulative necrosis, haemorrhage, inflammatory processes, presence of melano-macrophages and eosinophils. The results of the molecular analyses revealed that the myxozoan detected and identified in this study is sister to a group of other Sinuolinea species. Because this is the first report of this parasite in Nile tilapia, the parasite was named S. niloticus n. sp. This is the first report of a Sinuolinea species in Brazil and in tilapia.

First genome sequence of St. Louis encephalitis virus (SLEV) isolated from a human in Brazil.

St. Louis encephalitis virus (SLEV), a member of the family Flaviviridae, genus Flavivirus, is a causative agent of encephalitis in the Americas. In Brazil, sporadic cases of SLEV infection have been reported since 1953, but the first outbreak of SLEV in Brazil was identified only in 2007, concomitant with an outbreak of dengue virus (DENV) serotype 3. This finding, along with other reports, indicates that SLEV circulation in Brazil is largely unknown, and there may be epidemiological implications of the co-circulation of SLEV, DENV and other flaviviruses in Brazil. Here, we describe the first complete genome sequence of an SLEV strain isolated from a human patient in Brazil, strain BeH 355964. Phylogenetic analysis was performed to determine the genotype of BeH 355964 using the full-length genome and envelope (E) gene sequences separately. Both analyses showed that BeH 355964 could be classified as genotype V. Although the number of single gene sequences available is greater (such as for the E gene), the phylogenetic tree based on the complete genome sequence was better supported and provided further information about the virus.

Salmonella detection with LAMP and qPCR and identification of serovars of interest by multiplex qPCR in poultry carcasses.

Salmonella is present in the poultry production chain and is a major challenge in terms of food safety and animal health. The early Salmonella detection is one of the main tools to control and prevent the transmission of this pathogen. Microbiological isolation and serotyping to identify and differentiate Salmonella serovars are laborious processes, time-consuming, and expensive. Therefore, molecular diagnostic methods can be rapid and efficient alternatives to the detection of this pathogen. Thus, the aim herein was to standardize and evaluate the use of loop-mediated isothermal amplification (LAMP) in comparison with real-time PCR (qPCR) for detection of Salmonella associated with a multiplex qPCR for simultaneous identification and differentiation of S. Enteritidis, S. Typhimurium, S. Pullorum, and S. Gallinarum. The LAMP, qPCR, and multiplex qPCR assays were comparable in specificity. The three techniques were evaluated for specificity for 16 different serovars of Salmonella and for 37 strains of the serovars of interest. The limit of detection and the efficiency of the LAMP, qPCR, and multiplex qPCR reactions were determined. The techniques were applied to 33 samples of chicken carcasses and compared to the results of conventional microbiology for validation. As results, LAMP was specific in the detection of different Salmonella serovars but presented lower limit of detection ranging from 101 to 104 CFU/reaction. In comparison, qPCR could detect less cells (100 to 102 CFU/reaction), reaching equal specificity and better repeatability in the assays. The qPCR multiplexing for identification of the different serovars also showed good specificity, with the detection threshold between entre 101 and 102 CFU/reaction. The results obtained in the analyses on poultry carcasses suggested a correspondence between the results obtained in molecular methods and in conventional microbiology. Thus, the proposed assays are promising for the diagnosis of Salmonella in poultry carcasses, already proved to be faster and more efficient than conventional diagnostics techniques, being of great interest for poultry production, animal, and public health.

Molecular detection of Trypanosoma cruzi in equids in a semiarid zone of Pernambuco - Brazil.

Trypanosoma cruzi is the etiological agent of Chagas disease, a neglected and frequently occurring zoonosis in Central and South American countries. Wild mammals and domestic dogs are the main reservoirs of the parasite in the wild and domestic cycles, respectively. The vectors have a wide variety of food sources that can influence transmission cycles. The aim of this study was to determine the prevalence of T. cruzi infection in donkeys (Equus asininos) and mules (Equus mulus) living in rural areas of the Brazilian semi-arid region. Whole-blood samples from 72 equids (65 donkeys and 7 mules) were analyzed by nested polymerase chain reaction (nested PCR). A total of 51.39% of the samples (37/72) were positive. Phylogenetic analysis identified discrete typing units TcI and TcII, which suggested the possibility that donkeys and mules might be participating in domestic/peridomestic and wild transmission cycles. This was the first report of T. cruzi infection in donkeys and mules in Brazil, with high prevalence of positive animals. This places these animals as potential reservoirs for the parasite and the particular features of these hosts, the presence of vectors and the socioeconomic characteristics of the population under semiarid conditions create interactions that may favor transmission and overlapping T. cruzi infection cycles.

Strong Evidence of the Role of Donkeys in the Epidemiology of Leptospira spp. in Semiarid Conditions.

Donkeys (Equus asinus) are historically known for their close relationship to humanity, which raises the need to study zoonotic diseases that affect them. In this perspective, leptospirosis stands out as a disease with an economic and public health impact, and its occurrence is facilitated in times of higher rainfall indexes, especially in large urban centers. In view of the scarcity of information about leptospirosis in donkeys, the objective of this study was to detect the presence of Leptospira spp. and anti-leptospiral antibodies in donkeys rescued by a zoonosis center located in the Caatiga biome, Brazilian semiarid region. Overall, 30 donkeys of both sexes, aged between 4 months and 15 years, were used, from which 64 serum samples were collected and submitted to the microscopic agglutination test (MAT). In addition, 64 samples of urine, vaginal and preputial fluid, in duplicates, were subjected to the polymerase chain reaction (PCR) and microbiological. Sixteen (53.3%) animals tested positive in at least one diagnostic test, 12 (40%) of which were positive at MAT and seven (23.3%) in the molecular and bacteriological detection (urine, vaginal, and preputial fluid samples). This is the first report identifying donkeys infected with Leptospira spp. by molecular and bacteriological diagnosis in Brazil, and the first in the world to detect this agent in their genital fluids. The study also shows that donkeys are commonly exposed to leptospires in the Caatinga biome, and this constitutes a One Health-based concern, demonstrating the importance of broad studies where large numbers of humans and animals coexist when investigating zoonotic infections and when planning and implementing control measures for donkeys-associated leptospirosis.

The Discovery of a New Mimivirus Isolate in Association with Virophage-Transpoviron Elements in Brazil Highlights the Main Genomic and Evolutionary Features of This Tripartite System.

Mimiviruses are giant viruses of amoeba that can be found in association with virophages. These satellite-like viruses are dependent on the mimivirus viral factory to replicate. Mimiviruses can also be associated with linear DNA molecules called transpovirons. Transpovirons and virophages are important drivers of giant virus evolution although they are still poorly studied elements. Here, we describe the isolation and genomic characterization of a mimivirus/virophage/transpoviron tripartite system from Brazil. We analyzed transmission electron microscopy images and performed genome sequencing and assembly, gene annotation, and phylogenetic analysis. Our data confirm the isolation of a lineage A mimivirus (1.2 Mb/1012 ORFs), called mimivirus argentum, and a sputnik virophage (18,880 bp/20 ORFs). We also detected a third sequence corresponding to a transpoviron from clade A (6365 bp/6 ORFs) that presents small terminal inverted repeats (77 nt). The main genomic features of mimivirus argentum and of its virophage/transpoviron elements corroborates with what is described for other known elements. This highlights that this triple genomic and biological interaction may be ancient and well-conserved. The results expand the basic knowledge about unique and little-known elements and pave the way to future studies that might contribute to a better understanding of this tripartite relationship.

New insights on Leptospira sp. infection in ewes maintained in field semiarid conditions.

Leptospirosis is poorly studied in small ruminants raised in field semiarid conditions. In this study we compared serological, bacteriological and molecular diagnostic methods in ewes maintained in field Brazilian semiarid conditions. Blood, vaginal fluid and urine samples were collected from 60 Morada Nova ewes raised in a semi-intensive system in the Brazilian semiarid. Diagnostic tests performed were microscopic agglutination test (MAT), polymerase chain reaction (PCR) and bacterial isolation. Anti-Leptospira sp. antibodies were found in eight (13.33%) animals analyzed by MAT at reciprocal titer 25 (cut-off 25), while Leptospira sp. DNA was detected in urine or vaginal fluid of 56 animals (93.33%). There was growth of leptospires in 10 urine cultures and in 11 vaginal fluid cultures, however, two of urine (2/60-3.33%) and eight cultures of vaginal fluid (8/60-13.33%) were confirmed by PCR. Two samples of vaginal fluid (one of each animal) were submitted to sequencing demonstrating 99% similarity with L. santarosai and L. interrogans. The highest MAT sensitivities were obtained with reciprocal titer 25 (cut-off 25) compared to 50 and 100. The performance of different diagnostic techniques for leptospirosis in ewes raised in field semiarid conditions allowed a better evaluation of the herd, as well as made it possible to identify carrier animals. Genital route may be important for efficient transmission and without dependence on environmental factors in ewes from semiarid, as well as it's highlighted that titer 1:25 in serology was more efficient, indicating its use in ewes in field semiarid conditions.

Prediction of SARS-CoV Interaction with Host Proteins during Lung Aging Reveals a Potential Role for TRIB3 in COVID-19.

COVID-19 is prevalent in the elderly. Old individuals are more likely to develop pneumonia and respiratory failure due to alveolar damage, suggesting that lung senescence may increase the susceptibility to SARS-CoV-2 infection and replication. Considering that human coronavirus (HCoVs; SARS-CoV-2 and SARS-CoV) require host cellular factors for infection and replication, we analyzed Genotype-Tissue Expression (GTEx) data to test whether lung aging is associated with transcriptional changes in human protein-coding genes that potentially interact with these viruses. We found decreased expression of the gene tribbles homolog 3 (TRIB3) during aging in male individuals, and its protein was predicted to interact with HCoVs nucleocapsid protein and RNA-dependent RNA polymerase. Using publicly available lung single-cell data, we found TRIB3 expressed mainly in alveolar epithelial cells that express SARS-CoV-2 receptor ACE2. Functional enrichment analysis of age-related genes, in common with SARS-CoV-induced perturbations, revealed genes associated with the mitotic cell cycle and surfactant metabolism. Given that TRIB3 was previously reported to decrease virus infection and replication, the decreased expression of TRIB3 in aged lungs may help explain why older male patients are related to more severe cases of the COVID-19. Thus, drugs that stimulate TRIB3 expression should be evaluated as a potential therapy for the disease.

Serological, molecular and bacteriological approaches for detecting Leptospira sp. carrier rams maintained in semiarid conditions.

Even in the adverse environmental conditions of the semiarid region, leptospires can survive and spread by alternative routes of transmission, such as sexual in ewes, however, there is no data on rams. Thus, the present study aimed to evaluate the use of serological, molecular and microbial tools applied to diagnosis of Leptospira sp. Infection in rams maintained in semiarid conditions. Biological samples of urinary (urine, kidney and bladder) and genital (vas deferens, epididymis tail and vesicular gland) tracts were collected from 40 slaughtered rams for polymerase chain reaction (PCR) and bacterial isolation, as well as blood samples for antibody detection through microscopic serum agglutination test (MAT). Anti-Leptospira antibodies were found in five (12.5%) animals with antibody titer of 50 and 2 (5%) for the titer 100 for serogroups Pyrogenes, Ballum, Icterohaemorrhagiae and Australis. Leptospira sp. DNA was found in PCR of organs and urine of 30 (75%) animals. Overall, 240 fragments of organs from the urinary and genital tracts and urine were evaluated, with 93 (38.7%) positive samples, being 48/120 (40%) for the urinary tract and 45/120 (37.5%) for the genital. There was no statistically significant difference between the tracts. A bladder sample was sent for sequencing and showed 99% similarity with L. interrogans. Of the 240 cultures evaluated, 59 (24.5%) had leptospire growth, being that 23 (39%) were confirmed in PCR. Considering the PCR of organs and urine and bacterial growth as gold standards, the cut-off 50 in MAT showed greater sensitivity when compared to cut-off 100, regardless of the material used. The great proportion of leptospiral DNA in organs, urine and culture and bacterial growth from the genital tracts reinforce its importance as an extra-renal site and highlights the possible role of rams in venereal transmission, as well as the sensitivity of the cut-off 50 suggested its adoption in the serology of rams maintained in semiarid conditions.

Blocking drug-induced autophagy with chloroquine in HCT-116 colon cancer cells enhances DC maturation and T cell responses induced by tumor cell lysate.

Autophagy is an important mechanism for tumor escape, allowing tumor cells to recover from the damage induced by chemotherapy, radiation therapy, and immunotherapy and contributing to the development of resistance. The pharmacological inhibition of autophagy contributes to increase the efficacy of antineoplastic agents. Exposing tumor cells to low concentrations of select autophagy-inducing antineoplastic agents increases their immunogenicity and enhances their ability to stimulate dendritic cell (DC) maturation. We tested whether the application of an autophagy-inhibiting agent, chloroquine (CQ), in combination with low concentrations of 5-fluorouracil (5-FU) increases the ability of tumor cells to induce DC maturation. DCs sensitized with the lysate of HCT-116 cells previously exposed to such a combination enhanced the DC maturation/activation ability. These matured DCs also increased the allogeneic responsiveness of both CD4+ and CD8+ T cells, which showed a greater proliferative response than those from DCs sensitized with control lysates. The T cells expanded in such cocultures were CD69+ and PD-1- and produced higher levels of IFN-γ and lower levels of IL-10, consistent with the preferential activation of Th1 cells. Cocultures of autologous DCs and lymphocytes improved the generation of cytotoxic T lymphocytes, as assessed by the expression of CD107a, perforin, and granzyme B. The drug combination increased the expression of genes related to the CEACAM family (BECN1, ATGs, MAPLC3B, ULK1, SQSTM1) and tumor suppressors (PCBP1). Furthermore, the decreased expression of genes related to metastasis and tumor progression (BNIP3, BNIP3L, FOSL2, HES1, LAMB3, LOXL2, NDRG1, P4HA1, PIK3R2) was noted. The combination of 5-FU and CQ increases the ability of tumor cells to drive DC maturation and enhances the ability of DCs to stimulate T cell responses.

Didelphis albiventris as a carrier of Leptospira sp. in the central nervous tissue in the semiarid region of Northeast, Brazil.

Leptospirosis has been investigated in several species of wild animals. The white-eared opossum (Didelphis albiventris) is a mammal common in the brazilian semi-arid, so, this study aimed to investigate its role in the occurrence of the leptospirosis in the region Northeast of Brazil. 12 animals were used, from which samples were collected for the attempt of isolation, molecular detection and serological examination. There was no microbial growth, nor were any anti-Leptospira sp. antibodies found in the serological samples. The PCR detected leptospiric DNA in the central nervous system (CNS) of five animals (41.7 %). The gene in one of the samples was sequenced and showed identity with Leptospira interrogans. The presence of Leptospira sp. in the CNS of Didelphis albiventris does not allow the characterization of the studied animals as reservoirs with potential for transmission of the pathogen in the region, however it represents a site that needs to be further investigated.

Use of serological and molecular techniques for detection of Leptospira sp. carrier sheep under semiarid conditions and the importance of genital transmission route.

It is possible that there are peculiarities in the epidemiology of leptospirosis in regions with a semiarid climate, where the environment is often adverse, allowing the occurrence of alternative transmission routes. The objective of the work was to generate contributions to the diagnosis and epidemiology of Leptospira sp. infection in sheep reared in semiarid conditions, using serological, molecular and microbiological techniques for diagnosis in dry and rainy seasons. Samples of blood, vaginal fluid, urine, bladder, kidney, uterus, uterine tube, ovary and placenta were collected from 104 sheep (52 animals per season - dry and rainy) slaughtered in the Brazilian semiarid. Diagnostic tests performed were microscopic agglutination test (MAT), polymerase chain reaction (PCR) and bacterial isolation. Anti-Leptospira sp. antibodies were found in 26 (25%) of the animals analyzed by MAT at 1:50 dilution (cut-off 50), while 69 (66.3%) animals had at least one organ/fluid with the presence of Leptospira sp. DNA. Overall, PCR was performed on 758 fragments of organs/fluids from the genital and urinary tracts, and 519 (68.5%) samples tested positive. PCR-positivity was statistically different in dry (46.2%) and rainy (11.5%) seasons for vaginal fluid. It was possible to perform the DNA sequencing in nine samples with 99% similarity to L. interrogans and recovery of viable strains in three samples of vaginal fluid. Regardless of the biological material used in PCR to detect carrier animals and the season, the highest MAT sensitivity values were obtained with cut-off 50 compared to 100. The results obtained indicate that, even in the adverse environmental conditions of the semiarid region, leptospires may survive and propagate by alternative routes of transmission, such as sexual, and the presence of PCR-positive genital tracts in ewes suggests that sexual transmission may play an important role in the epidemiology of the disease in sheep in Brazilian semiarid. In addition, it is suggested the use of titer 50 as cut-off point at serology in semiarid conditions.

Zika detection: comparison of methodologies.

Many countries in the Americas have detected local transmission of multiple arboviruses that cause febrile illnesses. Therefore, laboratory testing has become an important tool for confirming the etiology of these diseases. The present study aimed to compare the sensitivity and specificity of three different Zika virus detection assays. One hundred serum samples from patients presenting with acute febrile symptoms were tested using a previously reported TaqMan® RT-qPCR assay. We used a SYBR® Green RT-qPCR and a conventional PCR methodologies to compare the results. Of the samples that were determined to be negative by the TaqMan® RT-qPCR assay, 100% (Kappa=0.670) were also found to be negative by SYBR® Green RT-qPCR based on Tm comparison; however, 14% (Kappa=0.035) were found to be positive by conventional PCR followed by agarose gel electrophoresis. The differences between the ZIKV strains circulating worldwide and the low viremia period can compromise diagnostic accuracy and thereby the accuracy of outbreak data. Therefore, improved assays are required to improve the diagnosis and surveillance of arbovirus.

Research on viral agents associated with feline reproductive problems reveals a high association with feline panleukopenia virus.

Although reproductive failures (RF) such as abortion, stillbirth and neonatal mortality in cats are still under researched, it is known that many RF are caused by viral agents. This research surveyed the viral agent prevalence in queens with RF. Queens were excluded from the study if their RF was caused by issues other than infection, such as genetic, traumatic, hormonal or nutritional problems, or if they had a history of RF. Blood samples from 26 pregnant females with RF were collected for complete blood counts (BCC), renal/hepatic biochemistry and glycaemic analysis. Ultrasonography was performed to evaluate gestational age and foetal viability. When possible, placentas, humours and foetal tissues were collected. Blood samples were tested by PCR and qPCR for feline leukaemia virus (FeLV), feline immunodeficiency virus (FIV), feline alphaherpesvirus 1 (FeHV-1) and carnivore protoparvovirus 1 (CPPV-1). All maternal samples were negative for FeLV, FIV and FeHV-1 and positive for CPPV-1. In addition, foetuses from one queen and three females were positive for CPPV-1 by qPCR and for feline panleukopenia virus (FPV) through DNA sequencing. The BCC and biochemistry results revealed significant neutrophilia, lymphopenia, monocytosis, and liver enzymes. These results provide the first description of an FPV agent causing only RF-related clinical signs in queens.

Propolis effects on pro-inflammatory cytokine production and Toll-like receptor 2 and 4 expression in stressed mice.

INTRODUCTION: Propolis is a beehive product and its immunomodulatory action has been well documented; however, little is known concerning its activity on the immune system of stressed mice. This work investigated a possible role of propolis against the immunosuppressive effects induced by stress in mice, assessing the pro-inflammatory cytokine (IL-1beta and IL-6) production and Toll-like receptor (TLR-2 and TLR-4) expression by spleen cells. METHODS: BALB/c mice were divided into 3 groups: G1 was considered control; G2 was submitted to restraint stress for 3 days, and G3 was treated with propolis and immediately submitted to stress. After sacrifice, spleens were removed and TLR-2 and TLR-4 gene expression was analyzed, as well as the pro-inflammatory cytokine production. Serum corticosterone levels were determined by radioimmunoassay as a stress indicator. RESULTS: Stressed mice, treated or not with propolis, produced higher corticosterone levels, whereas IL-1beta and IL-6 production was inhibited. TLR-2 and TLR-4 expression was inhibited in stressed mice, while propolis exerted an immunorestorative role in TLR-4 expression. The immunosuppressive effects on IL-1beta and IL-6 production and on TLR expression by stressed mice might have occurred due to a higher corticosterone production during stress. CONCLUSION: Propolis treatment did not antagonize the inhibitory effects on pro-inflammatory cytokine production, however it restored at least partially TLR2 mRNA expression and counteracted the inhibition on TLR-4 expression in stressed animals, contributing to the recognition of microorganisms during stressful conditions.

Detection of enterotoxin and toxic shock syndrome toxin 1 genes in Staphylococcus, with emphasis on coagulase-negative staphylococci.

The detection of staphylococcal enterotoxins is decisive for the confirmation of an outbreak and for the determination of the enterotoxigenicity of strains. Since the recognition of their antigenicity, a large number of serological methods for the detection of enterotoxins in food and culture media have been proposed. Since immunological methods require detectable amounts of toxin, molecular biology techniques represent important tools in the microbiology laboratory. In the present study, polymerase chain reaction (PCR) was used to identify genes responsible for the production of enterotoxins and toxic shock syndrome toxin 1 (TSST-1) in S. aureus and coagulase-negative staphylococci (CNS) isolated from patients and the results were compared with those obtained by the reverse passive latex agglutination (RPLA) assay. PCR detection of toxin genes revealed a higher percentage of toxigenic S. aureus strains (46.7%) than the RPLA method (38.3%). Analysis of the toxigenic profile of CNS strains showed that 26.7% of the isolates produced some type of toxin, and one or more toxin-specific genes were detected in 40% of the isolates. These results suggests the need for further studies in order to better characterize the pathogenic potential of CNS and indicate that attention should be paid to the toxigenic capacity of this group of microorganisms.