Comparison among three different serological methods for the detection of equine influenza virus infection.

The equine influenza virus (EIV) H3N8 subtype is responsible for all EIV outbreaks worldwide while the H7N7 subtype is less pathogenic and is considered extinct as it has not been confirmed in outbreaks since 1980. Although EIV is enzootic in Brazil, few reports describe the actual EIV antibody status in the country. The aims of this study were: - to evaluate the efficiency of different serum treatments described by the World Organisation for Animal Health (OIE) and the World Health Organization (WHO) to remove non-specific haemagglutination inhibitors for the haemagglutination inhibition (HI) assay for EIV - to evaluate the presence of EIV antibodies by HI, enzyme-linked immunosorbent assay and agar gel immunodiffusion in 83 non-vaccinated equines from São Paulo State - to evaluate a strategy to better analyse equine sera for EIV antibodies. Although there was no statistical difference among treatments, receptor-destroying enzyme treatment followed by chicken erythrocyte adsorption showed more consistent results, which corroborate the OIE and WHO recommendation to use this treatment preferentially. The HI results suggest equine H3N8 virus circulation among the animals tested from São Paulo State. The algorithm suggested here could be used to guide antibody detection against equine influenza virus in equines, improving the test specificity by aiming to avoid false positive results.

Tous les foyers de grippe équine dans le monde sont dus au sous-type H3N8 du virus. Le sous-type H7N7, moins pathogène, est considéré comme éteint, sa présence n'ayant été confirmée dans aucun des foyers enregistrés depuis 1980. Au Brésil, la grippe équine est enzootique mais la prévalence d'anticorps dans le pays est peu documentée. La présente étude avait trois objectifs : ­ évaluer l'efficacité de plusieurs traitements de sérums décrits par l'Organisation mondiale de la santé animale (OIE) et l'Organisation mondiale de la santé (OMS) sur la suppression des inhibiteurs d'hémagglutination non spécifiques, afin de pouvoir utiliser l'épreuve d'inhibition de l'hémagglutination pour la détection de la grippe équine, ­ évaluer la présence d'anticorps dirigés contre la grippe équine chez 83 chevaux non vaccinés de l'état de São Paulo en utilisant l'inhibition de l'hémagglutination, l'épreuve immuno-enzymatique (ELISA) et l'épreuve d'immunodiffusion en gélose (IDG) ; ­ évaluer une stratégie visant à améliorer les techniques sérologiques de détection des anticorps dirigés contre la grippe équine. S'il n'y a pas eu de différence statistique significative entre les traitements, celui faisant appel à l'enzyme de destruction du récepteur suivi d'une adsorption sur érythrocytes de poule a permis d'obtenir les résultats les plus cohérents, ce qui corrobore les recommandations de l'OIE et de l'OMS en faveur de ce traitement. Les résultats obtenus au moyen de l'inhibition de l'hémagglutination indiquent que le virus H3N8 est présent parmi les animaux testés de l'état de São Paulo. L'algorithme présenté par les auteurs pourrait servir de modèle pour détecter la présence d'anticorps dirigés contre le virus de la grippe équine chez les chevaux : en effet, il permet d'éviter les résultats faussement positifs, ce qui améliore la spécificité du test utilisé.

El subtipo H3N8 del virus de la gripe equina (VGE) es el agente etiológico de todos los brotes que se producen en el mundo, mientras que el subtipo H7N7, menos patogénico, se da por extinto, en la medida en que desde 1980 no se ha confirmado su intervención en brote alguno. Aunque en el Brasil el VGE es enzoótico, existen pocos trabajos que den cuenta de la situación real del país en cuanto a la presencia de anticuerpos contra el virus. Los autores describen un estudio que perseguía los siguientes objetivos: ­ evaluar la eficacia de distintos tratamientos séricos descritos por la Organización Mundial de Sanidad Animal (OIE) y la Organización Mundial de la Salud (OMS) para eliminar los inhibidores inespecíficos de la hemaglutinación con objeto de aplicar la técnica de inhibición de la hemaglutinación a la detección del VGE; ­ evaluar la presencia de anticuerpos contra el VGE por inhibición de la hemaglutinación, ensayo inmunoenzimático (ELISA) e inmunodifusión en gel de agar en 83 ejemplares equinos no vacunados del estado de São Paulo; ­ evaluar una estrategia encaminada a analizar más eficazmente sueros equinos para detectar en ellos anticuerpos anti-VGE. Aunque no se observaron diferencias estadísticamente significativas entre los tratamientos, el uso de enzimas destructores de receptores seguido de la técnica de adsorción de eritrocitos de pollo arrojó resultados más coherentes, cosa que avala la recomendación de la OIE y la OMS de privilegiar este tratamiento. Los resultados obtenidos por inhibición de la hemaglutinación parecen indicar que el virus H3N8 equino circula entre los animales analizados del estado de São Paulo. El algoritmo aquí propuesto podría servir de guía para detectar en equinos la presencia de anticuerpos contra el VGE. Puesto que apunta a evitar falsos positivos, su aplicación mejoraría la especificidad de la prueba.

An Avian coronavirus in quail with respiratory and reproductive signs.

An Avian coronavirus was detected in pools of lungs, tracheas, female reproductive tracts, kidneys, and enteric contents from quail (Coturnix coturnix japonica) and laying hen flocks, with and without infectious bronchitis (IB)-like signs, cohoused in farms located in two states of southeastern Brazil during 2009-2010. Although Avian metapneumovirus subtype B was found in two layers samples, Newcastle disease virus was not found in quail or in hens. Based on DNA sequences for the 3'-untranslated region and the gene encoding the RNA-dependent RNA polymerase, this avian coronaviruses in quail is an IB virus-like gammacoronavirus.

Detection of Genetic characterization of Porcine circovirus 2 (PCV2) in Brazilian wildlife boars.

A semi-intensive wildlife boars farm presented a clinical history of high mortality in 70 - 90 days-old pigs (> 50 %). Two 90 days-old animals with weight loss and wasting were necropsied and the samples tested for PCV2 by polymerase chain reaction (PCR). The genetic material of PCV2 was sequenced and classified into the PCV2a genotype together with PCV2 sequences obtained from samples of Poland, Brazil, Slovenia and Greece wild boars.

Rickettsial infections of dogs, horses and ticks in Juiz de Fora, southeastern Brazil, and isolation of Rickettsia rickettsii from Rhipicephalus sanguineus ticks.

The present study was performed in an area endemic for Brazilian spotted fever (BSF) in Juiz de Fora, state of Minas Gerais, Brazil, during the years 2007 and 2008, when fatal cases of BSF (caused by Rickettsia rickettsii) were reported. Adult ticks (Acari: Ixodidae) identified as Rhipicephalus sanguineus (Latreille) and Amblyomma cajennense (Fabricius) were collected from dogs and horses, respectively, and tested by polymerase chain reaction (PCR). Overall, 13.1% of the Rh. sanguineus ticks and none of the A. cajennense were found to be infected with R. rickettsii. Two isolates of R. rickettsii were successfully established in Vero cell culture from two Rh. sanguineus ticks. An indirect immunofluorescence assay (IFA) using R. rickettsii antigens detected blood serological reaction to R. rickettsii in 67.9% (53/78) of dogs and 41.0% (16/39) of horses living in the study area. Larval offspring from two Rh. sanguineus engorged females, naturally infected by R. rickettsii, were reared to adult stage in the laboratory. All active stages (larvae, nymphs, adults) remained 100% infected by R. rickettsii, which was efficiently transmitted to naïve rabbits. Overall, the results of the present study indicate a potential risk for transmission of R. rickettsii to humans by Rh. sanguineus, an occurrence yet to be documented in Brazil.

Bovine rotavirus in turkeys with enteritis.

Rotaviruses are the main agents responsible for diarrhea in different animal species and for infantile gastroenteritis. These viruses have been isolated from various avian species and have often been associated with poult enteritis and mortality syndrome. Nevertheless, the knowledge of rotavirus infection in turkeys is scarce. Six group A rotavirus strains obtained from pooled enteric contents of diarrheic turkeys were isolated in MA-104 cell culture and typed as G(6)P(1), a typical bovine rotavirus genotype. Additionally, the electropherotypes showed a migration pattern identical to the Nebraska calf diarrhea virus, and the complete NSP4 gene phylogeny showed that all six strains segregated in the genotype E2. Taken together, these results point toward a cattle-to-turkey rotavirus transmission. As a conclusion, bovine-origin rotavirus can be found in turkeys, and this transmission route must now be considered for the improvement of the health status in turkey farms.

First detection of the equine herpesvirus 1 neuropathogenic variant in Brazil.

This report describes the first detection of an equine herpesvirus 1 (EHV-1) neuropathogenic variant (G2254/D752) in Brazil from a case of fatal equine herpesvirus myeloencephalopathy (EHM) in a mare. The results of nucleotide sequencing of the EHV-1 ORF30 gene showed that two other Brazilian EHV-1 isolates from EHM cases are representatives of the non-neuropathogenic variant (A2254/N752), suggesting that other unidentified factors are probably also involved in the neuropathogenicity of EHV-1 in horses. These findings will contribute to the epidemiological knowledge of EHV-1 infection in Brazil.

Molecular epidemiology of avian infectious bronchitis in Brazil from 2007 to 2008 in breeders, broilers, and layers.

Multiple lineages of Brazilian strains from 2007 to 2008 of avian infectious bronchitis virus (IBV) were detected in flocks of breeders, broilers, and layers. Organs samples from 20 IBV-positive flocks with variable clinical signs were submitted to the partial amplification of S gene (nucleotides 726-1071) of IBV. Fifteen of the 20 sequenced strains segregated in a unique Brazilian cluster subdivided in three subclusters (Brazil 01, 02, and 03). Whereas three strains could be classified as Massachusetts (Mass) genotype, the remaining two strains, originating from flocks with reproductive and respiratory disorders, grouped within the 4/91-793B genotype, a genotype that has not been detected before in Brazil. The potential relevance of the findings to the poultry industry is discussed because the low level of identity of the sequenced part of the S gene from 17 of 20 detected field strains and the vaccines of the Massachusetts serotype used suggest that the level of cross-protection by the Massachusetts vaccines might be low.

Rickettsial infection in Amblyomma nodosum ticks (Acari: Ixodidae) from Brazil.

The rickettsial infections in 174 Amblyomma nodosum found on passeriform birds in the Atlantic forest, eastern Brazil, have recently been evaluated. Rickettsiae were successfully isolated from two ticks, using cultures of Vero cells. Both isolates were molecularly characterised, using the rickettsial genes gltA and htrA and, when possible, also ompA and ompB. Portions of the gltA and htrA genes from one of the rickettsial isolates were found be closely match the corresponding GenBank sequences for Rickettsia bellii, with 99.9% and 100% homology, respectively. This isolate was named R. bellii strain Pontal. Portions of the gltA, htrA and ompB genes from the second isolate most closely matched the corresponding sequences of R. parkeri, whereas a portion of the ompA gene from this isolate was closest to the relevant sequence of Rickettsia sp. strain COOPERI (which has been considered to be a strain of R. parkeri in Brazil). The second isolate was named R. parkeri strain NOD. Further investigation of the 172 ticks from which isolates were not recovered revealed R. parkeri strain NOD in 40 and R. bellii strain Pontal in nine, giving overall infection prevalences of 23.6% (41/174) and 5.7% (10/174), respectively. This appears to be the first report of R. bellii and R. parkeri in A. nodosum.

Evaluation of trypsin treatment on the inactivation of bovine herpesvirus type 1 on in vitro produced pre-implantation embryos.

The aim of this study was to evaluate the efficiency of trypsin treatment on the inactivation of bovine herpesvirus type 1 (BoHV-1) on in vitro produced by fertilization and artificially infected bovine embryos. Bovine embryos on day 7 were exposed with 10 microl of BoHV-1, Los Angeles strain 10(7.5) TCID. These embryos and control embryos were divided in two groups: submitted to the sequential washes or to the trypsin treatment according to the International Embryo Transfer Society (IETS) guidelines. The embryos and the last washing drop of each group were used as inoculum to infect Madin Darby bovine kidney (MDBK) cells and submitted to nested PCR reaction using the primer that encodes the gene conserved region of virus glycoprotein gB. The data have shown that the control embryos and their last washing drop were negative. The exposed embryos that were treated with trypsin have shown positive results on the n-PCR and MDBK culture, and their last washing drop were negative. Our data have demonstrated that the trypsin treatment was not able to eliminate the BHV-1 of the embryos, suggesting an interaction between virus and embryo.

Simultaneous presence of bovine papillomavirus in blood and in short-term lymphocyte cultures from dairy cattle in Pernambuco, Brazil.

Bovine papillomaviruses (BPV) are the causal agents of benign and malignant lesions; they can cause dramatic economic losses in cattle. Although 10 virus types have been described, three types are most common in tumors, namely BPV-1, -2 and -4. Previous studies have reported BPV in blood cells and the possibility of blood acting as a latent virus site and/or transmission agent of virus dissemination. We studied a Holstein dairy herd in Pernambuco, Brazil, in which several animals showed severe cutaneous papillomatosis, without previous determination of BPV types. Blood samples and short-term lymphocyte cultures were collected from 54 cows. We compared the BPV types detected in peripheral blood to those identified in the respective lymphocyte cultures: BPV-1 was detected in 74% and BPV-2 in 87% of the whole blood samples. Simultaneous virus presence (BPV-1 and BPV-2) was found in 65% of the blood samples. BPV-1 or BPV-2 were detected in the lymphocyte cultures in 93% of the samples, and both in 89%. The detection of viral DNA in whole blood and in lymphocyte cultures is evidence that this virus is carried by lymphocytes.

Detection of Leptospira spp. in semen and vaginal fluids of goats and sheep by polymerase chain reaction.

Thirteen goat herds and seven sheep flocks in the state of Rio de Janeiro, Brazil were screened for leptospirosis. From the three herds and three flocks with greatest seroreactivity, 19 goats (16 females and three bucks) and 40 sheep (26 ewes and 14 rams) that were seropositive (specific anti-Leptospira titres > or =400, based on a microscopic agglutination test), were selected for more detailed studies. From those animals, samples of vaginal fluids or semen were collected for bacteriological and molecular assays. For both species of animals, the most prevalent reactions were to serovars Hardjo, Shermani, and Grippotyphosa. Although leptospires were detected by darkfield microscopy in three vaginal fluid samples (from two goats and one ewe), pure isolates were not obtained by bacteriological culture of vaginal fluids or semen. However, seven vaginal fluid samples (from four goats and three ewes) and six semen samples (all from rams) were positive on polymerase chain reaction (PCR). Based on these findings, in addition to analogous findings in cattle, we inferred that there is potential for venereal transmission of leptospirosis in small ruminants.

Can established cultured papilloma cells harbor bovine papillomavirus?

Papillomaviruses have been reported to be very difficult to grow in cell culture. Also, there are no descriptions of cell cultures from lesions of bovine cutaneous papillomatosis, with identification of different bovine papilloma virus (BPV) DNA sequences. In the present report, we describe primary cell cultures from samples of cutaneous lesions (warts). We investigated the simultaneous presence of different BPV DNA sequences, comparing the original lesion to different passages of the cell cultures and to peripheral blood. BPV 1, 2 and 4 DNA sequences were found in lesion samples, and respective cell cultures and peripheral blood, supporting our previous hypothesis of the possible activity of these sequences in different samples and now also showing how they can be maintained in different passages of cell cultures.

A polymerase chain reaction for detection of Brucella canis in vaginal swabs of naturally infected bitches.

A PCR assay for the detection of Brucella canis in canine vaginal swab samples was evaluated, comparing its performance with that of bacterial isolation, serological tests, and a blood PCR assay. One hundred and forty-four female dogs were clinically examined to detect reproductive problems and they were tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR were performed on blood and vaginal swab samples. The results of the vaginal swab PCR were compared to those of the other tests using the Kappa coefficient and McNemar test. Of the 144 females that were examined, 66 (45.8%) were RSAT positive, 23 (15.9%) were 2ME-RSAT positive, 49 (34.02%) were blood culture positive, 6 (4.1%) were vaginal swab culture positive, 54 (37.5%) were blood PCR positive, 52 (36.2%) were vaginal swab PCR positive, and 50.69% (73/144) were positive by the combined PCR. The PCR was able to detect as few as 3.8 fg of B. canis DNA experimentally diluted in 54 ng of canine DNA, extracted from vaginal swab samples of non-infected bitches. In addition, the PCR assay amplified B. canis genetic sequences from vaginal swab samples containing 1.0 x 10(0) cfu/mL. In conclusion, vaginal swab PCR was a good candidate as a confirmatory test for brucellosis diagnosis in bitches suspected to be infected, especially those negative on blood culture or blood PCR; these animals may be important reservoirs of infection and could complicate attempts to eradicate the disease in confined populations.

A polymerase chain reaction for the detection of Brucella canis in semen of naturally infected dogs.

The objective was to evaluate a PCR assay for the detection of Brucella canis in canine semen, comparing its performance with that of bacterial isolation, serological tests and PCR assay of blood. Fifty-two male dogs were examined clinically to detect reproductive abnormalities and their serum was tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR assays were performed on blood and semen samples. The findings of the semen PCR were compared (Kappa coefficient and McNemar test) to those of blood PCR, culture of blood and semen, RSAT, and 2ME-RSAT. Nucleic acid extracts from semen collected from dogs not infected with B. canis were spiked with decreasing amounts of B. canis RM6/66 DNA and the resulting samples subjected to PCR. In addition, semen samples of non-infected dogs were spiked with decreasing amounts of B. canis CFU and the resulting suspensions were used for DNA extraction and amplification. Of the 52 dogs that were examined, the following tests were positive: RSAT, 16 (30.7%); 2ME-RSAT, 5 (9.6%); blood culture, 14 (26.9%); semen culture, 11 (21.1%); blood PCR, 18 (34.6%); semen PCR, 18 (34.6%). The PCR assay detected as few as 3.8 fg of B. canis DNA experimentally diluted in 444.9 ng of canine DNA (extracted from semen samples of non-infected dogs). In addition, the PCR assay amplified B. canis genetic sequences from semen samples containing as little as 1.0 x 10(0) cfu/mL. We concluded that PCR assay of semen was a good candidate as a confirmatory test for the diagnosis of brucellosis in dogs; its diagnostic performance was similar to blood culture or blood PCR. Furthermore, the PCR assay of semen was more sensitive than the 2ME-RSAT or semen culture. Examination of semen by PCR should be included for diagnosis of brucellosis prior to natural mating or AI; in that regard, some dogs that were negative on serological and microbiological examinations as well as blood PCR were positive on PCR of semen.

Diagnosis of canine brucellosis: comparison between serological and microbiological tests and a PCR based on primers to 16S-23S rDNA interspacer.

A pair of primers directed to 16S-23S rDNA interspacer (ITS) was designed directed to Brucella genetic sequences in order to develop a polymerase chain reaction (PCR) putatively capable of amplifying DNA from any Brucella species. Nucleic acid extracts from whole-blood from naive dogs were spiked with decreasing amounts of Brucella canis RM6/66 DNA and the resulting solutions were tested by PCR. In addition, the ability of PCR to amplify Brucella spp. genetic sequences from naturally infected dogs was evaluated using 210 whole-blood samples of dogs from 19 kennels. The whole-blood samples collected were subjected to blood culture and PCR. Serodiagnosis was performed using the rapid slide agglutination test with and without 2-mercaptoethanol. The DNA from whole blood was extracted using proteinase-K, sodium dodecyl sulphate and cetyl trimethyl ammonium bromide followed by phenol-chloroform purification. The PCR was capable of detecting as little as 3.8 fg of Brucella DNA mixed with 450 ng of host DNA. Theoretically, 3.8 fg of Brucella DNA represents the total genomic mass of fewer than two bacterial cells. The PCR diagnostic sensitivity and specificity were 100%. From the results observed in the present study, we conclude that PCR could be used as confirmatory test for diagnosis of B. canis infection.

Feline Coronavirus 3c Protein: A Candidate for a Virulence Marker?

Feline infectious peritonitis virus (FIPV) is highly virulent and responsible for the highly fatal disease feline infectious peritonitis (FIP), whereas feline enteric coronavirus (FECV) is widespread among the feline population and typically causes asymptomatic infections. Some candidates for genetic markers capable of differentiating these two pathotypes of a unique virus (feline coronavirus) have been proposed by several studies. In the present survey, in order to search for markers that can differentiate FECV and FIPV, several clones of the 3a-c, E, and M genes were sequenced from samples obtained from cats with or without FIP. All genes showed genetic diversity and suggested the presence of FCoV mutant spectrum capable of producing a virulent pathotype in an individual-specific way. In addition, all the feline coronavirus FIPV strains demonstrated a truncated 3c protein, and the 3c gene was the only observed pathotypic marker for FCoVs, showing that 3c gene is a candidate marker for the distinction between the two pathotypes when the mutant spectrum is taken into account.

Detection of porcine parvovirus DNA by the polymerase chain reaction assay using primers to the highly conserved nonstructural protein gene, NS-1.

Porcine parvovirus (PPV) infection is associated with reproductive losses in swine and its causative agent, the PPV, has been isolated worldwide. Serological surveys and virus isolation studies throughout Brazil confirm the occurrence of PPV infection in this country. The most common methods to detect PPV infection are fluorescent antibody staining of fetal tissues, hemagglutination assay of tissue extracts and virus isolation from fetal tissues. Non-specificity and low sensitivity are the major drawbacks of these techniques. The development of a polymerase chain reaction (PCR) and nested-PCR assays for PPV DNA detection from infected cell lines and clinical samples is described. The primers were designed to a highly conserved region of the PPV genome which codes for the non-structural protein, NS-1. Results showed that PCR could detect PPV in titres at least 10(6) higher than the hemagglutination assay. The PCR and nested-PCR assays were used to detect successfully PPV DNA in clinical samples.

Detection and differentiation of Leptospira spp. serovars in bovine semen by polymerase chain reaction and restriction fragment length polymorphism.

In view of the importance of venereal transmission of bovine leptospirosis, the objective of the present study was to apply the polymerase chain reaction (PCR) to 26 serovars of Leptospira interrogans, L. borgpetersenii, L. santarosai, L. noguchii and L. biflexa, to determine the detection threshold in semen samples and to evaluate the possibility of differentiation among serovars using 19 restriction endonucleases. The results showed that all serovars were amplified and the detection threshold in semen samples of a bull was 100 bacteria/ml. Using endonucleases we could classify the 26 serovars into eight groups. The present results show that PCR is a method of great potential for the detection of Leptospira spp. at bovine artificial insemination centers.

Immunodiagnosis of swine cysticercosis by indirect ELISA employing a heterologous antigen from Taenia crassiceps metacestode.

C. Larralde et al. (1990, Aoff. Pathol. Lab. Med., 114:926-928) demonstrated that heterologous antigen from the laboratory-adapted murine Taenia crassiceps metacestode may substitute those from Taenia solium in the immunodiagnosis of human cysticercosis by the indirect enzyme-linked immunosorbent assay (IE). This antigen is easily obtained at a laboratory level and solves the problem of T. solium cysticerci collection from naturally or experimentally infected swine. In this study an IE employing a heterologous antigen from the T. crassiceps metacestode was evaluated for the immunodiagnosis of swine cysticercosis. Sera from 300 swine free of T. solium cysticerci by post-mortem examination were employed to determine two IE cut-off values: 1) Mean ELISA values + 2 standard deviations (2 sigma cut-off) and 2) - Mean ELISA values + 3 standard deviations (3 sigma cut-off). The specificity of IE was 97% with the 2 sigma cut-off and 100% with the 3 sigma cut-off. When applied to ten sera from swine infected by cysticerci of T. solium by post-mortem examination, the sensitivity of IE was 100% independent of the cut off.