Expressão temporal de genes alfa, beta e gama durante a infecção pelo BoHV-5

Herpesvirus bovino 5 é um alfaherpesvírus causador de meningoencefalite não supurativa em bovinos. Esta doença possui ocorrência natural em surtos ou casos isolados, associadas a baixa morbidade e alta letalidade. Embora estudos anteriores tenham elucidado aspectos relacionados a patogenia da doença, há uma lacuna de conhecimento relacionado aos eventos moleculares que contribuem para a infecção e replicação do BoHV-5. O objetivo do presente estudo foi determinar a expressão gênica in vitro de genes virais (i.e., alfa, beta e gama) e das células hospedeiras (GAPDH e 18S) durante a infecção considerando diferentes momentos de infecção e quantidade de vírus utilizado. Três genes do BoHV-5 (bICP0, UL9, US4), um gene estrutural (GAPDH) e um gene constitutivo (18S) da célula bovina tiveram suas expressões avaliadas por PCR quantitativa (qPCR). Enquanto os genes virais tiveram sua expressão aumentada ao longo do tempo de infecção, o gene hospedeiro teve sua expressão diminuída, demonstrando a ação do vírus na expressão gênica de células bovinas in vitro. O gene constitutivo 18S teve sua expressão mantida durante todos os momentos do experimento. Nossos resultados claramente demonstraram que o GAPDH não deve ser usado como gene de referência em estudos com infecção por BoHV-5 pois é influenciado pela infecção viral. Entretanto, o 18S rRNA foi constitutivamente expresso e pode ser recomendado para normalização em células bovinas infectadas pelo vírus. A expressão de mRNA viral não foi alterada pela quantidade de vírus usada. Todos os genes virais demonstraram o mesmo padrão de expressão ao longo do tempo de infecção. Nossos resultados trazem importantes diferenças comparando aos estudos clássicos que avaliaram a expressão de genes alfa, beta e gama. Mais estudos são necessários para aumentar o conhecimento da biologia molecular do BoHV-5. Estudo utilizando sequenciamento de última geração (i.e., RNA-seq), usando modelos in vitro e in vivo, aparentam ser o próximo passo lógico para acessar as alterações do transcriptoma do hospedeiro e viral ao longo do curso da infecção.Bovine herpesvirus 5 is an alphaherpesvirus that causes nonsuppurative meningoencephalitis in cattle. This disease occurs naturally in either outbreaks or isolated cases, and exhibits low morbidity and high lethality. Although previous studies elucidated crucial aspects involved in the pathogenesis of the disease, there is a paucity of information regarding the molecular events contributing to infection and replication of BoHV-5. The objective of the present study was to determine the in vitro gene expression pattern of BoHV-5 (e.g., alpha, beta, and gamma genes) and host cells genes (GAPDH and 18S) over time utilizing different quantities of inoculated virus. Three BoHV-5 genes (bICP0, UL9, US4) and one structural bovine cell gene had their expression accessed by real-time PCR. While the expression of BoHV-5 genes increased during the course of infection, GAPDH gene expression decreased in the host cells, evidencing the effect of viral infection on the expression of bovine cell genes. The 18S ribosomal RNA (rRNA) gene was constitutively expressed throughout BoHV-5 infection. Our data clearly demonstrates that GAPDH gene should not be used as a reference gene in studies of BoHV-5 infection because it was influenced by viral infection. However, 18S rRNA was constitutively expressed and, therefore, is recommended for normalization of BoHV-5 infection studies in bovine cells. The expression of viral genes transcripts was not altered by increasing number of viral particles added to the culture. All viral genes included here demonstrated the same expression pattern over time and there was no difference in the expression of viral genes among the various time points. Our data show important differences comparing to classical studies regarding herpesvirus alpha, beta, and gamma genes expression. More research is necessary to improve our understanding about the BoHV-5 biology during infection. Studies employing next-generation sequencing (i.e., RNA-seq), using both in vitro and in vivo models, would be the next logical step to grasp the virus and host cell’s transcriptome changes over the course of infection

Pathological findings of post-anesthetic myopathy associated with type 1 polysaccharide storage myopathy in a Percheron horse

Background: Post-anesthetic myopathy is the most common complication associated with general anesthesia in horses. Polysaccharide storage myopathy (PSSM) is characterized by an abnormal accumulation of glycogen and glycogen-related polysaccharides in the skeletal muscle, which is categorized in type 1 (PSSM1) and type 2 (PSSM2). The purpose of this study is to report the clinical, pathological and molecular findings in a Percheron mare with post-anesthetic myopathy associated with a PSSM1. Case: A 9-year-old Percheron mare was submitted to a caesarean section due to clinical dystocia during labor. Xylazine was employed during pre-anesthesia, followed by induction with ketamine and diazepam, while anesthetic maintenance was obtained with isoflurane. The mare showed good recovery, however 24 h later, sternal recumbency and hyperthermia (41° C) were observed. The mare was euthanized, and a necropsy was performed. Samples of multiple tissues were collected and routinely processed for histology. At necropsy, segments of skeletal muscles had bilateral pale areas. The kidneys had old and recent infarcts. The heart had whitish areas in the myocardium. The brain showed focally extensive reddish areas, with flattening of gyri. Histologically, skeletal muscle fibers had in the sarcoplasm multiple homogeneous globular clear eosinophilic formations, in addition to mild hyaline necrosis. In the heart and in the kidney, there were extensive areas of acute coagulative necrosis. The brain showed marked multifocal fibrinoid degeneration of vessels and hemorrhage. Refrigerated liver samples were submitted to DNA extraction to detect mutations in the GYS1 (type 1 PSSM) and RyR1 genes (malignant hyperthermia). A positive result for a homozygous dominant mutation in GYS1 (type 1 PSSM) was observed, while the mutation responsible for malignant hyperthermia was not identified Discussion: The diagnosis of post-anesthetic myopathy associated with PSSM was obtained by the presence of amylase resistant polysaccharide complex inclusions, glycogen subsarcolemmal aggregates, and central cytoplasmic corpuscles containing glycogen through PAS-amylase resistant histochemical technique, associated to the myopathy microscopical features. Microscopic findings were related to clinical history, and the diagnosis of PSSM underlying post-anesthetic myopathy was determined. The predisposition of the Percheron horse has been described as an inherited predisposition leading to PSSM susceptibility, as was observed in the present case. We speculated that the anesthetic procedure resulted in the precipitation of the drug and a presentation of an acute anesthetic myopathy, while the muscle damage most likely occurred due to the ischemia caused by systemic hypotension. In addition to these lesions, other lesions were considered related to the use of the anesthetics, which may predispose to vasculogenic injuries. This horse was diagnosed as being homozygous dominant for the GYS1 gene, which causes a gain-offunction and results in glycogenolysis with glycogen accumulation in myofibers. Horses that are homozygous for the GYS1 gene may exhibit more severe histological changes in the skeletal muscle fibers, such as necrosis, anisocytosis, endomysial fibrosis, and fatty infiltration In PSSM, there is a bilateral involvement of the skeletal muscles with areas of degeneration of whitish or greyish coloration, as well as pale muscle with whitish streaks due to coagulative necrosis and edema. In our study, we observed bilateral skeletal muscle lesions and cardiomyocyte necrosis. Post-anesthetic myopathy, along with skeletal muscle lesions, may predispose to vasculogenic injuries, with kidney and brain lesions in horses. Dominant homozygosis for the GYS1 gene with consequent PSSM1 disease probably aggravated the condition in this Percheron, with more severe histological muscular lesions. Our study should bring attention to the use of anesthetics in horses with PSSM1, especially in the Percheron breed

Erythema Multiforme and its Clinicopathological Features in a Horse in Brazil

Background: Erythema multiforme (EM) is an immune-mediated skin disease which may manifest as cutaneous or mucocutaneous lesions. It is uncommon in horses. EM lesions have a symmetrical bilateral distribution; they are usually urticarial, necrotizing, and, less commonly, ulcerative. In equines, the trigger is usually unknown, and cases are often classified as idiopathic. Diagnosis is based on a thorough history and physical and histopathological examination of lesions. According to the clinical presentation and histopathological characteristics of the cutaneous lesions, this case is the first report to describe diagnosis and treatment of a horse with EM in Brazil. Case: A Quarter Horse filly was followed clinically for 12 months after sudden onset of skin lesions at 18 months of age. The initial lesions were non-alopecic papules with a symmetrical bilateral distribution. Six months after onset, the skin lesions maintained the original distribution pattern; however, they had progressed to papules and plaques with varying annular, arciform, serpiginous, targetoid, or alopecic appearance. At 8 months, the same distribution pattern and appearance remained, but the lesions had become more severe and extensive, with involvement of the labial commissures and perineal region, without any erosions/ulcerations, scaling/crusting, pain, or pruritus. At 12 months, new nodular lesions were found on the medial and lateral surfaces of the hind limbs, neck, bilateral trunk, and root of the tail. The lesions were firm, non-pruritic, and non-tender on palpation. Swabs were obtained from the papular lesions. Skin specimens were also obtained with a 6-mm punch and via incisional biopsy and histological sections were made. Bacterial and fungal cultures were negative. Appropriate stains did not identify fungal structures, were negative for acid-fast bacilli, and did not reveal any metachromatic granules in the sampled cell population. The histopathological findings were characteristic of immune-mediated disease, with a vacuolar interface dermatitis affecting the hair follicles more than the epidermis, necrotic keratinocytes, lymphocyte satellitosis, leukocytoclastic mixed vasculitis of the mid-dermis and deep dermis, and variable granulation tissue, consistent with erythema multiforme and leukocytoclastic vasculitis. Immunosuppressive therapy with corticosteroids and oral supplementation with omega-3 and omega-6 fatty acids and vitamin E were prescribed. After institution of therapy, no new lesions developed, the existing lesions remained stable (though permanent), and hair regrew in the previously alopecic areas. All physiological parameters remained normal throughout the follow-up period. Discussion: Erythema multiforme is rarely reported in horses. According to our literature review, this is the first description of EM in horses in Brazil. EM should be included in the differential diagnosis of horses that present with plaques in a diverse, geographic distribution and a negative initial dermatological screening examination. Further clinical investigation is warranted, with special attention to potential antigenic triggers. A thorough drug and dietary history and close attention to comorbidities are essential, as the suppression of potential culprit factors has important prognostic value and contributes to the elucidation of EM triggers

Concentração de imunoglobulinas G e M no soro sanguíneo de bezerros da raça Holandesa até os 90 dias de idade

This study evaluated the G and M concentrations in serum of 32 calves until 90 days old, considering the way (direct suckling from mother or by bottle) and the volume of ingested colostrum. The calves were divided into 4 groups. Group 1: four liters of colostrum fed by bottle; Group 2: two liters of colostrum fed by bottle; Group 3: direct suckling from mother and and Group 4: two liters of milk by bottle. Serum IgG concentrations obtained in this study had no significant difference in the way of colostrum feeding (direct suckling from mother or by bottle), but their levels were higher in the former way of administration of colostrum, followed by those which received 4 liters of colostrum. Those animals which were not fed with colostrum presented IgG and IgM levels earlier, from 30 and 15 days old, respectively.Avaliou-se os níveis de imunoglobulinas G e M no soro sangüíneo de 32 bezerros da raça Holandesa desde o nascimento até os 90 dias de vida, levando-se em consideração a forma de ingestão (mamada natural na mãe ou em mamadeira) e a quantidade de colostro administrado. Os animais foram distribuídos em quatro grupos. O grupo 1 recebeu quatro litros de colostro fornecido na mamadeira; o grupo 2 recebeu dois litros de colostro também fornecido através da mamadeira; o grupo 3 ingeriu colostro diretamente nas mães e o grupo 4 foi tratado exclusivamente com leite administrado através de mamadeira. Os resultados obtidos para as concentrações de IgG sérica não apresentaram diferenças estatísticas significativas quanto a forma de fornecimento do colostro (mamada na mãe ou em mamadeira), porém os níveis dessa imunoglobulina sempre foram mais elevados nos animais que mamaram nas mães, seguidos pelos que receberam quatro litros de colostro. Nos animais que não mamaram colostro, a produção de imunoglobulinas G e M é mais precoce, respectivamente, a partir de 30 e 15 dias de vida

Polioencephalomalacia in Sheep - Clinical and Magnetic Resonance Imaging Findings

Background: Polioencephalomalacia (PEM) is a neurological disease in ruminants, which is characterized by malacia of brain gray matter. Thiamine deficiency and sulfur intoxication are the most common causes of PEM in sheep. Affected animals present signs of cerebrocortical syndrome, including amaurosis, ataxia, head pressing, mental depression, seizures, and opisthotonus. The neurological examination aims to determine the neurolocalization of the lesions and advanced imaging techniques are useful for confirming the affected area(s) in the central nervous system. The aim of this study is to describe clinical features and ante-mortem diagnosis using magnetic resonance imaging (MRI) in a sheep with PEM. Case: An 18-month-old male Dorper sheep from a flock started receiving concentrate 7 days before. According to the owner, no clinical signs of abnormality were observed on the previous morning. However, in the afternoon, the animal became self-isolated and did not follow the flock to the sheepfold. The following day, he was found in recumbency. Physical examination revealed lateral recumbency, rectal temperature 39.5ºC, 52 bpm, 120 bpm, congested mucous membranes, capillary refill time 1 s, ruminal (4/5 min) and intestinal hypomotility. The assessment of the central nervous system revealed a decreased level of consciousness, focal seizures, opisthotonus, and absence of menace response. The following differential diagnoses were listed: PEM, head trauma, focal symmetrical encephalomalacia, bacterial encephalitis, and rabies. Treatment was composed of dexamethasone [0.2 mg/kg - i.v., SID (1st-3rd day), 0.1 mg/kg, i.v., SID (4th-6th day), and 0.05 mg/kg, i.v., SID (7th-9th day)]; mannitol [1 g/kg -  i.v. and diazepam 0.4 mg/kg, i.v. single dose at admission]; vitamin B1 [10 mg/kg - i.m., SID], furosemide [1 mg/kg - i.v., SID for 3 days] and sulfadoxine/trimethoprim [30 mg/kg -  i.m., SID for 10 days]. After the initial treatment, the patient showed mild clinical improvement; however, the amaurosis was still present. Magnetic resonance imaging of the brain was performed on the 2nd day of hospitalization, showing a symmetrical hypersignal in the parietal and occipital cortices, in the axial and sagittal sequences weighted in T2 and FLAIR. Discussion: This study aimed to describe the clinical signs and MRI findings in a sheep with PEM. In this case, the sudden change to the feed composition probably led to ruminal dysbiosis, inhibition of thiamine-producing microorganisms and proliferation of bacteria that synthesize thiaminase. Thiamine therapy proved to be effective and capable of reverting the clinical signs. The decrease in the level of consciousness, cortical blindness, and opisthotonus are due to alterations in the parietal cortex, in the occipital cortex, and in the cerebellum, respectively, which were demonstrated by hypersignal areas in the MRI. Therefore, the neurolocalization of the lesion based on neurologic examination and the MRI findings were related. The physicochemical and cytological evaluations of the cerebrospinal fluid, and dosage of thiamine and the concentration of hydrogen sulphide in the rumen were not performed. However, the response to thiamine treatment associated with the neurologic examination and MRI findings helped in determining the diagnosis. Additionally, MRI can be used as a useful tool for the ante mortem diagnosis of PEM. Keywords: cerebrocortical necrosis, diagnostic imaging, neurology, ruminants, thiamine deficiency