Brígida Kussumoto de Alcântara

Genetic characterization of a novel bovine papillomavirus member of the Deltapapillomavirus genus

This report describes the complete genomic sequence and taxonomic position of BPV type 13. The BPV13 genome was amplified using the multiply primed rolling-circle amplification technique and long-template PCR employing two specific primers. The two long PCR fragments obtained were cloned and sequenced via primer walking. The complete genomic sequence of the BPV13 contains 7961 bp encoding eight proteins, E1, E2, E4, E5, E6, E7, L1, and L2. Similarly to the E5 gene in BPVs 1 and 2, the putative BPV13 E5 ORF encodes a small transforming protein that contains a hydrophobic transmembrane domain. Meanwhile, the retinoblastoma tumor suppressor-binding domain is absent in the putative BPV13 E7 protein. The presence of these two specific molecular features has been recognized as a distinct marker for the development of fibropapilloma in artiodactyl PV-induced lesions. The phylogenetic analysis demonstrated that BPV13 is a new member of the Deltapapillomavirus genus, to be classified as the third representative of the Delta 4 species. The characterization of the genomic sequence of this novel PV will aid in the interpretation of the pathologies described to be related to this virus and provide support for the development of diagnostic tools for epidemiological surveillance of BPV13 in its potential natural hosts.

Bovine Papillomavirus Type 13 DNA in Equine Sarcoids

ABSTRACT Equine sarcoids are locally aggressive fibroblastic neoplasms considered to be the most common skin tumors of horses worldwide. Bovine papillomavirus types 1 and 2 have typically been associated with sarcoids in equids. Investigations aiming to identify papillomavirus strains, aside from bovine papillomaviruses 1 and 2, which might be associated with sarcoid lesions, have been lacking. The aim of this article is to report the identification of a third bovine papillomavirus type, bovine papillomavirus 13, associated with equine sarcoids. Six sarcoid lesions were collected from diverse anatomical sites on two horses from southern Brazil. To detect a broad spectrum of papillomavirus strains, eight degenerate primer pairs designed to detect conserved regions on the L1 and E1 genes were tested on the DNA samples. Direct sequencing was then performed on the obtained amplicons, and sequence identities were compared with sequences from all bovine papillomavirus types. The FAP59/FAP64, MY09/MY11, and AR-E1F2/AR-E1R4 sequences generated from the sarcoids were shown to present 99 to 100% identity with bovine papillomavirus 13, a new bovine papillomavirus type previously described in cattle. The results from this study suggest that there is a need to identify bovine papillomavirus type 13 and other papillomavirus strains that might be associated with sarcoids in diverse geographical areas; such investigations might establish the frequency of occurrence of this viral type in these common tumors of equids.

Clinical Manifestations of Senecavirus A Infection in Neonatal Pigs, Brazil, 2015

We identified new clinical manifestations associated with Senecavirus A infection in neonatal piglets in Brazil in 2015. Immunohistochemical and molecular findings confirmed the association of Senecavirus A with these unusual clinical signs and more deaths. Other possible disease agents investigated were not associated with these illnesses.

Cryptococcus gattii -Induced Infections in Dogs from Southern Brazil

Cryptococcus gattii-induced cryptococcosis is an emerging infectious disease of humans and animals worldwide, with rare descriptions of this infection in domestic animals from Brazil. This study presents the findings associated with C. gattii in dogs from Londrina, Paraná, Southern Brazil. Two dogs, a 3-year-old, female German shepherd and a 6-year-old, male Boxer, were evaluated by a combination of pathological, mycological, and molecular diagnostic techniques. Significant pathological alterations included cryptococcal lymphadenitis, meningoencephalitis, tonsillitis, and rhinitis with nasal cryptococcomas in the German shepherd dog, while cryptococcal lymphadenitis and pneumonia were observed in the Boxer; both dogs had pseudocystic cryptococcosis. The mucicarmine histochemical stain readily identified the intralesional cryptococcal budding organisms in all affected tissues. Mycological culture and isolation confirmed the yeasts as C. gattii due to positive reaction with the l-canavanine glycine bromothymol blue agar. A PCR assay using the internal transcribed spacers (ITS)1 and ITS2 primers, which target the ITS1 and 2 regions including the 5.8S rRNA gene, amplified the desired amplicons; direct sequencing confirmed the isolate as C. gattii. ITS nucleotide differentiation demonstrated that the isolate forms part of the ITS type 4 Cryptococcus organisms which corresponds to the C. gattii VGII molecular subtype or the RAPD type 2 Cryptococcus organisms. Collectively, these findings confirmed the participation of C. gattii in the etiopathogenesis of the lesions observed in these dogs and expanded the epidemiological niche of this important mycotic agent to include Southern Brazil. It is noteworthy to mention that previous epidemiological studies have suggested that C. gattii-induced cryptococcosis is more frequently diagnosed in Northern relative to Southern Brazil, so these findings might suggest an expansion of the distribution of this agent within continental Brazil.

Teat papillomatosis associated with bovine papillomavirus types 6, 7, 9, and 10 in dairy cattle from Brazil

This study describes the clinical, histopathological, and virological characterization of teat papillomatosis from Brazilian dairy cattle herds. Four types of bovine papillomavirus were identified (BPV6, 7, 9, and 10); one of these (BPV7) is being detected for the first time in Brazilian cattle.

Paracoccidioides brasiliensis-associated dermatitis and lymphadenitis in a dog

Paracoccidioidomycosis (PCM) is an endemic disease of humans from Latin America that is caused by Paracoccidioides brasiliensis and P. lutzii, with most cases of PCM in domestic animals being associated with P. brasiliensis. This study presents the clinical, cytological, mycological, serological, and molecular findings associated with P. brasiliensis in a dog from Southern Brazil. Fine needle biopsies were collected from the skin and several lymph nodes of a 5-year-old female Labrador dog that had enlargement of most superficial lymph nodes. Cytology of the skin and lymph nodes revealed pyogranulomatous dermatitis and lymphadenitis associated with fine-necked, budding fungal structures consistent with the Paracoccidioides genus of organisms; mycological culture derived from the lymph node aspirate demonstrated similar budding structures. Serological assays using exoantigens obtained from the fungal culture demonstrated that the fungal organisms derived from the lymph node were antigenically similar to P. brasiliensis by immunodiffusion and Western blot. A PCR assay, using the fungal culture as input, amplified a partial segment of the internal transcribed spacer 1 and 2 regions of P. brasiliensis; direct sequencing and phylogenetic analyses confirmed the PCR product as P. brasiliensis. The combined cytological, mycological, serological, and molecular findings confirmed a diagnosis of fungal dermatitis and lymphadenitis due to P. brasiliensis in this dog. This case represents the third description of clinical PCM in dogs and the first confirmation of mycotic dermatitis associated with P. brasiliensis in this species. The participation of dogs in the possible dissemination of PCM is reviewed, and it is proposed that dogs are probable accidental hosts in the epidemiological cycle associated with P. brasiliensis.

Caracterização molecular de DNA de Delta papillomavirus bovino (BPV1, 2 e 13) em sarcoides equinos

Sarcoids are fibroblastic lesions, which are considered as the most common skin tumors of horses; spontaneous regression rarely occurs. The bovine papillomavirus (BPV) types 1 and 2 may be involved in the pathogenesis of sarcoids, and probably the recently described BPV type (BPV13) might be associated with the pathogenesis of this lesion. This study characterized the DNA of BPVs in sarcoids from 15 horses from Brazil by analyzing 20 cutaneous lesions (12 recently collected; 8 from formalin-fixed paraffin-embedded (FFPE) tissues). Histopathology confirmed the proliferative lesions as sarcoids. Three PCRs were performed to amplify papillomavirus (PV) DNA. For screening, the primers IFNR2/IDNT2 were used to amplify a fragment of the PV L1 ORF. The second primer set was complementary to a common sequence of the E5L2 genomic region of BPV1, 2, and 13. The third primer pair (FAP59/FAP64) targeted a fragment of the PVs L1 ORF. The screening and E5L2 PCRs yielded amplicons in all samples evaluated. The FAP amplicons identified BPV1, 2, and 13 only from fresh tissue samples. The phylogenetic analyses of E5L2 resulted in the identification of BPV1, 2, and 13 in 14 (70%), 2 (10%), and 4 (20%) sarcoids, respectively. Two horses demonstrated multiple lesions: the sarcoids of one of these contained only BPV1 DNA and those of the other contained three types of bovine Deltapapillomavirus (BPV1, 2, and 13). This study confirmed the presence of BPV1, 2, and 13 DNA in equine sarcoids. Moreover, these findings represent the first description of three types of BPV diagnosed in the same horse, as well as the first confirmation of BPV1 and 2 in horses from Brazil.

Genetic diversity of bovine papillomavirus types, including two putative new types, in teat warts from dairy cattle herds

Teat papillomatosis affects dairy cows worldwide. Milking can become difficult due to teat warts, and maintaining affected cows in the herds may diminish economic profit in the dairy industry. Currently, 13 bovine papillomavirus (BPV) types have been fully characterized, and numerous putative BPV types have been identified through partial L1 gene PCR. In order to identify the viral types present in warts on the udders of dairy cows, 40 teat lesions from 24 cows from 13 cattle farms in three States of Brazil were evaluated by PV L1 gene PCR. The warts that were evaluated contained sequences from BPVs 6-10, the putative BPV types BAPV9 and BAPV4, and two unreported putative papillomavirus (PV) types, named BPV/BR-UEL6 and BPV/BR-UEL7. In addition, mixed infections and coinfections were identified, since more than one lesion was observed on the udders of 13 cows. Phylogenetic analysis showed that BPV/BR-UEL6 is closely related to BPVs belonging to the genus Xipapillomavirus, while BPV/BR-UEL7 clustered with the previously reported strains Cervus timorensis and Pudu puda PVs, which represent a putative new PV type, and it was only distantly related to xi-, epsilon-, delta- and dyoxi-PVs. These results provide information that will assist in the understanding of the association of BPVs 6, 7, 8, 9, and 10, as well as putative BPV types BAPV4 and BAPV9, with mammary papillomatosis. This is the first characterization of putative novel PV types BPV/BR-UEL6 and BPV/BR-UEL7 in teat warts of dairy cows, highlighting the high genetic diversity of BPVs associated with teat papillomatosis.

Identification of canine papillomavirus type 1 (CPV1) DNA in dogs with cutaneous papillomatosis

Canine oral papillomavirus (COPV), also known as Canine Papillomavirus type 1 (CPV1), induces papillomas at the mucous membranes of the oral cavity and at the haired skin of dogs. The classification of Papillomavirus (PV) types is based on the L1 capsid protein and nucleotide sequence; so far, 14 CPV types have been described in several countries, but the molecular characterization of CPV in Brazil is lacking. This study investigated the presence of the PV in seven papillomas from four mixed breed dogs from Londrina/PR, Southern Brazil, by partial sequencing of the L1 gene. Seven exophytic cutaneous lesions were surgically removed and processed for histopathological and molecular characterization. Histopathology confirmed the lesions as viral papillomas due to typical histological features. Polymerase Chain Reaction (PCR) assay using the FAP59 and FAP64 primers targeted the L1 gene followed by sequence analysis of the amplicons identified CPV1 in all evaluated papilloma samples. This study represents the first description of CPV1 DNA associated with canine papillomatosis in Brazil.