Applying a pan-flavivirus RT-qPCR assay in Brazilian public health surveillance.

The aim of this study was to improve flavivirus field monitoring in Brazil using a reliable probe-based RT-qPCR assay. Standard flavivirus strains were employed to evaluate the performance of the assay, and its applicability was evaluated using 235 stored pools of Culicidae samples collected between 1993 and 1997 and in 2016. Flavivirus species were identified by sequencing. Sixteen (6.8%) samples tested positive: Ilheus virus, Iguape virus, and Saint Louis encephalitis virus were identified in historical specimens from 1993-1994, while insect-specific flaviviruses were detected in the samples from 2016. This approach was demonstrated to be accurate for flavivirus detection and characterization, and it can be successfully applied for vector surveillance and for monitoring and discovery of insect specific flaviviruses.

The pathogenicity of two Neospora caninum goat strains in a BALB/c mouse model.

Goats are frequently described as an intermediate host for the protozoan Neospora caninum, manifesting the disease mainly by recurrent abortions with placentitis and encephalitis in fetuses. Several reports of natural and experimental infections in cattle and mice show differences in the immune response, and the outcome of the infection can be variable depending on the species affected and by the behavior of the infective strain. This study describes for the first time two Neospora caninum strains isolated from naturally infected goats from the state of Minas Gerais, Brazil. One placenta and one brain from different goats were processed for a first bioassay in gerbils (Meriones unguiculatus). Subsequently, a second bioassay was performed by inoculating the processed brain samples from gerbils into Interferon gamma (IFN-γ) knockout mice (KO mice). Tachyzoites collected from the peritoneal fluid of the KO mice were inoculated into VERO cell monolayers, where they presented a very slow growth rate. The tachyzoites were also inoculated into BALB/c mice with a dose of 106 tachyzoites per animal. After a 5-week follow up, the animals infected with both of the strains developed a strong polarized Th1 response with increased serum and spleen gene expression levels of pro-inflammatory cytokines (mainly IFN-γ and TNF-α) in the first week. Tissue lesions were mild in the animals infected with both strains. Despite the strong immune response preventing an infection in the visceral organs, the parasite was able to reach the brain, causing progressive brain lesions from the second to fifth week post infection. The NC-goat1-infected mice presented with severe meningoencephalitis, but the NC-goat2-infected animals had considerable histological brain lesions only at week 5. Immunohistochemical analysis of the mouse brains revealed a different pattern of inflammatory cells compared to the naturally infected goats. A severe inflammatory infiltrate of CD3+ T lymphocytes was found in the NC-goat1-infected mice. A more discrete infiltrate of CD3+ T cells was found in the NC-goat2-infected animals. Additionally, IBA1 IHC revealed an intense microglial reaction and monocyte perivascular cuffs in the NC-goat1-infected animals and lower microglia/monocyte infiltrates in the NC-goat2-infected mice. This work contributes knowledge on the pathogenicity of new Neospora caninum strains in mice, comparable with other well-established mouse models of the disease, and demonstrates the importance of studying goats as an intermediate host of this parasite.

Multiple infarcts and hemorrhages in the central nervous system of a dog with cerebral amyloid angiopathy: a case report.

BACKGROUND: ß-amyloid (Aß) can accumulate in the brain of aged dogs, and within vessels walls, the disease is called cerebral amyloid angiopathy (CAA). In humans, Alzheimer's disease and CAA are strongly correlated with cerebrovascular disease. However, in dogs, this association has not been extensively studied yet. The present report highlights the pathological and clinical features of a concomitant cerebrovascular disease and amyloid precursor protein (APP) accumulation in the brain of a dog. CASE PRESENTATION: A female, 16-year-old, Standard Poodle with a one-year history of cognitive deficits presented with an acute onset of right-sided postural reaction deficit and circling, left-sided head tilt, positional nystagmus, and ataxia. Due to poor prognosis the dog was euthanized, and pathological examination of the brain revealed an acute lacunar infarction within the thalamus extending to rostral colliculus. Additional findings included subacute and chronic areas of ischemia throughout the brain and areas of hemorrhage within the medulla. Immunolabeling revealed APP deposition within intraparenchymal vessels of frontal, temporal and occipital cortex, hippocampus, diencephalon, mesencephalon and myelencephalon, besides meningeal vessels walls. Glial fibrillary acidic protein (GFAP) immunolabeling showed marked astrocytosis around the acute area of infarction and within chronic areas of ischemia. Histological examination of the brain along with immunohistochemistry results showed a concomitant APP, which is an Aß precursor, accumulation within the neuroparenchyma and vessels (CAA) with histological evidences of a cerebrovascular disease in an aged dog. CONCLUSIONS: This report shows that APP accumulation in the brain can occur concomitantly to a severe cerebrovascular disease in a dog. Further studies are necessary to elucidate if cerebrovascular disease is associated with Aß accumulation in the brain of dogs.

A rabies virus vampire bat variant shows increased neuroinvasiveness in mice when compared to a carnivore variant.

Rabies is one of the most important zoonotic diseases and is caused by several rabies virus (RABV) variants. These variants can exhibit differences in neurovirulence, and few studies have attempted to evaluate the neuroinvasiveness of variants derived from vampire bats and wild carnivores. The aim of this study was to evaluate the neuropathogenesis of infection with two Brazilian RABV street variants (variant 3 and crab-eating fox) in mice. BALB/c mice were inoculated with RABV through the footpad, with the 50% mouse lethal dose (LD50) determined by intracranial inoculation. The morbidity of rabies in mice infected with variant 3 and the crab-eating fox strain was 100% and 50%, respectively, with an incubation period of 7 and 6 days post-inoculation (dpi), respectively. The clinical disease in mice was similar with both strains, and it was characterized initially by weight loss, ruffled fur, hunched posture, and hind limb paralysis progressing to quadriplegia and recumbency at 9 to 12 dpi. Histological lesions within the central nervous system (CNS) characterized by nonsuppurative encephalomyelitis with neuronal degeneration and necrosis were observed in mice infected with variant 3 and those infected with the crab-eating fox variant. However, lesions and the presence of RABV antigen, were more widespread within the CNS of variant-3-infected mice, whereas in crab-eating fox-variant-infected mice, RABV antigens were more restricted to caudal areas of the CNS, such as the spinal cord and brainstem. In conclusion, the results shown here demonstrate that the RABV vampire bat strain (variant 3) has a higher potential for neuroinvasiveness than the carnivore variant.

Histological and immunohistochemical characterization of the inflammatory and glial cells in the central nervous system of goat fetuses and adult male goats naturally infected with Neospora caninum.

BACKGROUND: Neospora caninum is an apicomplexan protozoan that is considered one of the main agents responsible for abortion in ruminants. The lesions found in the central nervous system (CNS) of aborted fetuses show multifocal necrosis, gliosis, and perivascular cuffs of mononuclear cells, but the inflammatory and glial cells have not been immunophenotypically characterized. The lesions in the CNS of infected adult animals have rarely been described. Therefore, in this study, we characterized the lesions, the immunophenotypes of the inflammatory and glial cells and the expression of MHC-II and PCNA in the CNS of goats infected with N. caninum. The CNS of eight aborted fetuses and six adult male goats naturally infected with N. caninum were analyzed with lectin histochemistry (RCA1) and immunohistochemistry (with anti-CD3, -CD79α, -GFAP, -MHC-II, and -PCNA antibodies). All animals were the offspring of dams naturally infected with N. caninum. RESULTS: The microscopic lesions in the CNS of the aborted fetuses consisted of perivascular cuffs composed mainly of macrophages (RCA1(+)), rare T lymphocytes (CD3(+)), and rare B lymphocytes (CD79α(+)). Multifocal necrosis surrounded by astrocytes (GFAP(+)), gliosis composed predominantly of monocytic-lineage cells (macrophages and microglia, RCA1(+)), and the cysts of N. caninum, related (or not) to the lesions were present. Similar lesions were found in four of the six male goats, and multinucleate giant cells related to focal gliosis were also found in three adult goats. Anti-GFAP immunostaining showed astrocytes characterizing areas of glial scarring. Cysts of N. caninum were found in three adult male goats. The presence of N. caninum was evaluated with histopathology, immunohistochemistry, and PCR. Immunohistochemistry demonstrated anti-PCNA labeling of macrophages and microglia in the perivascular cuffs and the expression of MHC-II by microglia and endothelial cells in the CNS of the aborted fetuses and adult male goats. CONCLUSIONS: Macrophages and microglia were the predominant inflammatory cells in the CNS of aborted fetuses and healthy adult male goats infected with N. caninum. Activated astrocytes were mainly associated with inflamed areas, suggesting that astrocytes were involved in the resolution of the lesions.

Brazil's landmark change on One Health, animal rights and protection.

The Sustainable Development Goals (SDG) launched by the United Nations in 2015 were a global challenge calling for ending poverty, protecting the environment, and guaranteeing peace and prosperity to world citizens by 2030. Brazil has changed gears, accepted the SDG challenge and moved one step forward. On the dawn of January 1st, 2023, the very first day of President Lula's office, Brazil issued the Decree no. 11,349/2023 and created an additional SDG itself on animal health by establishing the Department of Animal Protection and Rights (DAPR). The DAPR primarily aims to establish a Federal Animal Code and other nationwide standard procedures for pet population management and mass neutering/spaying programs, services against animal cruelty, welfare meat production, meat substitutes, and enforcement of native fauna protection. Meanwhile, Brazil's new government has reduced in 48% the Amazon deforestation and mining, enforced the inspections on national parks and preserved areas, limited wild boar hunting. On top of it, Lula's G20 Bloc presidency starting this coming December has shown the Brazil commitment to reestablish its historical prominence on international conversation and diplomacy. Finally, recognition of a clear and unquestionable nexus among animal welfare, environment, and sustainability, beyond the United Nations original proposition, in a country level, particularly with still-preserved nature areas, should be understood and invested as humanity heritage.

"Dying alone and being eaten": dog scavenging on the remains of an elderly animal hoarder-a case report.

Animal hoarding and human consumption by dogs have been important but often understudied aspects of the human-animal bond that can be addressed within a One Health framework. No scientific report has focused on dog scavenging on animal hoarders to date, despite isolated reports of dog scavenging on human remains, mostly due to starvation. The phenomenon has been approached as a confounding factor for human forensics. In 2014, the Animal Protection Department of Curitiba City was called to rescue and handle ten small dogs which had scavenged for a week on the human remains of their elderly owner, a potential animal hoarder. At inspection, three dead dogs in early putrefaction were also found in the household. Human autopsy revealed body putrefaction and lack of soft organs. Along with the dental arch, DNA testing was performed on the remains for official deceased identification. Due to the potential public health risks of aggression toward frail human beings and for the control of zoonotic diseases, all ten dogs were euthanized. Subsequent investigations by the crime scene police, homicide police, and autopsy services were unable to establish or rule out natural death, criminal or suicidal poisoning, zoonotic disease (rabies), fatal dog attack, or fatal accidental trauma. A general protocol has been proposed for future approaches to dog scavenging and suspicious killing of animal hoarders, as well an assessment for the potential adoption or euthanasia of animals owned by hoarders in these circumstances.

Cat rabies in Brazil: a growing One Health concern.

This review of human and cat rabies from 1986 to 2022 has shown mostly AgV3 variant in human cases with 29/45 (64.4%) reports including 23 from bats, four from cats, and two from unknown species, followed by 8/45 (17.8%) of AgV2 variant (all from dogs), 4/45 from marmoset variant (all from Callithrix jacchus), 2/45 samples compatible with wild canid variant (both from Cerdocyon thous), and one/45 of AgV1 variant from a domestic dog. Only one sample of human rabies was not typified, related to bat aggression. In addition, surveillance conducted in the state of São Paulo confirmed the presence of rabies in 7/23,839 cats (0.031%) and 3/106,637 dogs (0.003%) between 2003 and 2013, with a 10:1 overall cat-to-dog positivity ratio. This 10-fold higher infection rate for cat rabies may be explained by cats' hunting habits and predation. In addition, after 28 years of rabies-free status, a new cat rabies case was reported in the city of São Paulo in 2011. The rabid cat lived, along with other pets, in a household located near the largest downtown city park, whose owners presented animal hoarding behavior. Thus, animal hoarders and rescuers, public health agents, animal health professionals, and the general population with contact need to be aware of the risk of bat-borne rabies followed by spillover from cats to humans. In conclusion, cat rabies cases are becoming increasingly important in Brazil. This poses a One Health concern, given the overlapping of human, bat and cat populations within the same predisposed environment.

Transcranial Doppler sonographic findings in granulomatous meningoencephalitis in small breed dogs.

Granulomatous meningoencephalitis (GME) is an acute, progressive, and often fatal inflammatory disease of the central nervous system, affecting mainly small and toy dog breeds. A definitive diagnosis of GME can only be achieved through histopathologic examination of samples collected after death. This retrospective study describes transcranial Doppler ultrasonography (TDS) findings in dogs with confirmed clinical histopathology of GME. Eleven dogs were selected for this study. Sonographic findings in B-mode demonstrated diffuse decreased brain parenchyma echogenicity in 9 dogs, ventriculomegaly in 8 dogs, brain atrophy in 4 dogs, and hyperechoic focal lesions in 6 dogs. Color Doppler imaging revealed more obvious vessels of the arterial circle in 10 dogs. Spectral Doppler examination was performed in 10 dogs to detect the 6 major cerebral arteries of interest. The examination showed normal and high resistive index (RI) values in the outlined arteries. The TDS findings were consistent with pathology found on postmortem examination.

RésuméConstatations échographiques Doppler transcrâniennes pour la méningoencéphalite granulomateuse chez les chiens de petite race. La méningoencéphalite granulomateuse (MEG) est une maladie inflammatoire aiguë, progressive et souvent mortelle du système nerveux central qui touche surtout les chiens de petite race. Un diagnostic définitif de MEG peut seulement être obtenu par un examen histopathologique des échantillons recueillis après la mort. Cette étude rétrospective décrit les constatations d'une échographie Doppler transcrânienne (EDT) chez les chiens avec une histopathologie clinique confirmée de MEG. Onze chiens ont été sélectionnés pour cette étude. Les constatations échographiques en mode-B ont démontré une échogénécité diffuse réduite du parenchyme du cerveau chez 9 chiens, une ventriculomégalie chez 8 chiens, une atrophie du cerveau chez 4 chiens et des lésions focales hyperéchogènes chez 6 chiens. Une imagerie Doppler en couleur a révélé des vaisseaux plus évidents du cercle artériel chez 10 chiens. L'examen spectral Doppler a été réalisé chez 10 chiens pour détecter les 6 artères cérébrales majeures d'intérêt. L'examen a montré des valeurs d'indice de résistivité (IR) normales et hautes dans les artères indiquées. Les constatations de l'EDT étaient conformes à celles de la pathologie trouvées à l'examen postmortem.(Traduit par Isabelle Vallières).

Development, Optimization, and Validation of Forensic Analytical Method for Quantification of Anticholinesterase Pesticides in Biological Matrices from Suspected Cases of Animal Poisoning.

Anticholinesterase pesticides are a main cause of the intentional or accidental poisoning of animals. Anticholinesterases include several substances that cause the overstimulation of both central and peripheral acetylcholine-dependent neurotransmission. Forensic analyses of poisoning cases require high levels of expertise, are costly, and often do not provide reliable quantitative information for unambiguous conclusions. The purpose of the present study was to develop and validate a method of high-performance liquid chromatography with diode array detector (HPLC−DAD) for the identification and quantitation of n-methyl carbamates, organophosphates and respective metabolites from biological samples of animals that were suspected of poisoning. HPLC−DAD is reliable, fast, simplistic and cost-effective. The method was validated for biological samples obtained from stomach contents, liver, vitreous humor and blood from four different animal species. The validation of the method was achieved using the following analytical parameters: linearity, precision, accuracy, selectivity, recovery, and matrix effect. The method showed linearity at the range of 25−500 µg/mL, and the correlation coefficient (r2) values were >0.99 for all matrices. Precision and accuracy were determined by the (a) coefficient of variation (CV), (b) relative standard deviation low-quality control (LQC), (c) medium-quality control (QCM), and (d) high-quality control (QCA). The indicated parameters were all less than 15%. The recovery of analytes ranged from 31 to 71%. The analysis of results showed no significant interfering peaks due to common xenobiotics or matrix effects. The abovementioned method was used to positively identify pesticide analytes in 44 of the 51 animal samples that were suspected of poisoning, demonstrating its usefulness as a forensic tool.

Copper Toxicity in Horses: Does it Exist?

Copper toxicity is thought to be a rare condition in horses. However, the number of cases diagnosed in Brazil is growing. This article aims to describe cases of copper toxicity involving horses from different geographic locations and discuss findings of physical examinations, differential diagnoses and potential causes. Five cases referred from 4 different properties where at least 15 other horses were affected were described. Hemolytic anemia and hemoglobinuria, presence of Heinz bodies and elevated aspartate aminotransferase and gamaglutamil transferase levels were detected in all cases. The diagnosis was based on clinical history and signs, laboratory tests results, copper level determination in feed and/or soil and histopathological findings. Two horses progressed to acute death; remaining horses responded to clinical management with or without blood transfusion, depending on disease severity. However, one of these horses, after several returns to the veterinary hospital, was euthanized due to complications. One horse was treated with ammonium tetrathiomolybdate. Two horses had several recurring episodes over the course of several months, an uncommon presentation in ruminants suffering from copper toxicity. Excess copper was associated with soil fertilization with poultry litter or treatment of previous or neighbor crops with copper-containing products. It can be concluded that copper toxicity does occur in horses and may arise from several sources and/or be associated with predisposing dietary factors. Given the growing number of cases, the condition should be included in the differential diagnosis list and proper preventive dietary and pasture fertilization measures adopted.

Guapiaçu virus, a new insect-specific flavivirus isolated from two species of Aedes mosquitoes from Brazil.

Classical insect-flaviviruses (cISFVs) and dual host-related insect-specific flavivirus (dISFV) are within the major group of insect-specific flavivirus. Remarkably dISFV are evolutionarily related to some of the pathogenic flavivirus, such as Zika and dengue viruses. The Evolutionary relatedness of dISFV to flavivirus allowed us to investigate the evolutionary principle of host adaptation. Additionally, dISFV can be used for the development of flavivirus vaccines and to explore underlying principles of mammalian pathogenicity. Here we describe the genetic characterization of a novel putative dISFV, termed Guapiaçu virus (GUAPV). Distinct strains of GUAPV were isolated from pools of Aedes terrens and Aedes scapularis mosquitoes. Additionally, we also detected viral GUAPV RNA in a plasma sample of an individual febrile from the Amazon region (North of Brazil). Although GUAPV did not replicate in tested mammalian cells, 3'UTR secondary structures duplication and codon usage index were similar to pathogenic flavivirus.

Quantitative and qualitative analysis of lawsuits against veterinarians and correlation of potential risk factors with court decisions.

The increase in lawsuits against veterinarians based on recklessness, negligence and malpractice is a current trend since pets are increasingly viewed as part of the family, and legal action is sought if the owner is frustrated with the services rendered by the veterinarian. However, since scarce data exists on this issue, this study aims to quantify and qualify lawsuits filed against veterinarians in the state of São Paulo, Brazil to identify and correlate potential risk factors that may lead to conviction or acquittal of defendants. For this purpose, the methodology employed a search for lawsuits on the website of the São Paulo State Court of Justice, sorted by year; proceedings (summary or special civil court); plaintiff's claim (non-economic damages, economic damages, malpractice, negligence or recklessness); court decision (case granted or denied); reason for judgment (sufficient or insufficient evidence); reverse burden of proof (present or absent); type of evidence (deposition, documentary and expert testimony); value of damages claimed and awarded; location (greater São Paulo or inner state), and whether the lawsuit was filed against a veterinary clinic or hospital. These categories shed light, through bivariate and Poisson regression analyses, on the potential risk factors relative to court decisions. Results demonstrate that reverse burden of proof and insufficient evidence as the basis for judgment were, respectively, risk factors for conviction and acquittal of veterinary practitioners, and the number of lawsuits increased by 533 % over eight years.

Detection and characterization of Ilheus and Iguape virus genomes in historical mosquito samples from Southern Brazil.

In Brazil, flaviviruses have caused massive outbreaks. Surveillance programs designed to monitor virus activity in vectors provides a system for mapping disease distribution and for identifying specific vector species for targeted control. The present study aimed to describe the detection, whole genome characterization and phylogenetic analysis of Ilheus virus (ILHV) and Iguape virus (IGUV) strains obtained from historical mosquito's samples. Twelve isolates of pooled mosquito specimens (inoculated in neonate mouse brain) collected in the state of São Paulo, Brazil, in 1993, 1994 and 1997 were investigated. Viral RNA was extracted and analyzed by qRT-PCR using Flavivirus genus-specific primers. Positive samples were sequenced and underwent phylogenetic analyses. Flavivirus was detected in 50% of the specimens. Positive samples were successfully Sanger sequenced. Three Anopholes cruzii pools collected in 1994 were positive for IGUV. One Culex sp. pool, one Anopheles triannulatus pool, and one Coquillettidia juxtamansonia pool, collected in 1994, were positive for ILHV. Metagenomic sequencing successfully characterize one ILHV and four IGUV full genomes, and revealed a high degree of homology between the Brazilian ILHV and IGUV strains and isolates available in GenBank. Phylogenetic analysis of partial ILHV NS5 gene revealed three distinct lineages (clades), an indication of genetic heterogeneity in strains circulating in Brazil. Nucleotide insertions and a high-level of nucleotide diversity were observed in the NS1 protein and capsid region of IGUV strains, respectively. Detection of ILHV and IGUV in mosquitoes from Southeastern Brazil confirms the historical circulation of these viruses in this area. Furthermore, this first evidence of ILHV in Anopheles triannulatus suggests the potential importance of Anopheles mosquitoes in the IGUV transmission cycle. Genomic and phylogenetic analysis of these viruses provided insights into their diversity and evolution, which are important for the emergence patterns of flaviviruses and their evolutionary trends in Brazil, an endemic country for several arbovirus. in In-depth studies of ILHV and IGUV including vector competence and molecular studies are needed to shed light on their epidemiology and potential risk of future emergence.

Heat stress, Eimeria spp. and C. perfringens infections alone or in combination modify gut Th1/Th2 cytokine balance and avian necrotic enteritis pathogenesis.

Information on the dynamics of the chicken immune system during bacterial or parasite challenge in the presence or absence of stressful situations may provide a better understanding of the complex mechanisms behind these diseases. Necrotic enteritis (NE) had been controlled previously by the proper use of antimicrobial agents; however, more recently, NE has reemerged in many countries. The imposed restrictions on antimicrobial use and/or the intensive productive programs implemented by producers are challenges the birds, leading to large host adaptive responses that in many instances are like those elicited by stressors. This study analyses the effects of heat stress on Th1/Th2 cytokine balance, pathological features, and Toll-like receptor expression in the small intestine of broiler chickens infected with Clostridium perfringens type A in the presence or absence of Eimeria spp. co-infection. This co-infection model was experimentally used because it reproduces the findings commonly observed in the field during avian NE. For this purpose, broiler chickens infected with C. perfringens and/or Eimeria spp. were reared in isolator chambers subjected or not to heat stress intermittently. It was observed that heat stress directs the expression of Th2-type cytokines, increases Toll-like receptor 4 expression in the intestine and reduces the disease severity induced by Eimeria spp. and C. perfringens infections alone or in combination, most likely as a consequence of stress-induced changes in brain-gut axis activity.

A retrospective pathology study of two Neotropical deer species (1995-2015), Brazil: Marsh deer (Blastocerus dichotomus) and brown brocket deer (Mazama gouazoubira).

This retrospective study describes the biological and epidemiological aspects, gross and microscopical findings, and most likely causes of death (CD) in two species of Neotropical deer in Brazil. The animals were collected between 1995 and 2015 and represented 75 marsh deer (MD) and 136 brown brocket deer (BBD). Summarized, pneumonia was diagnosed microscopically in 48 MD and 52 BBD; 76 deer suffered trauma, involving dog attack (14 BBD) and vehicle-collision (14 BBD). Pulmonary edema (50 MD; 55 BBD) and congestion (57 MD; 78 BBD) were the most common findings for both species. Additionally, we diagnosed ruminal and myocardial mycosis in MD and BBD, respectively; ovarian dysgerminoma and pancreatic trematodiasis in BBD; and lesions suggestive of malignant catarrhal fever and orbiviral hemorrhagic disease in both species. The main CD in MD was: respiratory (41/75), alimentary, nutritional, trauma and euthanasia (3/75 each). Correspondingly, in BBD were: trauma (34/131), respiratory (30/131) and euthanasia (9/131). Respiratory disease was often defined by pulmonary edema and pneumonia. We provide evidence that respiratory disease, mainly pneumonia, is a critical pathological process in these Neotropical deer species. Although no etiological agents were identified, there is evidence of bacterial and viral involvement. Our results show trauma, mainly anthropogenic, as a common ailment in BBD. We propose to prioritize respiratory disease in future research focused on South American deer health aspects. We believe anthropogenic trauma may be a primary threat for populations of BBD.

Effect of Heat Stress on Sperm DNA: Protamine Assessment in Ram Spermatozoa and Testicle.

Sperm DNA fragmentation is considered one of the main causes of male infertility. The most accepted causes of sperm DNA damage are deleterious actions of reactive oxygen species (ROS), defects in protamination, and apoptosis. Ram sperm are highly prone to those damages due to the high susceptibility to ROS and to oxidative stress caused by heat stress. We aimed to evaluate the effects of heat stress on the chromatin of ejaculated and epididymal sperm and the activation of apoptotic pathways in different cell types in ram testis. We observed higher percentages of ejaculated sperm with increased chromatin fragmentation in the heat stress group; a fact that was unexpectedly not observed in epididymal sperm. Heat stress group presented a higher percentage of spermatozoa with DNA fragmentation and increased number of mRNA copies of transitional protein 1. Epididymal sperm presented greater gene expression of protamine 1 on the 30th day of the spermatic cycle; however, no differences in protamine protein levels were observed in ejaculated sperm and testis. Localization of proapoptotic protein BAX or BCL2 in testis was not different. In conclusion, testicular heat stress increases ram sperm DNA fragmentation without changes in protamination and apoptotic patterns.

Hepatic granulomas induced by Schistosoma mansoni in mice deficient for connexin 43 present lower cell proliferation and higher collagen content.

Granuloma formation involves a coordinated interaction between monocytes and macrophages, epithelioid cells, lymphocytes, eosinophils, neutrophils and fibroblasts. It has been established that extracellular communication via cytokines is important for the assembly of granulomas. However, the importance of gap junctions and intercellular communication to granuloma formation and development had never been assessed. Connexins are proteins that form gap junctions, and connexin 43 (Cx43) is present in macrophages, lymphoid cells, myelogenous cells, fibroblasts and others. We analyzed the effect of heterologous deletion of Gja1 (Cx43 gene) on the formation and development of hepatic granulomas induced by Schistosoma mansoni eggs. Heterozygous (Cx43(+/-)) and wild-type (Cx43(+/+)) mice were infected subcutaneously with S. mansoni cercarie and evaluated after 6, 8 and 12 weeks. Granuloma cells express Cx43, as revealed by real-time PCR in isolated granulomas, and by immunohistochemistry. Cx43 expression was reduced in Cx43(+/-) mice, as expected. No differences in the average area of granulomas or number of cells per granuloma were observed between mice of different genotypes. However, granuloma cells from Cx43(+/-) mice displayed a reduced index of the proliferating cell nuclear antigen (PCNA) labeling at 8 and 12 weeks post-infection. Moreover, Cx43(+/-) granulomas unexpectedly presented a higher degree of fibrosis, quantified by morphometric analysis in Sirius Red-stained slides. Our results indicate that the deletion of one allele of the Cx43 gene, and possibly the reduced gap junction intercellular communication capacity (GJIC), may impair the interactions between granuloma cells, reducing their proliferation and increasing their collagen content, thereby modifying the characteristics of S. mansoni granuloma in mice.

Evaluation of effects of prenatal exposure to the cyanide and thiocyanate in wistar rats.

A study was performed at term and at weaning to verify the toxic effects of the prenatal exposure to potassium cyanide (KCN) and potassium thiocyanate (KSCN) in pregnant Wistar rats. Females received daily in drinking water the doses: 1, 3 and 30mg KCN/kg or 0.8, 2.4 and 24mg KSCN/kg from GD 6 to GD 20 and were euthanized on GD 20 (trial A) or one day after weaning (trial B). Skeletal and visceral analyses of the fetuses (trial A) were performed and samples of blood and different organs, from both dams (trials A and B) and weaned pups (trial B), were collected in order to perform the biochemical evaluation and histopathology. Results showed high thiocyanate levels in dams of the different experimental groups from both trials. The intensity of the histological lesions observed in dams of trial B was similar to that of trial A, except those lesions found in the pancreas. The histopathological study of this organ revealed loss of cells in the Islets of Langerhans from dams of trial A which received the highest dose of cyanide (CN). There was an increase in the number of biliary ducts in animals treated with the highest doses of both thiocyanate and cyanide. The histopathological study of the spleen and the lungs of experimental and control groups did not reveal any significant alteration. In relation to fetuses (trial A), the visceral and skeletal evaluations did not reveal any significant malformation; on the other hand, pups from trial B showed some histological alterations similar to those observed in their dams. It is concluded that the cyanide and/or thiocyanate promoted toxic effects in the fetuses some of which could also be observed at weaning.

[Immunohistochemical staining of the astrocytic expression of glial fibrillary acidic protein and vimentin in the central nervous system of dogs with canine distemper]. / Marcação imunoistoquímica da expressão astrocitária de proteína glial fibrilar ácida e de vimentina no sistema nervoso central de cães com cinomose.

Considering that many aspects involved in the pathogenesis of the central nervous system (CNS) demyelinating diseases are still poorly understood and that astrocytes seem to mediate such processes, this study analyzed the participation of astrocytes in the demyelinating processes of CNS by using immunohistochemical staining of two astrocytic proteins--glial fibrillary acidic protein (GFAP) and vimentin (VIM)--comparing samples of cerebellum and brainstem from eight dogs with canine distemper and from two healthy dogs, from different breeds and ages varying from 1 to 4 years old. Histological sections were submitted to the avidin-biotin-peroxidase indirect method of immunohistochemical staining (ABC) and the astrocytic reactivity, observed in light microscopy, was quantified in a computer system for image analysis. It was possible to notice, on most of the sections from sick animals, degenerative lesions that indicate demyelination. The immunostaining for GFAP and VIM was more intense on animals with canine distemper, specially around the ventricules and near degenerated sites. There was no significant difference between the immunostaining (GFAP and VIM) of animals with canine distemper with and without inflammatory infiltrate of the cerebellar white matter. The increased immunoreactivity of astrocytes for GFAP and the VIM reexpression in injured areas indicate the astrocytic involvement on nervous tissue response to the demyelinating lesions induced by the canine distemper virus (CDV) in the CNS.