Phenotypic and molecular identification of Brucella suis biotype 1 in a pig from Brazil-case report.

Brucellosis is a zoonotic disease with a global impact. Brucella suis is one of the most pathogenic species to humans, requiring different measures for the control and/or eradication of the disease. The serological investigation for brucellosis was performed in pigs, horses, dogs, and cattle on a farm with a history of abortion in sows and necropsy of a boar with severe necrosuppurative orchitis. One sow, two cows, and two dogs reveled positive to Rose Bengal Test (RBT), although only the sow had a confirmatory outcome in 2-mercaptoethanol (2-ME). The 2-ME-positive sow was euthanized and microbiological culture of lymph nodes and liver followed by biochemical characterization allowed phenotypic characterization of Brucella suis biotype 1. PCR multiplex Bruce-ladder and Suis-ladder enabled molecular confirmation, respectively, of Brucella suis and biotype 1. The transmission aspects of B. suis to pigs and other domestic species, the combination of diagnostic procedures to diagnosis, as well as human health concerns of brucellosis are discussed.

Spatiotemporal expression pattern of alpha, beta, and gamma genes during BoHV-5 infection / Expressão temporal de genes alfa, beta e gama durante ainfecção pelo BoHV-5

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.(AU)

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.(AU)

Correlation of feline immunodeficiency virus status and the severity of feline chronic gingivostomatitis / Correlação do status sorológico para FIV e a gravidade da gengivoestomatite crônica felina

Feline chronic gingivostomatitis (FCGS) is a challenge for the veterinary practitioner since its etiology and treatments are still undefined. The present paper investigated the role of the feline immunodeficiency virus (FIV) in the severity of the FCGS. Oral mucosal biopsies obtained from 19 cats with FCGS were divided into two groups according to their FIV serology status. Later, the clinical lesion score was correlated with the histopathological grade of FCGS lesions and the degree of immunostaining in both groups. Most of the animals had significant histological changes; however, no correlation with FIV immunostaining intensity was observed. It was concluded that the presence of FIV infection or the animal's seropositivity status does not seem to interfere with the severity of clinical signs nor the degree of histopathological changes when compared to the seronegative group.(AU)

A gengivoestomatite crônica felina (FCGS) é um desafio para o veterinário, uma vez que a sua etiologia e tratamentos permanecem indefinidos. O presente trabalho investigou o papel do vírus da imunodeficiência felina (FIV) na gravidade do FCGS. Biópsias da mucosa oral de 19 gatos com FCGS foram divididas em dois grupos de acordo com o status sorológico de FIV. Mais tarde, o escore de lesão clínica foi correlacionado com o grau histopatológico das lesões FCGS e o grau de imunocoloração em ambos os grupos. A maioria dos animais apresentou alterações histológicas significativas, porém não foi observada correlação com a intensidade de imunocoloração para FIV. Concluiu-se que a presença de infecção por FIV ou o estado soropositivo dos animais não parece interferir com a gravidade dos sinais clínicos nem com o grau de alterações histopatológicas quando comparado ao grupo soronegativo.(AU)

Mycobacterium genavense infection in two species of captive snakes.

BACKGROUND: Mycobacterium is an important zoonotic agent with companion, livestock and wildlife animals reportedly playing a role as reservoirs. Although its association with reptiles has been described, the disease cycle remains to be fully established, particularly in snakes. Accordingly, this study aimed to report the occurrence of mycobacteriosis with clinical pneumonia in one exotic python snake (Python molurus) and one native green snake (Philodryas olfersii) from the Sorocaba Zoo, São Paulo state, Brazil. METHODS: Diagnosis was based on necropsy, histopathological examination, Ziehl-Neelsen stain and immunohistochemistry. RESULTS: Using a nested PCR followed by DNA sequencing and bioinformatics analysis, the causative Mycobacterium species was identified as Mycobacterium genavense. CONCLUSION: Mycobacterium genavense is an infectious zoonotic agent of animal and public health concerns.

Mycobacterium genavense infection in two species of captive snakes

Mycobacterium is an important zoonotic agent with companion, livestock and wildlife animals reportedly playing a role as reservoirs. Although its association with reptiles has been described, the disease cycle remains to be fully established, particularly in snakes. Accordingly, this study aimed to report the occurrence of mycobacteriosis with clinical pneumonia in one exotic python snake (Python molurus) and one native green snake (Philodryas olfersii) from the Sorocaba Zoo, São Paulo state, Brazil. Methods: Diagnosis was based on necropsy, histopathological examination, Ziehl-Neelsen stain and immunohistochemistry. Results: Using a nested PCR followed by DNA sequencing and bioinformatics analysis, the causative Mycobacterium species was identified as Mycobacterium genavense. Conclusion: Mycobacterium genavense is an infectious zoonotic agent of animal and public health concerns.

Mycobacterium genavense infection in two species of captive snakes

Background: Mycobacterium is an important zoonotic agent with companion, livestock and wildlife animals reportedly playing a role as reservoirs. Although its association with reptiles has been described, the disease cycle remains to be fully established, particularly in snakes. Accordingly, this study aimed to report the occurrence of mycobacteriosis with clinical pneumonia in one exotic python snake (Python molurus) and one native green snake (Philodryas olfersii) from the Sorocaba Zoo, São Paulo state, Brazil. Methods: Diagnosis was based on necropsy, histopathological examination, Ziehl-Neelsen stain and immunohistochemistry. Results: Using a nested PCR followed by DNA sequencing and bioinformatics analysis, the causative Mycobacterium species was identified as Mycobacterium genavense. Conclusion: Mycobacterium genavense is an infectious zoonotic agent of animal and public health concerns.(AU)

Comparative clinical sample preparation of DNA and RNA viral nucleic acids for a commercial deep sequencing system (Illumina MiSeq(®)).

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.

Rough virulent strain of Brucella ovis induces pro- and anti-inflammatory cytokines in reproductive tissues in experimentally infected rams.

The ovine brucellosis caused by Brucella ovis has tropism for reproductive tissues but until now the mechanism of bacterial persistence is not understood. Cytokine expression profiles were studied for 8 months in rams after being experimentally infected with the rough virulent strain of B. ovis (R-B. ovis) to study the pathogenesis of B. ovis and immune mechanism possibly associated to bacteria tropism and persistence. The messenger RNA (mRNA) expression levels of interleukin-1α (IL-1α), IL-1ß, IL-6, IL-10, IL-12, interferon-γ (INF-γ) and tumour necrosis factor-α (TNF-α) cytokines were quantified by real-time quantitative RT-PCR (qRT-PCR) in reproductive tissues (epididymus, testicles, ampolae, vesicular glands and bulbourethral glands), and non-reproductive (liver, spleen and kidneys) tissues at 30, 60, 120 and 240 days post infection (dpi). During the acute phase of infection at 30 dpi, the host immune response was most notable demonstrating an up-regulation of several cytokines in reproductive tissues, including the epididymus (IL-6, IL-1ß and IL-1α), testicles (INF-γ and IL-12), bulbourethral glands (IL-6 and TNF-α) and ampolae (INF-γ, IL-10, IL-1ß and IL-1α). During the development of infection, cytokine gene expression levels decreased, providing evidence of immunosuppression and evidence of immune evasion that favoured persistence of chronic R-B. ovis infection. During the chronic phase of R-B. ovis infection (120 and 240 dpi), cytokine production was down-regulated in the epididymus (IL-1ß and IL-1α), testicles (INF-γ and IL-12), and ampolae (INF-γ, IL-10, IL-1ß and IL-1α), with the exception of the bulbourethral glands (IL-6 and TNF-α) and epididymus (IL-6); in these tissues, R-B. ovis infection resulted in up-regulation of the pro-inflammatory cytokine IL-6. Herein, we report cytokine expression profiles in tissues of rams experimentally infected with the rough strain of B. ovis, which are associated with bacterial persistence and macrophage activation.

Canine distemper virus in a crab-eating fox (Cerdocyon thous) in Brazil: case report and phylogenetic analyses.

Although canine distemper is enzootic worldwide and has a wide host range, there are no reports of canine distemper virus in crab-eating foxes (Cerdocyon thous) that provide information on virus phylogeny and histopathologic lesions. The objective of this study is report and describe canine distemper in a crab-eating fox (C. thous), with a focus on the phylogeny of the virus strain and the histopathologic lesions in the animal.