Molecular evidence of bovine herpesvirus 1 and 5 in cattle with suspected rabies in Rio Grande do Sul state, Brazil.

Rabies and herpetic encephalitis are the main viral infections in bovines with neurological symptoms. Bovine rabies has a high prevalence in Central and South America, while bovine encephalitis associated with herpesvirus is especially important in South America. Viral isolation is the classical way to confirm herpesvirus infection, but molecular evidence of the presence of the virus in affected animals is gaining importance in the diagnosis of the disease in the laboratory. This study investigated the presence of herpesvirus type 1 and 5 (BoHV-1 and BoHV-5) in 182 encephalon of rabies-suspected cattle in Rio Grande do Sul state (RS), Brazil using multiplex real-time polymerase chain reaction (mRT-PCR). The rabies virus was investigated by direct fluorescent antibody assay and intracerebral suckling mouse inoculation. The genomes of BoHV-1 and BoHV-5 were detected in 17% of samples. BoHV-5 and BoHV-1 were detected in 100% and 19% of BoHV positive samples, respectively, indicating the circulation of the pathogens in cattle herds in RS. The high Ct values and the absence of isolation suggest viral latency. Coinfection of herpesvirus and the rabies virus was detected in 28% of samples, although no significant association between pathogens was observed. Rabies was detected in 57.7% of suspected samples, confirming the importance of the disease in the state. Concerning the method by which samples were conserved, no significant difference was observed between the number of positive results in frozen and refrigerated samples.

Effects of benign prostatic hyperplasia and finasteride therapy on prostatic blood flow in dogs.

Benign prostatic hyperplasia (BPH) is characterized by an enlargement of the prostate accompanied by an increase in prostatic blood perfusion and vascularization. The most indicated treatment is to perform orchiectomy, however, medical treatment with finasteride can be an option for breeding dogs or elderly animals with a critical health status. In dogs, the influence of medical treatment on prostatic hemodynamics is still unknown. Therefore, this study aimed to evaluate the effects of benign prostatic hyperplasia and finasteride therapy on hemodynamic and vascular features of the canine prostate. For this purpose, twenty dogs of different breeds, body weights (10-30 kg) and ages (5-13 years) were used, assigned for: Healthy-non treated group (n = 5), BPH-non treated group (n = 5), Healthy-finasteride treated group (n = 5) and BPH-finasteride treated group (n = 5). Dogs that presented hematospermia and at least one general clinical sign (tenesmus, hematuria or dysuria) were presumptively diagnosed with BPH. Dogs were evaluated ultrasonographycally by B-mode and Doppler of the prostatic artery in a monthly interval (day 0, 30 and 60) in order to measure prostate volume (PV), expected prostate volume (EPV), prostate vascularization score (scored as minimum, intermediary and maximum) and prostatic artery blood flow parameters with the use of spectral and color Doppler ultrasound. It was possible to observe a decrease in prostate vascularization score between Day 0 (intermediary degree) and 60 (minimum degree) in finasteride treated dogs. Moreover, non-treated dogs had higher score of vascularization at Day 60 compared to animals treated with finasteride, regardless of BPH diagnosis. Healthy-non treated animals presented higher peak systolic:diastolic velocity (S/D) than BPH-non treated dogs. Furthermore, BPH-non treated dogs had lower S/D than BPH-finasteride treated dogs. In 30 and 60 days, no difference on PV was observed between BPH-finasteride treated group and Healthy-non treated group. At day 60, no difference between PV and EPV was observed for the BPH-finasteride treated group. In conclusion, finasteride treatment reduces simultaneously the volume, local vascularization and blood flow of the prostate, thus, being considered an effective and additional choice of therapy for BPH. Moreover, the course of therapy in dogs can be followed by analyzing changes in prostatic artery.

Uterine haemodynamic, vascularization and blood pressure changes along the oestrous cycle in bitches.

Characterization of the bitch's reproductive physiology is of utmost importance for the development of new reproductive techniques and the diagnosis of reproductive diseases. In this respect, uterine B-mode ultrasonography has been employed in several studies; however, the focus on haemodynamic changes along the oestrous cycle is yet to be described. Thus, the aim of this study was to characterize haemodynamic changes (uterine vascularization, systemic arterial blood pressure and heart rate) throughout the oestrous cycle in bitches. For this purpose, ten Golden Retriever bitches were evaluated during an entire oestrous cycle, twice during each stage of the cycle. Uterine artery blood flow, velocity wave forms, haemodynamic parameters and vascularization were analysed by Doppler ultrasonography. Furthermore, uterine diameter, arterial blood pressure and heart rate were measured. Uterine artery pulsatility index at early prooestrus was significantly lower in comparison with early oestrus, mid- and late anoestrus. Uterine artery resistance index was higher at early oestrus when compared to late oestrus and uterine diameter was significantly higher during late prooestrus. Furthermore, mean arterial blood pressure was lower and heart rate was higher during late prooestrus in comparison with the other oestrous cycle stages. In conclusion, haemodynamic changes in the uterine artery, uterine diameter, systemic blood pressure and heart rate occur during the canine oestrous cycle. Specifically, there is an increase in uterine artery perfusion, uterine diameter and mean arterial blood pressure during prooestrus, while uterine blood flow diminishes during oestrus and anoestrus.

Chicken parvovirus and its associations with malabsorption syndrome.

Malabsorption syndrome (MAS) is a multifactorial syndrome which is characterized by enteric disorders and reduced growth rates of broilers. Such condition is responsible for significant economic losses to the poultry industry. A possible association between chicken parvovirus (ChPV) infections and the occurrence of MAS has been proposed. However, such association has not to date been elucidated in view that ChPV has been detected in healthy as well as in MAS-affected chickens. This study aimed to detect and quantify ChPV loads in sera and tissues of MAS-affected, as well as in healthy broilers. Fifty nine, 39-day-old broilers (50 diseased, 9 healthy birds), obtained from the same flocks, were examined. The highest ChPV DNA loads were detected in MAS-affected broilers, particularly in fecal samples and intestinal tissues (~5500 genomic copies/300ng of total DNA). The average viral genome load in serum in MAS-affected birds was 1134copies/mL, whereas no viral DNA was found in sera and thymus tissues from healthy animals. These findings reveal that MAS-affected broilers consistently carry ChPV DNA is serum, whereas healthy animals do not. In addition, viral loads in tissues (bursa of Fabricius, spleen, intestine and liver) of MAS-affected birds were significantly higher in comparison to the same tissues from healthy broilers. Although preliminary, the results obtained here indicate an association between the detection of ChPV DNA in serum, in addition to high ChPV viral loads in tissues, and the occurrence of MAS in broilers. Further experiments should be performed to confirm such results.

Swine Influenza Virus and Association with the Porcine Respiratory Disease Complex in Pig Farms in Southern Brazil.

Despite the putative endemic status of swine influenza A virus (swIAV) infections, data on the occurrence of swine influenza outbreaks are scarce in Brazil. The aim of this study was to detect and subtype swIAVs from six outbreaks of porcine respiratory disease complex (PRDC) in southern Brazil. Nasal swabs were collected from 66 piglets with signs of respiratory disease in six herds. Lung tissue samples were collected from six necropsied animals. Virus detection was performed by PCR screening and confirmed by virus isolation and hemagglutination (HA). Influenza A subtyping was performed by a real-time reverse transcriptase PCR (rRT-PCR) to detect the A(H1N1)pdm09; other swIAV subtypes were determined by multiplex RT-PCR. In lung tissues, the major bacterial and viral pathogens associated with PRDC (Pasteurella multocida, Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Haemophilus parasuis and PCV2) were investigated. In some affected pigs, clinico-pathological evaluations were conducted. Influenza A was detected by screening PCR in 46 of 66 swab samples and from five of six lungs. Virus was recovered from pigs of all six herds. Subtype A(H1N1)pdm09 was detected in four of six herds and H1N2 in the other two herds. In lung tissues, further agents involved in PRDC were detected in all cases; Pasteurella multocida was identified in five of six samples and Mycoplasma hyopneumoniae in three of six. Actinobacillus pleuropneumoniae (1/6), Haemophilus parasuis (1/6) and PCV2 (1/6) were also detected. These findings indicate that subtypes A(H1N1)pdm09 and H1N2 were present in pigs in southern Brazil and were associated with PRDC outbreaks.

Bovine Viral Diarrhoea Virus (BVDV) in Dairy Cattle: A Matched Case-Control Study.

Bovine viral diarrhoea virus (BVDV) causes one of the most important diseases of cattle in terms of economic costs and welfare. The aims were to estimate herd prevalence and to investigate the factors associated with antibodies in bulk tank milk (BTM) in dairy herds through a matched case-control study. To estimate herd prevalence, BTM samples were randomly selected (n = 314) from a population (N = 1604). The true prevalence of BVDV was 24.3% (CI 95% = 20.1-29.3%). For the case-control study, BVDV antibody-positive herds (high antibody titres) were classified as cases (n = 21) and matched (n = 63) by milk production with herds presenting low antibody titres (ratio of 1 : 3). Three multivariable models were built: 1) full model, holding all 21 variables, and two models divided according to empirical knowledge and similarity among variables; 2) animal factor model; and 3) biosecurity model. The full model (model 1) identified: age as a culling criteria (OR = 0.10; CI 95% = 0.02-0.39; P < 0.01); farms that provided milk to other industries previously (OR = 4.13; CI 95% = 1.17-14.49; P = 0.02); and isolation paddocks for ill animals (OR = 0.14; CI 95% = 0.01-0.26; P = 0.02). The biosecurity model revealed a significant association with the use of natural mating (OR = 9.03; CI 95% = 2.14-38.03; P < 0.01); isolation paddocks for ill animals (OR = 0.06; CI 95% = 0.05-0.83; P = 0.03); years providing milk for the same industry (OR = 0.94; CI 95% = 0.91-0.97; P = 0.02); and direct contact over fences among cattle of neighbouring farms (OR = 5.78; CI 95% = 1.41-23.67; P = 0.04). We recommend the application of grouping predictors as a good choice for model building because it could lead to a better understanding of disease-exposure associations.

Clinical Presentation Resembling Mucosal Disease Associated with 'HoBi'-like Pestivirus in a Field Outbreak.

The genus Pestivirus of the family Flaviviridae consists of four recognized species: Bovine viral diarrhoea virus 1 (BVDV-1), Bovine viral diarrhoea virus 2 (BVDV-2), Classical swine fever virus (CSFV) and Border disease virus (BDV). Recently, atypical pestiviruses ('HoBi'-like pestiviruses) were identified in batches of contaminated foetal calf serum and in naturally infected cattle with and without clinical symptoms. Here, we describe the first report of a mucosal disease-like clinical presentation (MD) associated with a 'HoBi'-like pestivirus occurring in a cattle herd. The outbreak was investigated using immunohistochemistry, antibody detection, viral isolation and RT-PCR. The sequence and phylogenetic analysis of 5'NCR, N(pro) and E2 regions of the RT-PCR positive samples showed that four different 'HoBi'-like strains were circulating in the herd. The main clinical signs and lesions were observed in the respiratory and digestive systems, but skin lesions and corneal opacity were also observed. MD characteristic lesions and a pestivirus with cytopathic biotype were detected in one calf. The present study is the first report of a MD like presentation associated with natural infection with 'HoBi'-like pestivirus. This report describes the clinical signs and provides a pathologic framework of an outbreak associated with at least two different 'HoBi'-like strains. Based on these observations, it appears that these atypical pestiviruses are most likely underdiagnosed in Brazilian cattle.

Herd-level risk factors for bovine viral diarrhea virus infection in dairy herds from Southern Brazil.

A cross-sectional study was carried out to identify risk factors for bovine viral diarrhea virus (BVDV) infection in 300 randomly selected dairy herds which were tested for antibodies in bulk tank milk (BTM) using a commercial indirect ELISA kit (SVANOVA). Results from the analysis were interpreted according to the Swedish BVDV control scheme. The testing revealed 129 (43%) BTM BVDV antibody-positive herds. Use of artificial insemination (AI) and herd size were significantly associated with BVDV serological status (P<0.05). Dairy herds that use AI had 2.82 increased odds of BVDV-seropositivity (95% CI: 1.02-7.24). Since the semen used in the studied population come from known selected sires, it was hypothesized that AI technicians should represent an important risk factor because the increasing number of visitors in the farm can introduce the virus through the clothes, shoes and contaminated equipment.

Pneumocystis sp. in bats evaluated by qPCR.

Molecular techniques have revealed a high prevalence of Pneumocystis colonization in wild mammals. Accurate quantification of Pneumocystis sp. is essential for the correct interpretation of many research experiments investigating this organism. The objectives of this study were to detect the presence of Pneumocystis sp. in bats by qPCR, and to distinguish colonization from infection. Probes and primers for real time PCR (qPCR) were designed based on the gene of major surface glycoprotein (MSG) of Pneumocystis sp., in order to analyze 195 lung tissue samples from bats captured (2007-2009). All samples were also analyzed by nested PCR, using oligonucleotide primers designed for the gene encoding the mitochondrial small subunit rRNA (mtSSU rRNA) to confirm the results. The qPCR assay was standardized using a standard curve made with the DNA extracted from bronchoalveolar lavage positive for Pneumocystis jirovecii. The average Ct was found to be between 13 and 14 (calibration curve) for the detection of infection with Pneumocystis sp. and above these values for colonization. It was considered as negative samples the ones that had Ct values equal to 50. Out of the total 195 samples, 47 (24.1%) bat lung DNA samples were positive for Pneumocystis sp. by qPCR. The most common bat species found were: Tadarida brasiliensis (23.4%), Histiotus velatus (17.0%), Desmodus rotundus (14.9%) and Molossus molossus (8.5%). The average cycle threshold of the positive samples (bats) was 25.8 and standard deviation was 1.7. The DNA samples with Ct values greater than 14 suggest that these animals might be colonized by Pneumocystis sp. Results obtained in this study demonstrated the usefulness of the qPCR procedure for identification of Pneumocystis sp. and for distinction between its colonizing or infectious status in bats.

Comparison of sputum induction with fiberoptic bronchoscopy in the diagnosis of tuberculosis: experience at an acquired immune deficiency syndrome reference center in Rio de Janeiro, Brazil.

Many patients with suspected pulmonary tuberculosis (PTB) do not produce sputum spontaneously or are smear-negative for acid-fast bacilli (AFB). We prospectively compared the yield of sputum induction (SI) and fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) for the diagnosis of PTB in a region with a high prevalence of tuberculosis and human immunodeficiency virus (HIV) infection. Fifty seven percent (143 of 251) of patients had diagnoses of PTB, of whom 17% (25 of 143) were HIV seropositive. There were no significant differences in the yields of AFB smears or cultures whether obtained via SI or BAL. Among 207 HIV-seronegative patients, the AFB smear and mycobacterial culture results from specimens obtained by SI and BAL were in agreement in 97% (202 of 207) (kappa test = 0.92) and 90% (186 of 207) (kappa test = 0.78), respectively. Among HIV-seropositive patients the agreements between AFB smear and culture results for SI and BAL specimens were 98% (43 of 44) (kappa test = 0.93) and 86% (38 of 44) (kappa test = 0.69), respectively. We conclude that SI is a safe procedure with a high diagnostic yield and high agreement with the results of fiberoptic bronchoscopy for the diagnosis of PTB in both HIV-seronegative and HIV-seropositive patients.

Differentiation of classical swine fever virus from ruminant pestiviruses by reverse transcription and polymerase chain reaction (RT-PCR).

A reverse transcriptase-polymerase chain reaction (RT-PCR) assay was designed to allow the differentiation of pestiviruses by the expected size of the amplified fragments. One oligonucleotide primer, conserved amongst pestiviruses, and two others specific for either classical swine fever virus (CSFV) or bovine viral diarrhea virus (BVDV), were designed from the 5' non-coding region of the genome. CSFV infected cultures (10 strains) amplified a fragment of an expected size of 200 bp; BVDV cultures (23 strains) or border disease virus (BDV) (2 strains) amplified a fragment of an expected size of 260 bp. The specificity of the amplified fragments was confirmed by restriction enzyme analysis. The threshold of sensitivity was 100 TCID50 for CSFV and 1 TCID50 for BVDV. The RT-PCR described here provides a rapid and sensitive diagnostic tool for the detection and differentiation of CSFV from ruminant pestiviruses.