Aline S. Hora

Phylogenetic Analyses of the VP4 and VP7 Genes of Porcine Group A Rotaviruses in São Paulo State, Brazil: First Identification of G5P[23] in Piglets

ABSTRACT This study determined the group A rotavirus occurrence in pig farms from 7 different cities in São Paulo State, Brazil. Out of 143 samples, 70 tested positive. Sequence analyses of 37 strains indicated that the strains had the G3, G5, G9, and P[6], P[13]/P[22]-like, and P[23] genotypes.

Felid Herpesvirus 1 as a Causative Agent of Severe Nonsuppurative Meningoencephalitis in a Domestic Cat

ABSTRACT Felid herpesvirus 1 is an important respiratory pathogen of domestic cats. This report presents the first case of severe nonsuppurative meningoencephalitis caused by this virus in a cat.

Glial response in the central nervous system of cats with feline infectious peritonitis.

Objectives The aim of the study was to evaluate central nervous system (CNS) lesions in non-effusive and effusive cases of feline infectious peritonitis (FIP) regarding aspects related to astrocytic and microglial reactions. Methods Five necropsied cats that were naturally infected with FIP virus, confirmed by reverse transcriptase polymerase chain reaction and immunohistochemistry, with different intensities of CNS lesions, were studied. Brain and cerebellum were evaluated by light microscopy and immunohistochemistry for glial fibrillary acidic protein (GFAP) and vimentin to assess astrocytic morphology, and lectin histochemistry for Ricinus communis agglutinin-I (RCA-I) to detect microglia was performed to evaluate the glial response in the CNS of cats with FIP. Results An important astrocytic response in many areas of the CNS of all cats, including the periventricular areas of lateral ventricles and fourth ventricle, the molecular layer of the cerebellum and cerebral cortex, was visualized. This astrocytic reactivity was associated with areas of granulomatous or pyogranulomatous vasculitis/perivasculitis in most cases, and it was characterized by multifocal to coalescing astrocytosis and astrogliosis with an increase in the expression of intermediate filaments, such as GFAP. However, astrocytes exhibited strong vimentin expression in neuroparenchyma with severe inflammatory and necrotic changes, but GFAP expression was mild or absent in these cases. A microglial response was present only in severe lesions, and RCA-I expression was detected primarily in gitter cells and resting microglia. Conclusions and relevance The present study indicates a strong astrocytic response, including the presence of many less differentiated vimentin-positive astrocytes and gitter cells positive for RCA-1 in severe lesions in the CNS of cats with FIP.

Simultaneous Detection of Group A Rotavirus in Swine and Rat on a Pig Farm in Brazil

This study investigated the occurrence of rotavirus in porcine and Rattus norvegicus, at the same time, on a pig farm in the city of Jaguariúna, São Paulo, Brazil. Swine (n = 21) and rat (n = 6) fecal samples were analyzed by nested RT-PCR assay. Rotavirus occurred in seven porcine and two rat samples. A total of three pig and one rat samples were further submitted to genetic sequencing. The partial NSP5 gene phylogeny showed that all strains were segregated in the genotype H1. These results point toward a cross-species transmission between rats and pigs on the surveyed farm and represent the first detection of rotavirus in Rattus norvegicus in Brazil.

Intrahost Diversity of Feline Coronavirus: A Consensus between the Circulating Virulent/Avirulent Strains and the Internal Mutation Hypotheses?

To evaluate the most controversial issue concerning current feline coronavirus (FCoV) virology, the coexisting hypotheses of the intrahost and interhost origins of feline infectious peritonitis virus (FIPV) in regard to the pathogenesis of feline infectious peritonitis (FIP), this study aimed to assess the molecular diversity of the membrane gene FCoVs in 190 samples from 10 cats with signs of FIP and in 5 faecal samples from cats without signs of FIP. All samples from the non-FIP cats and 25.26% of the samples from the FIP cats were positive for the FCoV membrane (M) gene. Mutations in this gene consisted of SNP changes randomly scattered among the sequences; few mutations resulted in amino acid changes. No geographic pattern was observed. Of the cats without FIP that harboured FECoV, the amino acid sequence identities for the M gene were 100% among cats (Cats 1–3) from the same cattery, and the overall sequence identity for the M gene was ≥91%. In one cat, two different lineages of FCoV, one enteric and one systemic, were found that segregated apart in the M gene tree. In conclusion, the in vivo mutation transition hypothesis and the circulating high virulent-low virulent FCoV hypothesis have been found to be plausible according to the results obtained from sequencing the M gene.

Alphacoronavirus in urban Molossidae and Phyllostomidae bats, Brazil.

BackgroundBats have been implicated as the main reservoir of coronavirus (CoV). Thus the role of these hosts on the evolution and spread of CoVs currently deserve the attention of emerging diseases surveillance programs. On the view of the interest on and importance of CoVs in bats the occurrence and molecular characterization of CoV were conducted in bats from Brazil.FindingsThree hundred five enteric contents of 29 bat species were tested using a panCoV nested RT-PCR. Nine specimens were positive and eight was suitable for RdRp gene sequencing. RdRp gene phylogeny showed that all CoVs strains from this study cluster in Alphacoronavirus genus, with one Molossidae and one Phlyllostomidae-CoV specific groups. Phylogenetic analyses of two S gene sequences showed a large diversity within the Alphacoronavirus genus.ConclusionsThis study indicated a CoV-to-host specificity and draws attention for CoV detection in Cynomops sp, a potential new reservoir. The phylogenetic analyses indicate that diversity of CoV in bats is higher than previously known.

Experimental Neospora caninum infection in chickens (Gallus gallus domesticus) with oocysts and tachyzoites of two recent isolates reveals resistance to infection

The importance of birds in the biological cycle of Neospora caninum is not clear. We report unsuccessful Neospora infection in chickens (Gallus gallus domesticus) using two isolates of N. caninum. In experiment #1, 30 White Leghorn chickens were orally inoculated with viable N. caninum oocysts (NC-SP1 isolate, 200 oocysts per bird) via the crop at 21days of age. Groups of three birds were euthanised at intervals of 7days (a total of 9weeks) and one group was challenged with the same oocyst dose at 37daysp.i. and observed for 11weeks. Blood samples were collected weekly, and sera were tested using IFAT. Chicken tissues were collected for PCR, quantitative PCR and immunohistochemistry. Two dogs approximately 45days of age were fed with tissues from chickens euthanised at 138 and 159daysp.i. The results indicated that the chickens were resistant to neosporosis as revealed by failure to seroconvert, to detect parasite DNA or N. caninum antigen by immunohistochemistry in inoculated bird tissues, and by no oocyst excretion by the dogs fed avian tissues. Similar results were obtained in experiment #2, in which 34 1-week-old chickens were each s.c. inoculated with 100,000 tachyzoites of the NcWTDMn1 isolate of N. caninum. The chickens were euthanised on days 7, 15, 22, 28, 36 and 60p.i. At necropsy, all tissues and serum from each bird were collected. All chickens remained asymptomatic, and N. caninum antigen was not detected by immunohistochemistry. Seven chickens euthanised at day 60p.i. demonstrated low (1:25 dilution) levels of antibodies by using the Neospora agglutination test. Two 12-week-old dogs fed tissues pooled from 10 inoculated chickens euthanised at day 60p.i. did not excrete N. caninum oocysts. This investigation indicates that chickens are resistant to experimental infection by N. caninum.

Development and validation of a modified TaqMan based real-time PCR assay targeting the lipl32 gene for detection of pathogenic Leptospira in canine urine samples

A modified TaqMan real-time polymerase chain reaction targeting a 138 bp fragment within the lipl32 gene was developed to identify exclusively pathogenic Leptospira spp. in dog urine samples. Thirty-five samples from dogs with suspected clinical leptospirosis and 116 samples from apparently healthy dogs were tested for presence of leptospiral DNA using the TaqMan-based assay. The results were compared with those from a well-established conventional PCR targeting the 16S RNA encoding gene associated with nucleotide sequencing analysis. The overall agreement between the assays was 94.8% (confidence interval [CI] 95% 88–100%). The newly developed assay presented 91.6% (CI 95% 71.5–98.5%) relative sensitivity (22[+] lipl32 PCR/24[+] 16S RNA and sequencing), 100% (CI 95% 96.3–100%) relative specificity and 98.7% accuracy (CI 95% 94.8–100%). The lipl32 assay was able to detect and quantify at least 10 genome equivalents/reaction. DNA extracted from 17 pathogenic Leptospira spp., 8 intermediate/saprophytic strains and 21 different pathogenic microorganisms were also tested using the lipl32 assay, resulting in amplification exclusively for pathogenic leptospiral strains. The results also demonstrated high intra and inter-assay reproducibility (coefficient of variation 1.50 and 1.12, respectively), thereby qualifying the newly developed assay as a highly sensitive, specific and reliable diagnostic tool for leptospiral infection in dogs using urine specimens.

Report of the first clinical case of intestinal trichomoniasis caused by Tritrichomonas foetus in a cat with chronic diarrhoea in Brazil

BackgroundTritrichomonas foetus is an emergent and important enteric pathogen of cats, which causes prolonged diarrhoea in cats.Case presentationThis study describes a T. foetus infection in a seven-month-old, entire male domestic shorthair kitten with a six-month history of persistent large intestinal diarrhoea, faecal incontinence, prostration, apathy and weight loss. Parasites were microscopically observed and confirmed by PCR and DNA sequencing. Molecular analyses were carried out comparing the sequence obtained in this study with T. foetus and T. suis. Retrieved from GenBank. After treatment with ronidazole, the cat showed resolution of clinical signs.ConclusionsThis is the first clinical case of T. foetus infection in a chronic diarrheic cat in Brazil and South America, confirming the presence of this pathogen in this part of the world and highlighting the importance of this protozoa being considered in the differential diagnosis of cats presenting diarrhoea of the large intestine. Our case report enriches our knowledge on the geographical distribution of T. foetus in cats in Brazil and provides further understanding of the clinical significance of feline intestinal trichomoniasis in this country.

Prospective study of canine leptospirosis in shelter and stray dog populations: Identification of chronic carriers and different Leptospira species infecting dogs

Dogs are highly susceptible to the leptospiral infection, notably stray and sheltered dogs. Unsanitary conditions often observed in dog shelters may predispose the introduction and spread of leptospires among sheltered populations, potentially increasing the chances for the inadvertent adoption of asymptomatically infected animals. The present work describes a longitudinal study using a multidisciplinary approach for the identification of chronically infected dogs and the characterization of potentially pathogenic strains circulating among stray and sheltered dog populations in São Paulo, Brazil. A total of 123 dogs from three populations were included. The initial evaluation consisted of blood and urine quantitative PCR testing (qPCR), the detection of specific antibodies by microscopic agglutination test (MAT), physical examination and hematological and serum biochemistry analyses. The qPCR-positive dogs were prospectively examined, and reevaluations also included culture from urine samples. Positive qPCR samples were subjected to 16S rRNA and secY gene phylogenetic analysis. The recovered strains were characterized by Multilocus Sequence Typing, polyclonal serogroup identification and virulence determination. Leptospiruria was detected in all populations studied (13/123), and phylogenetic analysis revealed that 10 dogs had L. interrogans infection. Three dogs (3/13) had L. santarosai infection. The secY phylogenetic analysis revealed that the L. santarosai sequences clustered separately from those obtained from other hosts. Ten leptospiruric dogs were reevaluated, and three dogs presented persistent leptospiruria, allowing culturing from two dogs. The strains were characterized as L. interrogans serogroup Canicola (virulent) and L. santarosai serogroup Sejroe (not virulent). Serum samples were retested by MAT using the DU92 and DU114 strains as antigens, and no increased seroreactivity was detected. Asymptomatic L. santarosai infection was observed in all populations studied, suggesting a possible role of dogs in the chain of transmission of this leptospiral species. The results suggest a genetic distinction between lineages of Brazilian L. santarosai maintained by dogs and other animal hosts. Our findings revealed that dogs could act as maintenance hosts for distinct pathogenic Leptospira, highlighting also that asymptomatically infected dogs can be inadvertently admitted and adopted in dog shelters, potentially increasing the risks of zoonotic transmission.