Canine ringworm caused by Trichophyton mentagrophytes - detection by SYBR-Green real-time PCR

Background: Dermatophytes, fungi of universal distribution, invade semi or fully keratinized structures, such as skin, fur/ hair and nails. The various species of dermatophytes are classified into three genera anamorphic: Microsporum, Trichophyton and Epidermophyton. The genus Epidermophyton includes only E. floccosum, that rarely affects animals. The main species responsible for the disease in dogs and cats are Microsporum canis, M. gypseum and Trichophyton mentagrophytes, which were characterized through conventional mycological methodology (microscopic examination with KOH and culture). Molecular methodologies, such as real-time PCR, can contribute to a rapid laboratory diagnosis, helping clinicians to initiate an early antifungal treatment. This case report describes a case of canine dermatophytosis due to Trichophyton mentagrophytes detected from a clinical sample by SYBR-Green real-time PCR. Case: A 8-year-old dog, rescued from the street, was referred to a private veterinary clinic in the city of Canoas, RS, Brazil, presenting generalized lymphadenomegaly, crusted lesions all over the body, generalized alopecia, signs of excoriation and epistaxis. Initially, were administered prednisone [1 mg/kg every 48 h, BID] and cephalexin [30 mg/kg, BID]. Weekly baths with benzoyl peroxide were also given. The therapy was not clinically successful. Wood's Lamp Test was negative. As a differential diagnosis, PCR for detection of Leishmania was negative. Complete blood count and serum biochemical assay were also performed. For mycological diagnosis, hair specimen was clarified and examined microscopically using 10% potassium hydroxide (KOH) for the visualization of chains of arthroconidia (ectothrix invasion of hair). The infected hair was plated onto MycoselTM Agar, incubated at 28°C for 15 days. Microscopy of hyphae/ conidia and macroscopic colony characteristics (colors and texture) were conducted for the differentiation of the species within the genus Microsporum and Trichophyton. In addition, real-time PCR was applied for direct analysis of the fungal DNA obtained from the hair sample. Microscopic examination was negative. The dermatophyte present in the hair sample was confirmed as Trichophyton mentagrophytes by culture and qPCR (melting-point analysis). The patient was treated with systemic itraconazole [10 mg/ kg SID - 90 days]. Twice-weekly application of 2.5 % miconazole and 2% chlorhexidine shampoo until complete cure. Discussion: Dermatophytosis is often listed as self-limiting infection; however, animal dermatophytosis can spread between pets, as well as a zoonotic transmission to humans. The literature on dermatophytosis indicates that Microsporum canis is the predominant etiological agent, followed by M. gypseum. Trichophyon mentagrophytes that appear in a lower percentage of isolation. The culture of hair, even with specific medium containing chloramphenicol and cyclohexamide, may present contaminating fungi, not related to dermatophytosis, which can inhibit or override the growth of dermatophytes. The use of real-time PCR provided a faster and specific diagnosis of dermatophytosis when compared to the conventional mycological methodology for detection and identification of T. mentagrophytes, which takes around 10 to 15 days for culture. It is possible to use this technique as an alternative diagnosis for dermatophytes associated to clinical hair samples of dogs.

Antifungal susceptibility profile of Aspergillus fumigatus isolates from avian lungs / Perfil de suscetibilidade antifúngica de isolados de Aspergillus fumigatus provenientes de pulmões de aves

Susceptibility testing is essential to inform the correct management of Aspergillus infections. In this study we present antifungal susceptibility profile of A. fumigatus isolates recovered from lungs of birds with and without aspergillosis. Fifty three isolates were tested for their antifungal susceptibility to voriconazole (VRC), itraconazole (ITZ), amphotericin (AMB) and caspofungin (CSP) using the M38-A2 broth microdilution reference method. Five isolates were resistant to more than one antifungal drug (CSP + AMB, VRC + ITZ and AMB + ITZ). Fifteen (28%) isolates with susceptible increased exposure (I) to ITZ were sensible to VRC. Resistance to AMB (>2µg/mL) was observed in only four isolates. Eleven (21%) A. fumigatus present resistance to ITZ (13%) and VRC (8%). Fungal isolation from respiratory samples has been regarded as being of limited usefulness in the ante mortem diagnosis of aspergillosis in birds. However, the results suggest that the detection and antifungal susceptibility profile may be helpful for monitoring of therapy for avian species and where antifungal resistance might be emerging and what conditions are associated to the event.(AU)

Os testes de suscetibilidade são essenciais para informar o correto manejo das infecções por Aspergillus. Neste estudo apresentamos o perfil antifúngico de isolados de A. fumigatus provenientes de pulmões de aves com e sem aspergilose. Cinqüenta e três isolados foram testados quanto à susceptibilidade antifúngica ao voriconazol (VRC), itraconazol (ITZ), anfotericina B (AMB) e caspofungina (CSP) pelo método de referência de microdiluição do caldo M38-A2. Cinco isolados foram resistentes a mais de um antifúngico (CSP + AMB, VRC + ITZ e AMB + ITZ). Quinze (28%) isolados suscetíveis - com exposição aumentada (I) ao ITZ foram sensíveis ao VRC. A resistência ao AMB (>2µg/mL) foi observada em apenas quatro isolados. Onze (21%) A. fumigatus apresentaram resistência a ITZ (13%) e VRC (8%). O isolamento de fungos de amostras respiratórias tem sido considerado de utilidade limitada no diagnóstico ante mortem de aspergilose em aves. No entanto, os resultados sugerem que a detecção e o perfil de suscetibilidade a antifúngicos podem ser úteis para o monitoramento da terapia de espécies aviárias, assim como a emergência da resistência antifúngica e quais condições podem estar associadas ao evento.(AU)

Antifungal susceptibility profile of Aspergillus fumigatus isolates from avian lungs / Perfil de suscetibilidade antifúngica de isolados de Aspergillus fumigatus provenientes de pulmões de aves

Susceptibility testing is essential to inform the correct management of Aspergillus infections. In this study we present antifungal susceptibility profile of A. fumigatus isolates recovered from lungs of birds with and without aspergillosis. Fifty three isolates were tested for their antifungal susceptibility to voriconazole (VRC), itraconazole (ITZ), amphotericin (AMB) and caspofungin (CSP) using the M38-A2 broth microdilution reference method. Five isolates were resistant to more than one antifungal drug (CSP + AMB, VRC + ITZ and AMB + ITZ). Fifteen (28%) isolates with susceptible increased exposure (I) to ITZ were sensible to VRC. Resistance to AMB (>2µg/mL) was observed in only four isolates. Eleven (21%) A. fumigatus present resistance to ITZ (13%) and VRC (8%). Fungal isolation from respiratory samples has been regarded as being of limited usefulness in the ante mortem diagnosis of aspergillosis in birds. However, the results suggest that the detection and antifungal susceptibility profile may be helpful for monitoring of therapy for avian species and where antifungal resistance might be emerging and what conditions are associated to the event.(AU)

Os testes de suscetibilidade são essenciais para informar o correto manejo das infecções por Aspergillus. Neste estudo apresentamos o perfil antifúngico de isolados de A. fumigatus provenientes de pulmões de aves com e sem aspergilose. Cinqüenta e três isolados foram testados quanto à susceptibilidade antifúngica ao voriconazol (VRC), itraconazol (ITZ), anfotericina B (AMB) e caspofungina (CSP) pelo método de referência de microdiluição do caldo M38-A2. Cinco isolados foram resistentes a mais de um antifúngico (CSP + AMB, VRC + ITZ e AMB + ITZ). Quinze (28%) isolados suscetíveis - com exposição aumentada (I) ao ITZ foram sensíveis ao VRC. A resistência ao AMB (>2µg/mL) foi observada em apenas quatro isolados. Onze (21%) A. fumigatus apresentaram resistência a ITZ (13%) e VRC (8%). O isolamento de fungos de amostras respiratórias tem sido considerado de utilidade limitada no diagnóstico ante mortem de aspergilose em aves. No entanto, os resultados sugerem que a detecção e o perfil de suscetibilidade a antifúngicos podem ser úteis para o monitoramento da terapia de espécies aviárias, assim como a emergência da resistência antifúngica e quais condições podem estar associadas ao evento.(AU)

Pneumonia by Cryptococcus neoformans in a goat in the Southern region of Brazil / Pneumonia por Cryptococcus neoformans em um caprino na região Sul do Brasil

This report describes the clinical, pathological, and molecular aspects of a pneumonia by Cryptococcus neoformans in a goat in the Southern region of Brazil. A goat with a history of prolonged transportation presented dyspnea, nasal discharge and cough, and was subjected to necropsy, histopathology, and mycological evaluation. Grossly, cranio-ventral pulmonary consolidation was observed, characterized by firm and grayish areas interspersed with dark red foci. Histopathology of the lungs showed areas of parenchymal necrosis, containing blastoconidia with a slightly basophilic central cell, surrounded by an unstained capsule. It was associated with moderate granulomatous inflammatory infiltrate and peripheral fibrosis. The capsule and fungus cell exhibited marked Alcian Blue and periodic acid-Schiff staining, respectively. Diagnosis of fungal pneumonia by C. neoformans was based on clinical, pathological, and molecular findings.(AU)

Este relato objetiva descrever os aspectos clínicos, patológicos e moleculares de pneumonia por Cryptococcus neoformans em um caprino na região Sul do Brasil. Um caprino com histórico de transporte prolongado apresentou dispneia, secreção nasal e tosse e foi encaminhado para necropsia, análise histopatológica e micológica. Macroscopicamente, observou-se consolidação pulmonar cranioventral, caracterizada por áreas firmes e acinzentadas, entremeadas por focos vermelho-escuros. Na análise histopatológica dos pulmões foram evidenciadas áreas de necrose do parênquima, que continham blastoconídeos com célula central levemente basofílica, circundada por cápsula não corada, associados a moderado infiltrado inflamatório granulomatoso e fibrose periférica. A cápsula e a célula do fungo foram fortemente coradas pelo azul alciano e pelo ácido periódico de Schiff, respectivamente. O diagnóstico de pneumonia fúngica por C. neoformans foi baseado nos achados clínicos, patológicos e moleculares.(AU)

Esporotricose óssea em gato causada por Sporothrix brasiliensis / Bone sporotrichosis in a cat caused by Sporothrix brasiliensis

Background: Sporotrichosis is a deep cutaneous mycosis caused by the Sporothrix species complex, dimorphic fungi of which at least five are of clinical importance: S. brasiliensis, S. globosa, S. luriei, S. mexicana, and S. schenckii sensu stricto. The disease affects humans and animals, especially cats, which can manifest a wide spectrum of clinical sings, from cutaneous-lymphatic involvement to disseminated form. Infection usually results from direct inoculation of the fungus into skin. Zoonotic transmission is associated with scratching or biting of sick cats. The aim of this work was to report an atypical case of bone sporotrichosis in a cat.Case: A 5-year-old, male, neutered, mongrel and indoor cat was present at the Veterinary Clinic Hospital, Federal University of Rio Grande do Sul (HCV-UFRGS), Porto Alegre, Brazil, with lameness and increased volume in the left hindlimb. The animal had been treated intermittently with itraconazole during the last three years due to another cutaneous lesion which was recurrent and undiagnosed. A firm and painful mass was found in tarsal region of left hindlimb, that had approximately 5 cm in diameter. Radiographic examination of the left tibial-tarsal joint revealed bone lysis in the fifth metatarsal calcaneus, in addition to periosteum proliferation in calcaneus, tibio-tarsal subluxation, presence of osteophytes in tarsal bones and increase in soft tissue volume. Histopathological analysis of the biopsied tissue showed piogranulomatous inflammation. No yeast-like structures were observed in cytopathological exam. Tissue fragments were plated and Sporothrix sp. complex growth in mycological culture (Sabouraud Cycloheximide Chloramphenicol Agar). Physiological tests (growth rate at different temperatures and assimilation of sucrose and raffinose) were conducted for the differentiation of the species of complex. Molecular identification was performed using panfungal primers (ITS3-F / ITS4-R)...

Esporotricose óssea em gato causada por Sporothrix brasiliensis / Bone sporotrichosis in a cat caused by Sporothrix brasiliensis

Background: Sporotrichosis is a deep cutaneous mycosis caused by the Sporothrix species complex, dimorphic fungi of which at least five are of clinical importance: S. brasiliensis, S. globosa, S. luriei, S. mexicana, and S. schenckii sensu stricto. The disease affects humans and animals, especially cats, which can manifest a wide spectrum of clinical sings, from cutaneous-lymphatic involvement to disseminated form. Infection usually results from direct inoculation of the fungus into skin. Zoonotic transmission is associated with scratching or biting of sick cats. The aim of this work was to report an atypical case of bone sporotrichosis in a cat.Case: A 5-year-old, male, neutered, mongrel and indoor cat was present at the Veterinary Clinic Hospital, Federal University of Rio Grande do Sul (HCV-UFRGS), Porto Alegre, Brazil, with lameness and increased volume in the left hindlimb. The animal had been treated intermittently with itraconazole during the last three years due to another cutaneous lesion which was recurrent and undiagnosed. A firm and painful mass was found in tarsal region of left hindlimb, that had approximately 5 cm in diameter. Radiographic examination of the left tibial-tarsal joint revealed bone lysis in the fifth metatarsal calcaneus, in addition to periosteum proliferation in calcaneus, tibio-tarsal subluxation, presence of osteophytes in tarsal bones and increase in soft tissue volume. Histopathological analysis of the biopsied tissue showed piogranulomatous inflammation. No yeast-like structures were observed in cytopathological exam. Tissue fragments were plated and Sporothrix sp. complex growth in mycological culture (Sabouraud Cycloheximide Chloramphenicol Agar). Physiological tests (growth rate at different temperatures and assimilation of sucrose and raffinose) were conducted for the differentiation of the species of complex. Molecular identification was performed using panfungal primers (ITS3-F / ITS4-R)...(AU)

Phylogenetic analysis: basic concepts and its use as a tool for virology and molecular epidemiology / Análise filogenética: conceitos básicos e suas utilizações como ferramenta para virologia e epidemiologia molecular

Background: Phylogenetic analyses are an essential part in the exploratory assessment of nucleic acid and amino acid sequences. Particularly in virology, they are able to delineate the evolution and epidemiology of disease etiologic agents and/or the evolutionary path of their hosts. The objective of this review is to help researchers who want to use phylogenetic analyses as a tool in virology and molecular epidemiology studies, presenting the most commonly used methodologies, describing the importance of the different techniques, their peculiar vocabulary and some examples of their use in virology. Review: This article starts presenting basic concepts of molecular epidemiology and molecular evolution, emphasizing their relevance in the context of viral infectious diseases. It presents a session on the vocabulary relevant to the subject, bringing readers to a minimum level of knowledge needed throughout this literature review. Within its main subject, the text explains what a molecular phylogenetic analysis is, starting from a multiple alignment of nucleotide or amino acid sequences. The different software used to perform multiple alignments may apply different algorithms. To build a phylogeny based on amino acid or nucleotide sequences it is necessary to produce a data matrix based on a model for nucleotide or amino acid replacement, also called evolutionary model. [...](AU)

Phylogenetic analysis: basic concepts and its use as a tool for virology and molecular epidemiology / Análise filogenética: conceitos básicos e suas utilizações como ferramenta para virologia e epidemiologia molecular

Background: Phylogenetic analyses are an essential part in the exploratory assessment of nucleic acid and amino acid sequences. Particularly in virology, they are able to delineate the evolution and epidemiology of disease etiologic agents and/or the evolutionary path of their hosts. The objective of this review is to help researchers who want to use phylogenetic analyses as a tool in virology and molecular epidemiology studies, presenting the most commonly used methodologies, describing the importance of the different techniques, their peculiar vocabulary and some examples of their use in virology. Review: This article starts presenting basic concepts of molecular epidemiology and molecular evolution, emphasizing their relevance in the context of viral infectious diseases. It presents a session on the vocabulary relevant to the subject, bringing readers to a minimum level of knowledge needed throughout this literature review. Within its main subject, the text explains what a molecular phylogenetic analysis is, starting from a multiple alignment of nucleotide or amino acid sequences. The different software used to perform multiple alignments may apply different algorithms. To build a phylogeny based on amino acid or nucleotide sequences it is necessary to produce a data matrix based on a model for nucleotide or amino acid replacement, also called evolutionary model. [...]

Phylogenetic characterization of the first Ungulate tetraparvovirus 2 detected in pigs in Brazil

Ungulate tetraparvovirus 2 (UTV2) , formerly known as porcine hokovirus due to its discovery in Hong Kong, is closely related to a Primate tetraparvovirus (human PARV-4) and Ungulate tetraparvovirus 1 (bovine hokovirus). Until now, UTV2 was detected in European, Asian and North American countries, but its occurrence in Latin America is still unknown. This study describes the first report of UTV2 in Brazil, as well as its phylogenetic characterization. Tissue samples (lymph node, lung, liver, spleen and kidney) of 240 piglets from eight different herds (30 animals each herd) were processed for DNA extraction. UTV2 DNA was detected by PCR and the entire VP1/VP2 gene was sequenced for phylogenetic analysis. All pigs from this study displayed postweaning multisystemic wasting syndrome (PMWS). UTV2 was detected in 55.3% of the samples distributed in the variety of porcine tissues investigated, as well as detected in almost all herds, with one exception. The phylogenetic analysis demonstrated that Brazilian UTV2 sequences were more closely related to sequences from Europe and United States.(AU)

Molecular characterization of SINEs integrated in endogenous retrovirus sequences from Leopardus geoffroyi and Puma concolor

Background: Short interspersed nuclear elements (SINEs) are transposable elements which are transcribed by RNA polymerase III and widespread in mammalian genomes. Can-SINE is a family of SINE sequences specific to carnivores, predominant in their genomes and present in high copy numbers. The aim of this study was to characterize sequences of Can-SINEs integrated into sequences of endogenous retroviruses (ERVs) from Brazilian wild cats Puma concolor and Leopardus geoffroyi. Additionally, these sequences are considered from some perspectives of their evolution. Material, Methods and Results: By using PCR and sequencing to screen for ERVs within the genomes of L. geoffroyi and P. concolor, two new ERV sequences were amplified with an insertion around 220 nucleotides long, similar to published carnivore SINEs. The sequences were further identified and characterized using a combination of BLAST, BLAT searches and phylogenetic analyses. The results showed that SINE sequences integrated into the ERV from P. concolor (SINE_Pco) and L. geoffroyi (SINE_Lg) are lysine-tRNA derived. These sequences presented a typical RNA polymerase III-specific internal promoter sequence followed by a microsatellite region (TC)n and by an A/T-rich tail with the polyadenylation signal AATAAA. BLAST searches using the whole sequence of L. geoffroyi clone as query (ERV plus SINE) detected two sequences which were highly similar to the cougar (P. concolor) and the domestic cat. However, the SINE from Leopardus geoffroyi is not present in these related sequences. On the other hand, during searches using the whole sequence of the P. concolor clone as query, we found the same SINE insertion in a very similar ERV from domestic cat. All insertions occurred in the RT domain, but SINE_Lg was integrated in a distinct site when compared to SINE_Pco. Another interesting difference between these SINE sequences was that the statistics reported in BLAST searches recovered a much higher number of hits from the domestic cat genome using SINE_Lg as seed than in searches for sequences related to SINE_Pco. The phylogenetic tree based on the SINE fragment grouped these new SINE sequences with Can-SINEs from felids. Within this major clade SINE_Lg and SINE_Pco are related to different lineages of felids Can-SINEs. Discussion: In this study we showed that two different sequences from felid endogenous retrovirus harbor Can-SINE sequences. These insertions are not surprising taking account that ~11% of domestic cat genome is composed of SINE sequences and they are ubiquitous in felid genomes. Furthermore, the insertions of SINEs into the ERV sequences reported here are not unique events. However, they are curious insertions representing genomic fossils and a little piece of felid history. Based on the results of the phylogenetic analyses and position of the integration sites, we suggest that SINE_Lg and SINE_Pco represent independent integration events originated by derived copies from different progenitors. We hypothesized that SINE_Lg is a "young" integration due to the absence of highly similar ERVs from Puma concolor and Felis catus. This lineage may be recently active in felid genomes given that we found very similar MegaBLAST hits at EST database from domestic cat. Instead, SINE_Pco seems to be "old", sharing an identical insertion site to ERVs from domestic cat and its lineage could be inactive in felids considering that any MegaBLAST hits resulted from EST database searches. The latter suggests an integration event in an ancestor species at least 6.7 million years ago, which represents the split between puma and domestic cat lineages.

Análise comparativa de métodos de extração de DNA genômico de células do sangue e do leite de pequenos ruminantes / Comparative analysis of genomic DNA extraction methods from small ruminant blood and milk cells

Genomic DNA (gDNA) extraction allows genetic material accessible to apply molecular techniques for diagnostic and/or genomic characterization. The main goal of this work was to analyze comparatively three manual methods of genomic DNA extraction - DNAzol, FTA and Silica - evaluating quality and yield of the nucleic acids obtained.[...]

Análise comparativa de métodos de extração de DNA genômico de células do sangue e do leite de pequenos ruminantes / Comparative analysis of genomic DNA extraction methods from small ruminant blood and milk cells

Genomic DNA (gDNA) extraction allows genetic material accessible to apply molecular techniques for diagnostic and/or genomic characterization. The main goal of this work was to analyze comparatively three manual methods of genomic DNA extraction - DNAzol, FTA and Silica - evaluating quality and yield of the nucleic acids obtained.[...](AU)

Mixed mycotic rhinitis and pneumonia in wild boars

Background: Wild boar population is present worldwide. Contact between wild boars and domestic pigs may occur occasionally, and several diseases, as well as the occurrence of opportunistic infections are observed in both species. Mycotic rhinitis and pneumonia were reported before in pig herds, mainly associated with immunosuppression caused by viral infection. This study reports the occurrence of mycotic rhinitis in two wild boars due to Aspergillus fumigatus, A. fl avus and Candida albicans, together with Pneumocystis sp. in the lungs, originating from a herd infected with PCV2. Cases: In a commercial wild boar herd, poor body condition, sneezing and diarrhea were observed. Three animals were euthanized and, in two of them, yellow and green plaque-like masses of fungal growth in the mucosal and in cartilage surface and accentuated atrophy of nasal turbinates were observed. Additionally, multifocal subcutaneous abscesses in the maxillary area and bilateral reddening of the ocular mucosa with muco-purulent discharge were noted. Microscopically, in fragments from the nasal cavity of the two affected wild pigs, massive ulceration of the mucosal surface and presence of hyphae with septations and dichotomous branching and pseudohyphae were observed. Multifocal moderated interstitial pneumonia and alveolar edema were the main histological lesions founded in the lungs of 3 animals. In the lymph nodes multifocal moderated lymphoid depletion and lymphohistiocytic infi ltrated was the main microscopical lesion. Aspergillus fumigatus, A. fl avus and Candida albicans were isolated in nasal cavity. Pseudomonas aeruginosa was isolated from the subcutaneous abscesses and Staphylococcus hyicus and Streptococcus equisimilis from ocular swab. Pneumocystis was detected in lungs from the three wild boars by nested PCR, Grocott´s staining and immunohistochemistry (IHC). Porcine circovirus 2 (PCV2) was detected in lungs by PCR. Virus detection by IHC was only confi rmed in one wild boar. Discussion: Diagnostic of mycotic rhinitis and pneumonia was based on macroscopical and microscopical fi ndings, as well as mycological analysis, IHC and Groccott ´s methenamine staining. Pneumocystis carinii, Aspergillus spp. and Candida spp. are considered as opportunistic fungal pathogens commonly associated with immunosuppression in animals and humans and have been found in lungs and in muco-cutaneous tissue of PMWS affected pigs. Clinically, immunodeficiency is usually associated with illness caused by organisms of low pathogenicity or well-know secondary pathogens, among other factors. Besides immunodefi ciency, prolonged antimicrobial therapy is another predisposing factor to the development of mycotic infections, well described in animals. In the present report, antimicrobial therapy was performed when respiratory signs were noted in therapeutic doses, suggesting that massive antibiotic use was not the trigger of mycotic rhinitis. PCV2 IHC result positive only in one wild pig, although all the samples were positive by PCR. This fi nding could indicate a subclinical infection or a recovery phase of the disease in the IHC negative cases, as previously suggested for domestic and wild pigs using in situ hybridization. PCV2 load in wild boar was lower when compared with domestic pigs. A viral load higher than 108 PCV2 genomes per 500 ng DNA was required to give a visible IHC staining in swine. Although quantitative PCR it was not used in order to detect PCV2 in the present report, the viral load could be another possible explanation for the IHC negative cases observed. The role of PCV2 as a cause of immunosupression, facilitating the infection with secondary agents as Aspergillus, Candida and Pneumocystis cannot be ruled out.(AU)

Mixed mycotic rhinitis and pneumonia in wild boars

Background: Wild boar population is present worldwide. Contact between wild boars and domestic pigs may occur occasionally, and several diseases, as well as the occurrence of opportunistic infections are observed in both species. Mycotic rhinitis and pneumonia were reported before in pig herds, mainly associated with immunosuppression caused by viral infection. This study reports the occurrence of mycotic rhinitis in two wild boars due to Aspergillus fumigatus, A. fl avus and Candida albicans, together with Pneumocystis sp. in the lungs, originating from a herd infected with PCV2. Cases: In a commercial wild boar herd, poor body condition, sneezing and diarrhea were observed. Three animals were euthanized and, in two of them, yellow and green plaque-like masses of fungal growth in the mucosal and in cartilage surface and accentuated atrophy of nasal turbinates were observed. Additionally, multifocal subcutaneous abscesses in the maxillary area and bilateral reddening of the ocular mucosa with muco-purulent discharge were noted. Microscopically, in fragments from the nasal cavity of the two affected wild pigs, massive ulceration of the mucosal surface and presence of hyphae with septations and dichotomous branching and pseudohyphae were observed. Multifocal moderated interstitial pneumonia and alveolar edema were the main histological lesions founded in the lungs of 3 animals. In the lymph nodes multifocal moderated lymphoid depletion and lymphohistiocytic infi ltrated was the main microscopical lesion. Aspergillus fumigatus, A. fl avus and Candida albicans were isolated in nasal cavity. Pseudomonas aeruginosa was isolated from the subcutaneous abscesses and Staphylococcus hyicus and Streptococcus equisimilis from ocular swab. Pneumocystis was detected in lungs from the three wild boars by nested PCR, Grocott´s staining and immunohistochemistry (IHC). Porcine circovirus 2 (PCV2) was detected in lungs by PCR. Virus detection by IHC was only confi rmed in one wild boar. Discussion: Diagnostic of mycotic rhinitis and pneumonia was based on macroscopical and microscopical fi ndings, as well as mycological analysis, IHC and Groccott ´s methenamine staining. Pneumocystis carinii, Aspergillus spp. and Candida spp. are considered as opportunistic fungal pathogens commonly associated with immunosuppression in animals and humans and have been found in lungs and in muco-cutaneous tissue of PMWS affected pigs. Clinically, immunodeficiency is usually associated with illness caused by organisms of low pathogenicity or well-know secondary pathogens, among other factors. Besides immunodefi ciency, prolonged antimicrobial therapy is another predisposing factor to the development of mycotic infections, well described in animals. In the present report, antimicrobial therapy was performed when respiratory signs were noted in therapeutic doses, suggesting that massive antibiotic use was not the trigger of mycotic rhinitis. PCV2 IHC result positive only in one wild pig, although all the samples were positive by PCR. This fi nding could indicate a subclinical infection or a recovery phase of the disease in the IHC negative cases, as previously suggested for domestic and wild pigs using in situ hybridization. PCV2 load in wild boar was lower when compared with domestic pigs. A viral load higher than 108 PCV2 genomes per 500 ng DNA was required to give a visible IHC staining in swine. Although quantitative PCR it was not used in order to detect PCV2 in the present report, the viral load could be another possible explanation for the IHC negative cases observed. The role of PCV2 as a cause of immunosupression, facilitating the infection with secondary agents as Aspergillus, Candida and Pneumocystis cannot be ruled out.

Co-infection by porcine circovirus type 2 and porcine parvovirus in aborted fetuses and stillborn piglets in southern Brazil / Co-infecção por circovírus suíno tipo 2 e parvo-vírus suíno em fetos abortados e natimortos suínos no Sul do Brasil

Porcine circovirus types 1 and 2 (PCV1, PCV2) and porcine parvovirus (PPV) are widespread in pig populations around the world. Nevertheless, only PCV2 has been associated with different clinical syndromes, thus representing a major problem to the pig industry. The association of cases of swine abortions and stillborns with PCV1 and PCV2 and PPV was studied retrospectively (2005-2007). Additional pathogens were also investigated in lesioned fetuses. The studied litters included stillborn piglets and several mummified fetuses of varied sizes. Ventricular dilatation, myocardial pale areas, and mesocolic edema were the gross lesions. Escherichia coli was detected as co-infecting with PCV2 the cases in which mesocolic edema was seen. Microscopic lesions included non-suppurative myocarditis, myocardial necrosis and fibrosis, mineralization foci and intranuclear inclusion bodies in cardiomyocytes, and interstitial mononuclear pneumonia. Samples from 7 (5.78 per cent) of 121 aborted fetuses and stillborn piglets had lesions consistent with a viral cause and showed both positive anti-PCV2 immunostaining as well as PCV2-PCR. In samples from 3 (2.47 per cent) of these 7 fetuses, co-infection with PPV was confirmed by Nested-PCR. Both viruses were detected in fetuses at different stages of gestation. Viral antigens of PCV2 were detected by immunohistochemistry mainly in macrophages and myocytes. PCV1 individually was not detected in any of these affected fetuses, but it was associated with PCV2 and/or PPV in some of them. These findings indicate that PCV2 alone or in association with PPV should be kept in mind when investigating causes of infectious abortion in pigs in Brazil.(AU)

Estudou-se retrospectivamente (2005-2007) a associação de casos de abortos e natimortos suínos com infecções por circovírus suíno (PCV) tipos 1 e 2 e parvovírus suíno (PPV). Outros agentes patogênicos foram pesquisados em amostras de fetos com lesões. O estudo incluiu natimortos e fetos mumificados de tamanhos variados. Dilatação ventricular, áreas pálidas miocárdicas e edema de mesocólon foram as lesões macroscópicas observadas. Escherichia coli co-infectou com PCV2 as amostras dos casos com edema de mesocólon. Lesões microscópicas incluíram miocardite não supurativa, necrose e fibrose miocárdicas, focos de mineralização e corpúsculos de inclusão em cardiomiócitos e pneumonia intersticial mononuclear. Entre os 121 fetos suínos abortados ou natimortos analisados, sete (5,78 por cento) tinham lesões compatíveis com origem viral e foram positivos pelas técnicas de imunoistoquímica e PCR para PCV2. Além disso, três (2.47 por cento) desses sete casos também foram confirmados como co-infectados com PPV através da PCR. Antígenos de PCV2 foram observados principalmente em macrógafos e no interior de miócitos dos fetos suínos abortados e natimortos. PCV2 e PPV foram detectados em diferentes estágios de gestação. PCV1 não foi associado isoladamente com feto ou natimorto afetado, mas estava presente em associação com PCV2 e/ou PPV em alguns desses produtos. Esses achados indicam que a infecção por PCV2, isoladamente ou em associação com PPV, deve ser considerada no diagnóstico de aborto infeccioso suíno no Brasil.(AU)

Imunofluorescência utilizando isolados brasileiros no diagnóstico sorológico de infecção por lentivírus em caprinos / Immunofluorescence using Brazilian isolates for serological diagnosis of lentivirus infection in goats

Os lentivírus de pequenos ruminantes (SRLV) têm distribuição mundial e causam infecções persistentes em ovinos e caprinos. O objetivo deste trabalho foi desenvolver um teste de imunofluorescência indireta (IFA), utilizando isolados brasileiros de SRLV, para o diagnóstico sorológico de infecção por estes agentes em caprinos. A técnica de IFA foi comparada, quanto à sensibilidade e à especificidade, ao teste de AGID com antígeno do vírus Maedi-Visna WLC-1. Cultivos celulares secundários de membrana sinovial ovina infectadas com dois isolados de SRLV de origem caprina (CAEV Br/UFRGS-2 e CAEV Br/UFRGS-5) foram utilizados para o teste de IFA. Duzentas e trinta e nove amostras de soro caprino foram submetidas aos dois testes. O teste de AGID detectou 129 (53.9 por cento) amostras de soro caprino com anticorpos para SRLV. O teste de IFA detectou mais amostras reagentes, sendo que resultados diferentes foram observados de acordo com o isolado de SRLV empregado. Quando o isolado CAEV Br/UFRGS-2 foi utilizado como antígeno, 216 (90.3 por cento) amostras de soro caprino foram reagentes, enquanto que o isolado CAEV Br/UFRGS-5 detectou 213 (89.1 por cento) amostras de soro positivas. Não houve diferença estatisticamente significativa entre esses dois isolados. O teste de IFA desenvolvido teve sensibilidade de 94.6 por cento e 96.9 por cento e especificidade 14.5 por cento e 20 por cento, quando os isolados CAEV Br/UFRGS-2 e CAEV Br/UFRGS-5 foram usados como antígeno, respectivamente. O aprimoramento da técnica, assim como sua comparação com um teste mais sensível, ainda se fazem necessários. No entanto, os resultados demonstraram que a técnica de IFA, utilizando isolados brasileiros de SRLV como antígeno, apresenta potencial como um teste alternativo e complementar para o diagnóstico sorológico de infecção por estes agentes (AU)

Detecção do provirús da imunodeficiência felina em gatos domésticos pela técnica de reação em cadeia da polimerase / Detection of feline immunodeficiency provirus in domestic cats by polymerase chain reaction

A infecção de gatos domésticos pelo Vírus da Imunodeficiência Felina (FIV) é um dos modelos mais promissores para o estudo da infecção pelo vírus da imunodeficiência humana (HIV) que causa a Síndrome de Imunodeficiência Adquirida (AIDS). O FIV causa, em gatos, uma enfermidade similar àquela observada em pacientes com AIDS, sobretudo no que diz respeito ao aumento da susceptibilidade a infecções oportunistas. No presente estudo, utilizou-se a Reação em Cadeia da Polimerase (PCR), com o objetivo de detectar o provírus do FIV em gatos com sinais clínicos de imunodeficiência. O fragmento de DNA escolhido como alvo para amplificação situa-se no gene gag do lentivírus felino, o qual é conservado entre as diferentes amostras do vírus. O DNA utilizado foi extraído a partir de amostras de sangue e de tecidos de animais com suspeita clínica de imunodeficiência. Das 40 amostras analisadas, 15 foram positivas, das quais 4 foram submetidas à hibridização, confirmando a especificidade dos fragmentos amplificados. Esses resultados demonstram a presença do FIV na população de gatos domésticos do Rio Grande do Sul, Brasil. (AU)