Feline herpesvirus 1 viral load related to environmental factors in sheltered cats / Relação entre carga viral de herpesvirus felino 1 e fatores causadores de estresse em felinos de abrigo

ABSTRACT: Shelter environment stress factors are related to FHV-1 viral reactivation. However, comparisons between conjunctival viral load and environmental factors have not been commonly evaluated. The aim of this study was to correlate FHV-1 viral load in domestic cats with and without clinical signs of conjunctivitis to shelter design in order to use FHV-1 viral load as a parameter of "health management". Cats from four different shelters underwent an ophthalmological examination. Samples were collected by rolling a DNA/RNAse-free cytobrush over the ventral conjunctival fornix and were stored in 1.5 mL sterile microtubes in 500 μL of Eagle's minimum essential medium and kept at 4 ºC. Molecular procedures were performed up to 48 hours after collection. Different routines regarding new arrivals were directly related to FHV-1 viral load. Shelters where new arrivals occurred on daily basis had the highest viral load (2.69x108 copies/µL), while those shelters where new arrivals had not occurred in the few months prior to the beginning of the study had the lowest rate (1.63x103 copies/µL). Environmental factors directly influenced FHV-1 DNA viral load. This study highlighted the need to improve the management approach in the animal shelter environment to reduce stressful situations responsible for FHV-1 reactivation and higher viral load quantification.

RESUMO: No ambiente do abrigo encontram-se fatores que geram estresse nos animais que ali residem. Esses fatores acabam por provocar a reativação do FHV-1. No entanto, comparações entre carga viral conjuntival e fatores ambientais não foram ainda avaliadas. Objetivo deste estudo foi correlacionar a carga viral de FHV-1 em felinos domésticos com e sem sinais clínicos de conjuntivite com as características dos abrigos. Assim, pode-se usar carga viral de FHV-1 como parâmetro de sanidade. Todos os gatos foram submetidos a exame clínico oftalmológico. Amostras foram coletadas com uso de escova citológica, acondicionadas em microtubos estéreis de 1,5mL contendo 500 μL de meio Eagle essencial mínimo e mantidas em 4 ºC. Análises moleculares foram realizadas no prazo de 48 horas após coleta. A rotina de entrada de novos animais estava diretamente relacionada a carga viral de FHV-1. Abrigos com entrada diária apresentaram carga viral maior (2.69x108 cópias/µL), do que abrigo onde novos animais não chegaram nos meses que antecederam a coleta (1.63x103 cópias/µL). Fatores ambientais influenciam diretamente carga viral de FHV-1. Esse estudo evidencia a necessidade de aprimorar o sistema de manejo dos abrigos de forma a reduzir situações de estresse responsáveis pela reativação de FHV-1 e consequente aumento na carga viral.

Molecular characterization of feline calicivirus variants from multicat household and public animal shelter in Rio de Janeiro, Brazil

ABSTRACT The aim of this study was to perform the molecular characterization of conserved and variable regions of feline calicivirus capsid genome in order to investigate the molecular diversity of variants in Brazilian cat population. Twenty-six conjunctival samples from cats living in five public short-term animal shelters and three multicat life-long households were analyzed. Fifteen cats had conjunctivitis, three had oral ulceration, eight had respiratory signs (cough, sneeze and nasal discharge) and nine were asymptomatic. Feline calicivirus were isolated in CRFK cells and characterized by reverse transcription PCR target to both conserved and variable regions of open reading frame 2. The amplicons obtained were sequenced. A phylogenetic analysis along with most of the prototypes available in GenBank database and an amino acid analysis were performed. Phylogenetic analysis based on both conserved and variable region revealed two clusters with an aLTR value of 1.00 and 0.98 respectively and the variants from this study belong to feline calicivirus genogroup I. No association between geographical distribution and/or clinical signs and clustering in phylogenetic tree was observed. The variants circulating in public short-term animal shelter demonstrated a high variability because of the relatively rapid turnover of carrier cats constantly introduced of multiple viruses into this location over time.

Correlation between clinical signs of feline conjunctivitis and molecular detection of felid herpesvirus-1, feline calicivirus, chlamydophila felis and mycoplasma felis in cats from shelters in Rio de Janeiro / Correlação entre sinais clínicos da conjuntivite felina e a detecção molecular de felid herpesvirus-1, feline calicivirus, chlamydophila felis e mycoplasma felis em gatos de abrigos no Rio de Janeiro

Objectives: To perform molecular diagnosis of microbial agents (FHV-1, FCV, Mycoplasma felis, and Chlamydophila felis) in kittens with conjunctivitis and correlate the clinical signs with clinical severity. Material and Methods: A total of 108 conjunctival swab were collected from kittens without (G1; n = 40) and with (G2; n = 68) clinical signs of conjunctivitis. Animals from G2 group were scored from 1 (mild) to 4 (severe) according to the severity of conjunctivitis. All samples were submitted to PCR and RT-PCR. Results: FHV-1 was detected in 62/108 (57.4%) of samples, FCV in 40/108 (37.0%), M. felis in 11/108 (10.2%) and C. felis in 26/108 (24.1%). Mixed infections were detected in 39/108 (36.1%). In G1, 28/40 (70.0%) were positive for one or more agents, in G2, 58/68 (85.3%) were positive (P = 0.03). In 1, single infections by FHV-1were found in 21/40 (52.5%) samples, FCV in 2/40 (5.0%), C. felis in 1/40 (2.5%), and no pathogens were detected in 12/40 (30%) of samples, while mixed infections accounted for 29/40 (72.5%) of the cases. In G2, single FHV-1 infections were found in 31/68 (45.6%) samples, FCV in 10/68 (14.7 %), M. felis in 2/68 (3.0%) and C. felis also in 2/68 (3.0%), and no pathogens were detected in 10/68 (14.7%) samples, while mixed infections accounted for 36/68 (52.0%) of the cases. They were categorized as grade 1, 20/68 (29.4%), grade 2, 14/68 (20.6%), grade 3, 21/68 (30.9%) and grade 4, 13/68 (19.1%). The presence of FHV-1 and FCV is equally distributed among the four categories. More severe clinical signs, scores 3 and 4, are related to coinfections by C. felis and M. felis. Conclusions: FHV-1, FCV, C. felis and M. felis were identified in feline conjunctivitis. Co-infections are related to more severe cases of conjunctivitis.Molecular diagnosis is helpful to detect asymptomatic carriers and is a rapid and accurate method to determine the pathogen of feline conjunctivitis.(AU)

O objetivo deste estudo foi realizar diagnóstico molecular de agentes microbiológicos (FHV-1, FCV, Mycoplasma felis e Chlamydophila felis) em gatos filhotes e associar a presença dos patógenos à gravidade dos sinais clínicos de conjuntivite. Foram coletadas um total de 108 amostras de suabe conjuntival de filhotes felinos assintomáticos (G1; n = 40) e sintomáticos (G2; n = 68). Animais do G2 foram categorizados de 1 (leve) até 4 (grave), de acordo com o quadro clínico de conjuntivite. As 108 amostras foram submetidas à PCR e RT-PCR. O FHV-1 foi detectado em 57,4% das amostras, o FCV em 37%, o M. felis em 10,2% e o C. felis em 24,1%. Coinfecções, por sua vez, foram detectadas em 36,1%. No G1, 70% das amostras foram positivas para um ou mais patógenos. No G2, 85,3% apresentavam infecções (P = 0,03). No G1, monoinfecções por FHV-1 foram diagnosticadas em 52,5% das amostras, por FCV em 5%, por C. felis em 2,5%, e em 30% das amostras analisadas nenhum dos patógenos estudados foi encontrado. Coinfecções, por sua vez, estavam presentes em 72,5% das amostras. No G2, monoinfecções por FHV-1 foram encontradas em 45,6% das amostras, por FCV em 14,7 %, por M. felis em 3% e por C. felis também em 3%. Nenhum dos patógenos estudados foi encontrado em 14,7% das amostras analisadas. Coinfecções, responsáveis por 52% dos casos, foram categorizados como Grau 1 (29,4%), Grau 2 (20,6%), Grau 3 (30,9%) e Grau 4 (19,1%). A presença de FHV-1 e FCV está igualmente distribuída entre as quatro categorias. Os sinais clínicos mais graves (graus 3 e 4) estão relacionados a coinfecções por C. felis e M. felis. Os agentes microbiológicos FHV-1, FCV, C. felis e M. felis foram encontrados em animais com conjuntivite. Coinfecções estão relacionadas aos casos mais graves. Por fim, concluiu-se que o diagnóstico molecular, além de detectar portadores assintomáticos, é um método rápido e acurado para o diagnóstico do patógeno causador da conjuntivite felina.(AU)