Tatiana Xavier de Castro

Clinical and epidemiological aspects of canine parvovirus (CPV) enteritis in the State of Rio de Janeiro: 1995 - 2004

This paper relates the clinical and epidemiological aspects of canine parvovirus infection (CPV) in the State of Rio de Janeiro from April 1995 to March 2004. A total of 341 fecal samples were collected from up to 6-months-old puppies with gastroenteritis. The diagnosis of CPV infection was confirmed by hemagglutination/ hemagglutination inhibition tests, enzyme immunoassay, virus isolation in cell culture or polymerase chain reaction. One hundred and fifty-seven samples (46%) were positive for CPV. No correlation among sex, breed or age and the occurrence of CPV infection was observed. The classical signs of parvoviral enteritis (anorexia, lethargy, vomiting and hemorrhagic fluid diarrhea) were observed in 70% of CPV-positive and in 60% of CPV-negative puppies. Although CPV could be detected throughout the studied period, its occurrence was significantly higher from June to September and November to December. These results show that CPV is still circulating in the State of Rio de Janeiro.

Monitoring of canine parvovirus (CPV) strains detected in vaccinated puppies in Brazil.

The objective of this study was to investigate, by partial sequencing of VP2 protein, the variability of CPV detected in 37 fecal samples collected from vaccinated puppies with enteritis. Laboratorial diagnosis of CPV was confirmed by HA/HI and PCR and, for sequencing analyses, two different regions of the VP2 gene were amplified by PCR. From 1995 to 2004, all strains were characterized as CPV-2a. After that, both CPV-2a and CPV-2b were detected. All CPV-2a showed a non-synonymous mutation in the residue 297 (Ser→Ala). A synonymous substitution at the AA 574 was also observed in 15/37 samples. Our findings indicate that the cases of vaccine failure are most likely not associated to the mutations detected in the sequenced regions. However, the monitoring of genotyping mutations that led to new CPV strains is essential to determinate if current vaccines will keep providing protection against all new future variants.

Molecular characterization of canine coronavirus strains circulating in Brazil

Abstract To characterize canine coronavirus (CCoV) circulating in diarrheic puppies in Brazil, 250 fecal samples collected between 2006 and 2012 were tested. By using RT-PCR to partially amplify the M gene, CCoV RNA was detected in 30 samples. Sequence analysis of the M protein grouped eight strains with CCoV-I and another 19 with CCoV-II prototypes. To genotype/subtype the CCoV strains and assess the occurrence of single or multiple CCoV infections, RT-PCR of the S gene was performed, and 25/30 CCoV-positive strains amplified with one or two primer pairs. For 17/25 samples, single infections were detected as follows: six CCoV-I, nine CCoV-IIa and two CCoV-IIb. Eight samples were positive for more than one genotype/subtype as follows: seven CCoV-I/IIa and one CCoV-I/IIb. Sequence analysis revealed that the CCoV-I and IIa strains shared high genetic similarity to each other and to the prototypes. The Brazilian strains of CCoV-IIb displayed an aminoacid insertion that was also described in CCoV-IIb-UCD-1 and TGEV strains. Among the 25 CCoV-positive puppies, five had a fatal outcome, all but one of which were cases of mixed infection. The current study is the first reported molecular characterization of CCoV-I, IIa and IIb strains in Brazil. To characterize canine coronavirus (CCoV) circulating in diarrheic puppies in Brazil, 250 fecal samples collected between 2006 and 2012 were tested. By using RT-PCR to partially amplify the M gene, CCoV RNA was detected in 30 samples. Sequence analysis of the M protein grouped eight strains with CCoV-I and another 19 with CCoV-II prototypes. To genotype/subtype the CCoV strains and assess the occurrence of single or multiple CCoV infections, RT-PCR of the S gene was performed, and 25/30 CCoV-positive strains amplified with one or two primer pairs. For 17/25 samples, single infections were detected as follows: six CCoV-I, nine CCoV-IIa and two CCoV-IIb. Eight samples were positive for more than one genotype/subtype as follows: seven CCoV-I/IIa and one CCoV-I/IIb. Sequence analysis revealed that the CCoV-I and IIa strains shared high genetic similarity to each other and to the prototypes. The Brazilian strains of CCoV-IIb displayed an aminoacid insertion that was also described in CCoV-IIb-UCD-1 and TGEV strains. Among the 25 CCoV-positive puppies, five had a fatal outcome, all but one of which were cases of mixed infection. The current study is the first reported molecular characterization of CCoV-I, IIa and IIb strains in Brazil.

Molecular characterisation of calicivirus and astrovirus in puppies with enteritis.

Canine parvovirus (CPV) and canine coronavirus (CCoV) are considered to be the main pathogens responsible for acute gastroenteritis in young dogs Greene and Decaro 2012). Recently, canine caliciviruses (CaCV), genus Norovirus and canine astroviruses (CaAstV) have been associated with outbreaks of enteritis in puppies that were either singly infected or coinfected with CCoV and/or CPV Martella and others 2008,Mesquita and others 2010,Ntafis and others 2010,Zhu and others 2011,Grellet and others 2012,Martella and others 2012). Previous reports have shown that CPV and/or CCoV are responsible for approximately 48 per cent of the enteritis cases in puppies in the state of Rio de Janeiro, Brazil Castro and others 2012). However, cases involving CaCV or CaAstV infections have not been described yet in South America. The purpose of this study was to perform the molecular characterisation of CaCV in faecal samples from puppies showing clinical signs of gastroenteritis using RT-PCR and sequence analysis. In addition, the unexpected detection of CaAstV in puppies with enteritis was reported. Faecal samples collected between February 2005 and May 2012 from 232 client-owned puppies with diarrhoea, that were less than one year of age, from private animal hospitals in the city of Rio de Janeiro, Brazil, were analysed. This trial was licensed by the Ethics Committee of Animal Research-PROPP/ UFF-CEPA/ NAL, No. 0223/12. For genome detection, nucleic acids were extracted from 10 per cent faecal suspensions in 0.01 M Tris/HCl/Ca2+ using …

Characterization of parvoviruses from domestic cats in Brazil

To characterize Feline parvovirus (FPV) circulating in domestic cats in Brazil, 51 fecal samples from unvaccinated domestic cats were collected during 2004–2005. Six parvoviruses were characterized by hemagglutination (HA) assay at different pH values and temperatures and by polymerase chain reaction (PCR) using different pairs of primers. However, data obtained from HA and PCR did not allow the discrimination between FPV and Canine parvovirus (CPV). Two regions of the VP2 capsid gene (1,171-bp fragment) involved in controlling canine and feline host range were sequenced; 9 synonymous and 10 non-synonymous nucleotide substitutions were detected. All samples were confirmed as FPV by nucleotide sequencing, but 3 feline samples had amino acid changes at residues 93, 375, and 426, which are present in canine strains. The phylogenetic tree built based on nucleotide sequences showed that Brazilian feline samples form a cluster distinct from other parvoviruses deposited in GenBank. Taken together, the findings reinforce the importance of monitoring the continuous evolution of CPV and FPV in the feline population in Brazil.

Molecular diversity of human parvovirus B19 during two outbreaks of erythema infectiosum in Brazil

This study was conducted to provide information on the genetic diversity of human parvovirus B19 (B19V) circulating in the municipality of Niterói, Rio de Janeiro, Southeast Brazil during 1996-2006, a period with two distinct outbreaks of B19V infection: 1999-2000 and 2004-2005. A total of 27 sera from patients with erythema infectiosum and five sera from HIV-infected patients that tested positive for B19V DNA during the study period were analyzed. To genotype B19V strains, a semi-nested PCR for partial amplification of the capsid gene was performed and sequence analysis revealed that 31 sequences belonged to subgenotype 1a (G1a) of the main genotype 1 and one sequence was characterized as subgenotype 3b (G3b). The phylogenetic tree supported the division of the G1a into two well-defined clades with 1.3% of divergence. The low diversity of the G1a strains may be explained by the fact that all patients had acute B19V infection and 30/32 sera were collected during two distinct outbreaks. The G3b strain was from an HIV-infected patient who seroconverted to anti-B19 IgG antibodies in September/2005. This is the first report of G3b in the state of Rio de Janeiro.

Detection and molecular characterization of caliciviruses (vesivirus and norovirus) in an outbreak of acute diarrhea in kittens from Brazil.

Abstract Feline caliciviruses (FCVs) have occasionally been described in cats in association with enteric disease, but an etiological role for these viruses in acute gastroenteritis is still unclear. In this study, molecular characterization of FCV and feline norovirus (FNoV) was undertaken using real-time PCR (RT-PCR) and sequence analysis of the ORF1 region in fecal specimens from 29 diarrheic cats. The specimens were also screened for parvovirus, coronavirus, astrovirus and group A rotavirus. A quantitative one step RT-PCR was also performed to detect and quantitate NoV genogroup IV and the role of these animal caliciviruses in feline gastroenteritis was investigated. This is the first description of enteric FCV and FNoV in South America.

Genetic diversity of Ehrlichia canis strains from naturally infected dogs in Rio de Janeiro, Brazil.

The aim of this study was to characterize Ehrlichia canis strains from naturally infected dogs in Rio de Janeiro, Brazil. In addition, all the clinical and hematological findings observed in these dogs were reported. PCR targeting the 16S rRNA gene was used for diagnostic purposes, and the TRP19 and TRP36 genes were sequenced to evaluate the genetic diversity. Fifteen samples were positive for E. canis. The polymerase chain reaction for the TRP19 gene resulted in 11 amplicons (11/15), which were cloned into the pGEM-T easy vector for sequencing. The complete sequence of TRP19 gene was compared to those in the GenBank, revealing high identicalness. Phylogenetic analysis on the TRP36 gene sequences demonstrated two distinct strains from two dogs, named 56C and 70C. The 56C strain was grouped with the strain Cuiaba 16, which is a hybrid strain formed by Brazilian and US genogroups; and the 70C strain was grouped with other strains of the US genogroup, thus suggesting that there are at least two genogroups of E. canis in Rio de Janeiro (US and Brazilian). Those animals, in which the 70C and 56C strains were isolated, showed distinct clinical and hematological manifestations of the disease. The appearance of different genotypes may express new phenotypes, thus resulting in different forms of presentation of the disease and making its diagnosis more complex.

Comparison of three laboratorial tests for diagnosis of canine parvovirus infection

The aim of this study was to evaluate the rapid tests currently used for canine parvovirus (CPV) diagnosis: hemagglutination test (HA), enzyme immunoassay (EIA) and polymerase chain reaction (PCR). A total of 112 fecal samples collected from diarrheic puppies up to one year of age were tested. The EIA was able to detect CPV antigen in 44 samples. By HA, 32 samples tested highly positive with titers >128, eight tested weakly positive (titers 32 and 64) and 72 were negative (titers <16). Using PCR, 57 samples were found positive including 13 EIA-negative and 19 HA-negative samples. The best correlation was observed between EIA and PCR (88.4%). These tests were able to detect all types of CPV, including CPV-2c. Considering that 23%-33% of dogs presenting enteritis did not show infection by EIA nor HA, negative results from the antigen detection tests should be confirmed through molecular methods.

Clinical aspects and weight gain reduction in swine infected with porcine circovirus type 2 and torque teno sus virus in Brazil

Simultaneous Porcine circovirus type 2 (PCV-2) and Torque teno sus virus (TTSuV) infections have been reported around the world, generally linked to severe infections. In the present study, 257 swine plasma samples from 31 swine herds located in Brazil, were PCR screened for PCV-2 and TTSuV-1/2 and correlated with clinical data. PCV-2 was detected in 25%, followed by 38.1% and 42.4% of TTSuV-1 and TTSuV-2, respectively. Co-infections of two or three viruses were found in 32.3% of samples. PCV-2 was more frequently detected in the growing (p=0.030) and finishing phases (p=0.0005) while TTSuV-2 in the nursery (p=0.009). Only TTSuV-1 was statistically associated to clinical disease (multiple signs), in combination or not with PCV-2 or TTSuV-2 (p=0.015). PCV-2/TTSuV co-infections were more frequently related to weight gain reduction in comparison to mono-infections (p=0.049) and no-infections (p=0.027), and also in animals with (p=0.011) or without (p=0.037) clinical signs, being the nursery the most affected phase (p=0.025). Our results uphold the pathogenic potential of TTSuV in naturally infected pigs and the clinical/economical impact of this agent, especially in co-infections. Studies addressing the physiopathological mechanisms of simultaneous infections are needed.