RESUMO
OBJECTIVE: As biologic augmentation methods emerge, objective measures of soft tissues are necessary for developmental study. The purpose of this study was to develop a quantitative MRI mapping protocol for the ACL. The objectives were (1) to provide age-based T2 relaxation, T2* relaxation, and volume values in healthy individuals, (2) to establish the intra-rater and inter-rater reliability of ACL mapping, and (3) to determine whether 3-T or 7-T MRI is more appropriate for future clinical trials. MATERIALS AND METHODS: Thirty healthy participants, aged 18-62, asymptomatic for knee pathology and without history of knee injury underwent both a 3-T and 7-T MRI. Manual image mapping of the anterior cruciate ligament was performed by two observers and processed to obtain T2, T2*, and volume values. Analysis of variance and two-way random effects model were used to calculate statistical significance and intraclass correlation coefficients. RESULTS: Across all participants, 3-T and 7-T mean T2, T2* and volume values were 37.1 ± 7.9 and 39.7 ± 6.2 ms (p = 0.124), 10.9 ± 1.3 and 10.9 ± 0.9 ms (p = 0.981), and 2380 ± 602 and 2484 ± 736 mm3 (p = 0.551), respectively. The T2, T2*, and volume did not vary between age cohorts (p > 0.05). Excellent inter-rater and intra-rater reliability regarding T2 and T2* values was found. While ACL volume exhibited good inter-rater reliability and excellent intra-rater reliability. CONCLUSIONS: T2 relaxation values and ACL volume do not vary with age and therefore can be used as a quantifiable, non-invasive method to assess ACL graft maturation. 7-T MRI analysis was not superior to 3-T MRI analysis, suggesting that 3-T MRI is practical and capable for future comparative studies.
Assuntos
Ligamento Cruzado Anterior/diagnóstico por imagem , Imageamento por Ressonância Magnética/métodos , Adolescente , Adulto , Ligamento Cruzado Anterior/anatomia & histologia , Feminino , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos TestesRESUMO
PURPOSE: To determine the cellular composition of a product created with peripheral blood harvested after systemic mobilization with filgrastim and processed with one point-of-care blood concentrating system, i.e., a platelet-rich plasma (PRP) system. The second purpose was to compare mobilized platelet-rich plasma (M-PRP) with a concentrated bone marrow aspirate (cBMA) and a PRP created from the same subjects with the same PRP system. METHODS: Ten healthy volunteer subjects were recruited for collection and analysis of 3 tissue sources: non-treated peripheral blood, bone marrow aspirate, and filgrastim-mobilized peripheral blood, involving 4 doses of weight-based filgrastim. One point-of-care blood and bone marrow concentrating system was used to create 3 products: PRP, cBMA, and M-PRP. Automated hematologic analysis was performed on all products to quantify total red blood cells, white blood cells (WBCs), monocyte, platelet, and hematopoietic progenitor cell (HPC) concentrations. Flow cytometry was used to determine hematopoietic and mesenchymal progenitor cell populations. Lastly, concentrates were cultured and fibroblast colony-forming units (CFU-F) and morphology of adherent cells were evaluated. RESULTS: M-PRP contained a greater concentration of WBC (mean difference = 53.2 k/µL; P < .0001), monocytes (mean difference = 8.3 k/µL; P = .002), and a trend toward a greater concentration of HPC (mean difference = 200.5 /µL; P = .060) when compared with PRP. M-PRP contained a greater concentration of monocytes (mean difference = 5.5 k/µL; P = .017) and a trend toward a greater concentration of platelets (mean difference = 348 k/µL; P = .051) and HPC (mean difference = 193.4 /µL; P = .068) when compared with cBMA. M-PRP had a similar concentration of platelets to PRP (mean difference = 110 k/µL; P = .051) and PRP had a greater concentration than cBMA (mean difference = 458 k/µL; P = .003). cBMA remained the only product capable of producing CFU-Fs (446 ± 247 /mL) as neither the M-PRP nor PRP produced CFU-Fs. M-PRP produced colonies consistent with WBC. CONCLUSIONS: M-PRP, produced with filgrastim mobilized blood and a proprietary PRP system, contained more total WBCs, monocytes, platelets, and HPCs than cBMA and more WBCs, monocytes, and HPCs than PRP. CLINICAL RELEVANCE: Filgrastim mobilized PRP may be an alternative to cBMA for use as a point-of-care product for orthopaedic treatments.
Assuntos
Plaquetas/citologia , Células da Medula Óssea/citologia , Filgrastim/farmacologia , Células-Tronco Mesenquimais/citologia , Plasma Rico em Plaquetas , Adulto , Adesão Celular , Contagem de Células , Citometria de Fluxo , Humanos , Masculino , Adulto JovemRESUMO
Nonprimate hepacivirus (NPHV) is the closest known relative of hepatitis C virus (HCV) and its study could enrich our understanding of HCV evolution, immunity, and pathogenesis. High seropositivity is found in horses worldwide with â¼ 3% viremic. NPHV natural history and molecular virology remain largely unexplored, however. Here, we show that NPHV, like HCV, can cause persistent infection for over a decade, with high titers and negative strand RNA in the liver. NPHV is a near-universal contaminant of commercial horse sera for cell culture. The complete NPHV 3'-UTR was determined and consists of interspersed homopolymer tracts and an HCV-like 3'-terminal poly(U)-X-tail. NPHV translation is stimulated by miR-122 and the 3'-UTR and, similar to HCV, the NPHV NS3-4A protease can cleave mitochondrial antiviral-signaling protein to inactivate the retinoic acid-inducible gene I pathway. Using an NPHV consensus cDNA clone, replication was not observed in primary equine fetal liver cultures or after electroporation of selectable replicons. However, intrahepatic RNA inoculation of a horse initiated infection, yielding high RNA titers in the serum and liver. Delayed seroconversion, slightly elevated circulating liver enzymes and mild hepatitis was observed, followed by viral clearance. This establishes the molecular components of a functional NPHV genome. Thus, NPHV appears to resemble HCV not only in genome structure but also in its ability to establish chronic infection with delayed seroconversion and hepatitis. This NPHV infectious clone and resulting acute phase sera will facilitate more detailed studies on the natural history, pathogenesis, and immunity of this novel hepacivirus in its natural host.
Assuntos
Hepacivirus/fisiologia , Regiões 3' não Traduzidas , Clonagem Molecular , DNA Complementar , Hepacivirus/genética , Dados de Sequência Molecular , Biossíntese de Proteínas , Carga Viral , Replicação ViralRESUMO
The recent identification of nonprimate hepaciviruses in dogs and then in horses prompted us to look for pegiviruses (GB virus-like viruses) in these species. Although none were detected in canines, we found widespread natural infection of horses by a novel pegivirus. Unique genomic features and phylogenetic analyses confirmed that the tentatively named equine pegivirus (EPgV) represents a novel species within the Pegivirus genus. We also determined that EPgV causes persistent viremia whereas its clinical significance is undetermined.
Assuntos
Infecções por Flaviviridae/veterinária , Flaviviridae/classificação , Doenças dos Cavalos/virologia , Cavalos/virologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Flaviviridae/genética , Infecções por Flaviviridae/epidemiologia , Infecções por Flaviviridae/virologia , Doenças dos Cavalos/epidemiologia , Dados de Sequência Molecular , Filogenia , Prevalência , Análise de Sequência de DNA , Especificidade da Espécie , Estados Unidos/epidemiologia , Viremia/epidemiologia , Viremia/virologiaRESUMO
Rabies is the deadliest viral infection known, with no reliable treatment, and although it is entirely preventable, rabies continues to kill more than 60,000 people every year, mostly children in countries where dog rabies is endemic. America is only 1 generation away from the time when rabies killed more than 10,000 animals and 50 Americans every year, but 3 to 5 Americans continue to die annually from rabies. Distressingly, > 50,000 Americans undergo rabies prevention therapy every year after exposure to potentially rabid animals. While enormous progress has been made, more must be done to defeat this ancient but persistent, fatal zoonosis. In the US, lack of public awareness and ambivalence are the greatest dangers imposed by rabies, resulting in unnecessary exposures, anxiety, and risk. Veterinarians have a special role in informing and reassuring the public about prevention and protection from rabies. This summary of current facts and future advances about rabies will assist veterinarians in informing their clients about the disease.
Assuntos
Doenças do Cão , Vacina Antirrábica , Raiva , Médicos Veterinários , Animais , Cães , Humanos , Raiva/epidemiologia , Raiva/prevenção & controle , Raiva/veterinária , Zoonoses , Ansiedade , Transtornos de Ansiedade , Vacina Antirrábica/uso terapêutico , Doenças do Cão/prevenção & controle , Doenças do Cão/epidemiologiaRESUMO
Economic losses due to infection with Bovine viral diarrhea virus (BVDV) have prompted introduction of organized control programs. These programs primarily focus on the removal of persistently infected (PI) animals, the main source of BVDV transmission. Recently, persistent BVDV infection was demonstrated experimentally in white-tailed deer, the most abundant wild ruminant in North America. Contact of cattle and white-tailed deer may result in interspecific BVDV transmission and birth of persistently infected offspring that could be a threat to control programs. The objective of this study was to assess the potential for interspecific BVDV transmission from persistently infected cattle cohabitated with pregnant white-tailed deer. Seven female and one male white-tailed deer were captured and bred in captivity. At approximately 50 days of gestation, two cattle persistently infected with BVDV 1 were cohabitated with the deer. In a pen of approximately 0.8 ha, both species shared food and water sources for a period of 60 days. Transmission of BVDV as indicated by seroconversion was demonstrated in all exposed adult deer. Of the seven pregnancies, four resulted in offspring that were infected with BVDV. Persistent infection was demonstrated in three singlet fawns by immunohistochemistry and ELISA on skin samples, PCR, and virus isolation procedures. Furthermore, two stillborn fetuses were apparently persistently infected. This is the first report of BVDV transmission from cattle to white-tailed deer using a model of natural challenge. Under appropriate circumstances, BVDV may efficiently cross the species barrier to cause transplacental infection and persistently infected offspring in a wildlife species.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/transmissão , Cervos , Vírus da Diarreia Viral Bovina/fisiologia , Animais , Bovinos , Suscetibilidade a Doenças , Feminino , Transmissão Vertical de Doenças Infecciosas , Gravidez , Especificidade da EspécieRESUMO
Bovine viral diarrhea virus (BVDV) is one of the most relevant pathogens affecting today's cattle industries. Although great strides have been made in understanding this virus in cattle, little is known about the role of wildlife in the epidemiology of BVDV. While persistently infected cattle are the most important reservoir, free-ranging ungulates may become infected with BVDV as demonstrated by serosurveys and experimental infections. Therefore, free-ranging wildlife may maintain BVDV as the result of an independent cycle and may serve as a reservoir for the virus. Systematic studies on prevalence of BVDV-specific antibodies or frequency of persistent BVDV infection in North American wildlife are sparse, and no information is available from the southeastern United States. The objective of this study was to evaluate blood and skin samples from hunter-harvested white-tailed deer (Odocoileus virginianus) for evidence of BVDV infection. Virus-neutralizing antibodies were detected in 2 of 165 serum samples. Skin biopsy immunohistochemistry (IHC) was performed on samples from 406 deer using a BVDV-specific monoclonal antibody (MAb) (15c5), and BVDV antigen was detected in one sample. A similar IHC staining pattern was obtained using a second BVDV MAb (3.12F1). Viral antigen distribution in the skin sample of this deer resembled that found in persistently infected cattle and in a previously described persistently infected white-tailed deer; thus, the deer was presumed to be persistently infected. Evidence of BVDV infection in free-ranging white-tailed deer should encourage further systematic investigation of the prevalence of BVDV in wildlife.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Cervos/virologia , Vírus da Diarreia Viral Bovina/isolamento & purificação , Reservatórios de Doenças/veterinária , Alabama/epidemiologia , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/análise , Doença das Mucosas por Vírus da Diarreia Viral Bovina/sangue , Doença das Mucosas por Vírus da Diarreia Viral Bovina/epidemiologia , Bovinos , Reservatórios de Doenças/virologia , Imuno-Histoquímica/veterinária , Testes de Neutralização/veterinária , Prevalência , Pele/virologiaRESUMO
Bovine viral diarrhea virus (BVDV) infections cause substantial economic losses to the cattle industries. Persistently infected (PI) cattle are the most important reservoir for BVDV. White-tailed deer (Odocoileus virginianus) are the most abundant species of wild ruminants in the United States and contact between cattle and deer is common. If the outcome of fetal infection of white-tailed deer is similar to cattle, PI white-tailed deer may pose a threat to BVDV control programs. The objective of this study was to determine if experimental infection of pregnant white-tailed deer with BVDV would result in the birth of PI offspring. Nine female and one male white-tailed deer were captured and housed at a captive deer isolation facility. After natural mating had occurred, all does were inoculated intranasally at approximately 50 days of pregnancy with 10(6) CCID(50) each of a BVDV 1 (BJ) and BVDV 2 (PA131) strain. Although no clinical signs of BVDV infection were observed or abortions detected, only one pregnancy advanced to term. On day 167 post-inoculation, one doe delivered a live fawn and a mummified fetus. The fawn was translocated to an isolation facility to be hand-raised. The fawn was determined to be PI with BVDV 2 by serial virus isolation from serum and white blood cells, immunohistochemistry on skin biopsy, and RT-PCR. This is the first report of persistent infection of white-tailed deer with BVDV. Further research is needed to assess the impact of PI white-tailed deer on BVDV control programs in cattle.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Cervos/virologia , Vírus da Diarreia Viral Bovina/patogenicidade , Transmissão Vertical de Doenças Infecciosas/veterinária , Complicações Infecciosas na Gravidez/veterinária , Animais , Animais Selvagens/virologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/patologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/prevenção & controle , Bovinos , Cervos/imunologia , Vírus da Diarreia Viral Bovina/imunologia , Vírus da Diarreia Viral Bovina/isolamento & purificação , Reservatórios de Doenças/veterinária , Feminino , Sangue Fetal/virologia , Morte Fetal/veterinária , Morte Fetal/virologia , Imuno-Histoquímica/veterinária , Masculino , Testes de Neutralização/veterinária , Gravidez , Complicações Infecciosas na Gravidez/patologia , Complicações Infecciosas na Gravidez/prevenção & controle , Complicações Infecciosas na Gravidez/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Especificidade da EspécieRESUMO
Bovine viral diarrhea virus (BVDV) affects cattle populations causing clinical signs that range from subclinical immunosuppression to severe reproductive and respiratory problems. Detection and removal of persistently infected (PI) calves is the single most important factor for control and eradication of BVDV. Current testing strategies to detect PI calves rely heavily on immunohistochemistry (IHC) and a commercially available antigen capture ELISA (ACE) assay. These viral assays depend on 1 or 2 monoclonal antibodies which target the E(rns) glycoprotein of BVDV. The sensitivity and specificity of these two tests have been reported previously. The purpose of this research was to characterize a strain of BVDV (AU501) that was undetectable using IHC and ACE based on a single monoclonal antibody, but was consistently detected in samples from a Holstein steer using virus isolation and PCR testing. Sequencing of this AU501 viral isolate revealed a unique mutation in the portion of the genome coding for the E(rns) glycoprotein. This unique field strain of BVDV demonstrates the risk of relying on a single monoclonal antibody for detection of BVDV. Multiple testing strategies, including polyclonal or pooled monoclonal antibodies that detect more than one viral glycoprotein may be necessary to detect all PI calves and facilitate eradication of BVDV.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Vírus da Diarreia Viral Bovina/isolamento & purificação , Glicoproteínas de Membrana/análise , Proteínas do Envelope Viral/análise , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Variação Antigênica , Sequência de Bases , Bovinos , Vírus da Diarreia Viral Bovina/genética , Orelha Externa/virologia , Ensaio de Imunoadsorção Enzimática/veterinária , Imuno-Histoquímica/veterinária , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase/veterinária , Alinhamento de Sequência , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologiaRESUMO
In this study, equine source polyclonal anti-Bacillus anthracis immunoglobulins were generated and utilized to demonstrate passive protection of mice in a lethal challenge assay. Four horses were hyper-immunized with B. anthracis Sterne strain for approximately one year. The geometric mean anti-PA titer in the horses at maximal response following immunization was 1:77,936 (Log2 mean titer 16.25, SEM ± 0.25 95% CI [15.5 -17.0]). The geometric mean neutralizing titer at maximal response was 1:128 (Log2 mean titer 7, SEM ± 0.0, 95% CI 7). Treatment with hyper-immune plasma or purified immunoglobulins was successful in passively protecting A/J mice from a lethal B. anthracis Sterne strain challenge. The treatment of mice with hyper-immune plasma at time 0 h and 24 h post-infection had no effect on survival, but did significantly increase mean time to death (p < 0.0001). Mice treated with purified immunoglobulins at time 0 h and 24 h post-infection in showed significant increase in survival rate (p < 0.001). Bacterial loads in lung, liver and spleen tissue were also assessed and were not significantly different in mice treated with hyper-immune plasma from placebo treated control mice. Mice treated with purified antibodies demonstrated mean colony forming units/gram tissue fourfold less than mice receiving placebo treatment (p < 0.0001). Immunotherapeutics harvested from horses immunized against B. anthracis Sterne strain represent a rapidly induced, inexpensive and effective expansion to the arsenal of treatments against anthrax.
RESUMO
OBJECTIVE: To determine whether viral involvement with platelets obtained from cattle persistently infected (PI) with bovine viral diarrhea virus (BVDV) is associated with altered platelet function or decreased platelet counts. SAMPLE POPULATION: Platelets obtained from 8 cattle PI with BVDV and 6 age-, sex-, and breed-matched uninfected control cattle. PROCEDURE: Manual platelet counts were determined, and platelet function was assessed through optical aggregometry by use of the aggregation agonists ADP and platelet-activating factor. Identification of BVDV in serum and preparations of purified platelets was determined by use of virus isolation tests. RESULTS: No significant difference in platelet counts was detected between cattle PI with BVDV and control cattle. In response to the aggregation agonists, maximum aggregation percentage and slope of the aggregation curve were not significantly different between cattle PI with BVDV and control cattle. We isolated BVDV from serum of all PI cattle and from purified platelets of 6 of 8 PI cattle, but BVDV was not isolated from serum or platelets of control cattle. CONCLUSIONS AND CLINICAL RELEVANCE: Isolation of BVDV from platelets in the peripheral circulation of cattle immunotolerant to BVDV does not result in altered platelet function or decreases in platelet counts.
Assuntos
Plaquetas/virologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/sangue , Vírus da Diarreia Viral Bovina Tipo 1/isolamento & purificação , Vírus da Diarreia Viral Bovina Tipo 2/isolamento & purificação , Animais , Contagem de Células Sanguíneas , Bovinos , Vírus da Diarreia Viral Bovina Tipo 1/metabolismo , Vírus da Diarreia Viral Bovina Tipo 2/metabolismo , Feminino , Masculino , Fator de Ativação de Plaquetas/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/fisiologiaRESUMO
The objective of this study was to determine the abundance and distribution of γδ T lymphocytes in lymphoid tissue during acute infection with high (HV) or low virulence (LV) non-cytopathic bovine viral diarrhea virus (BVDV) in beef calves. This study was performed using tissue samples from a previous experiment in which thirty beef calves were randomly assigned to 1 of 3 groups: LV [n=10; animals inoculated intranasally (IN) with LV BVDV-1a (strain SD-1)], HV [n=10; animals inoculated IN with HV BVDV-2 (strain 1373)], and control (n=10; animals inoculated with cell culture medium). On day 5 post inoculation, animals were euthanized, and samples from spleen and mesenteric lymph nodes (MLN) were collected to assess the abundance of WC1(+) γδ T cells. A higher proportion of calves challenged with BVDV showed signs of apoptosis and cytophagy in MLN and spleen samples compared to the control group. A significantly lower number of γδ T cells was observed in spleen and MLN from calves in HV and LV groups than in the control calves (P<0.05). In conclusion, acute infection with HV or LV BVDV resulted in depletion of WC1(+) γδ T cells in mucosal and systemic lymphoid tissues at five days after challenge in beef calves. This reduction in γδ T cells in the studied lymphoid tissues could be also due to lymphocyte trafficking to other tissues.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/imunologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Vírus da Diarreia Viral Bovina Tipo 1/patogenicidade , Vírus da Diarreia Viral Bovina Tipo 2/patogenicidade , Subpopulações de Linfócitos T/imunologia , Doença Aguda , Animais , Antígenos de Superfície/metabolismo , Doença das Mucosas por Vírus da Diarreia Viral Bovina/patologia , Bovinos , Efeito Citopatogênico Viral , Vírus da Diarreia Viral Bovina Tipo 1/imunologia , Vírus da Diarreia Viral Bovina Tipo 2/imunologia , Contagem de Linfócitos , Tecido Linfoide/imunologia , Tecido Linfoide/patologia , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Subpopulações de Linfócitos T/patologia , Virulência/imunologiaRESUMO
Washing procedures (without trypsin treatment) recommended by the International Embryo Transfer Society (IETS) for use on in vivo-derived embryos effectively removed a cytopathic strain (NADL) of bovine viral diarrhea virus (BVDV) after artificial exposure. However, these washing procedures have not been evaluated using other isolates of BVDV, including representative non-cytopathic strains. Thus, the objective of this study was to evaluate the efficacy of the IETS procedures following artificial exposure of in vivo-derived bovine embryos to two different strains and biotypes of BVDV. One hundred and twenty-nine zona pellucida-intact (ZP-I) morulae and blastocysts (MB) and 56 non-fertile and degenerated (NFD) ova were collected 7 days following exposure to bulls from 32, BVDV-negative, superovulated cows. After collection, all MB and NFD ova were washed according to IETS standards. Subsequently, half of the MB and NFD ova were exposed for 1h to approximately 10(6)-cell culture infective doses (50% endpoint) per milliliter of viral strain SD-1, and the other half were exposed to the same concentration of CD-87. After exposure, groups of > or =3 and < or = 10 MB or NFD ova were washed using methods that met or exceeded IETS standards. Then, the washed groups were sonicated, and sonicate fluids were assayed for presence of virus using virus isolation and a reverse transcription nested polymerase chain reaction. No virus was detected in any group of MB or NFD ova that had been exposed to the CD-87 isolate. However, virus was detected in association with 50% of the groups of MB and 33% of the groups of NFD ova that had been exposed to the SD-1 isolate. Therefore, standard embryo-washing procedures recommended by the IETS are more effective for removal of some isolates of BVDV than for others. It remains to be determined if the quantity of a high-affinity isolate of BVDV associated with individual washed embryos would infect recipients via the intrauterine route. Further, it should be determined if an alternative embryo processing procedure, washing and trypsin treatment, would be more effective for removal of high-affinity isolates.
Assuntos
Bovinos/embriologia , Vírus da Diarreia Viral Bovina/fisiologia , Embrião de Mamíferos/virologia , Animais , Vírus da Diarreia Viral Bovina/isolamento & purificação , Feminino , Masculino , Reação em Cadeia da Polimerase , Sonicação , Especificidade da Espécie , Coleta de Tecidos e Órgãos/veterináriaRESUMO
Routine quality controls in production of bovine embryos by in vitro fertilization (IVF) should include screening all materials of animal origin for the presence of bovine viral diarrhea virus (BVDV). Using a reverse transcription nested polymerase chain reaction (RT-nPCR) assay, we detected BVDV in primary cultures of uterine tubal cells (UTC) that had been used during IVF procedures. The goal of our ensuing investigation was to determine its source and assess risks associated with the identified contaminant. Sequencing of the amplified 5' nontranslated region (NTR) of the viral genome confirmed a Genotype I BVDV contaminant. This viral contaminant was also identified by RT-nPCR in multiple samples of the same lot of fetal bovine serum (FBS) that was used in transport media by the laboratory that harvested the UTC. Both routine and enhanced roller bottle methods for virus isolation failed to detect BVDV in the FBS. Furthermore, virus neutralization assays did identify antibodies to Genotype I strains of BVDV in the FBS. After 7 days of co-incubation, neither cultured, washed UTC nor exposed, washed embryos were RT-nPCR positive for BVDV. Eight embryos produced in the contaminated system were nonsurgically transferred into eight seronegative cows. None of the embryo recipients seroconverted to BVDV. Thus, contamination of cell culture medium with BVDV did not result in transmission of the virus when IVF embryos were transferred. Failure to transmit disease was likely aided by serendipitous control from anti-BVDV antibodies in the FBS. However, a diagnostic dilemma was created when the RT-nPCR assays used to screen for BVDV were positive, yet attempts to isolate the virus were negative. This case study illustrates that if molecular assays are to be used to confirm the pathogen-free status of IVF embryo production systems, media components of animal origin (e.g. FBS) should be screened with molecular assays for BVDV as well as traditional virus isolation techniques.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Bovinos/embriologia , Vírus da Diarreia Viral Bovina/isolamento & purificação , Fertilização in vitro/veterinária , Regiões 5' não Traduzidas/química , Regiões 5' não Traduzidas/genética , Animais , Sequência de Bases , Doença das Mucosas por Vírus da Diarreia Viral Bovina/diagnóstico , Doença das Mucosas por Vírus da Diarreia Viral Bovina/transmissão , Células Cultivadas , Técnicas de Cocultura/veterinária , Vírus da Diarreia Viral Bovina/genética , Transferência Embrionária/veterinária , Feminino , Fertilização in vitro/efeitos adversos , Masculino , Dados de Sequência Molecular , Testes de Neutralização/veterinária , Oócitos/virologia , Gravidez , RNA Viral/química , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Alinhamento de Sequência , Soroalbumina Bovina/efeitos adversosRESUMO
OBJECTIVE: To evaluate persistence of bovine viral diarrhea virus (BVDV) in semen after inoculation of postpubertal bulls. ANIMALS: Three 2-year-old bulls and five 6-month-old calves. PROCEDURE: 3 seronegative 2-year-old bulls were inoculated intranasally with BVDV. Serum and semen samples were obtained at regular intervals until 7 months after inoculation. Serum samples were tested for BVDV by use of virus isolation (VI) and reverse transcription-nested polymerase chain reaction (RT-nPCR) tests. Semen samples were tested for virus by use of VI and RT-nPCR tests. Testicular biopsy specimens were obtained 7 months after inoculation and tested for BVDV by use of immunohistochemical analysis and VI and RT-nPCR tests. Semen samples collected from 1 bull immediately before and 5 and 7 months after inoculation were administered IV to seronegative calves, which were monitored for subsequent viremia and seroconversion. RESULTS: Use of VI and RT-nPCR tests detected transient virus in serum of all bulls. The VI test detected BVDV in semen of 2 bulls for < 21 days after inoculation, whereas RT-nPCR assay detected BVDV until 7 months after inoculation. Virus was detected in testicular biopsy specimens of these 2 bulls by use of immunohistochemical analysis and RT-nPCR assay but could only be isolated from the biopsy specimen of 1 bull. Of the calves administered semen IV to detect infectious virus, only the recipient of semen collected 5 months after inoculation of the adult bull was viremic and seroconverted. CONCLUSIONS AND CLINICAL RELEVANCE: Bovine viral diarrhea virus can persist in semen of acutely infected bulls for several months after exposure.
Assuntos
Bovinos/virologia , Vírus da Diarreia Viral Bovina/isolamento & purificação , Vírus da Diarreia Viral Bovina/fisiologia , Sêmen/virologia , Maturidade Sexual , Animais , Sequência de Bases , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Vírus da Diarreia Viral Bovina/genética , Imuno-Histoquímica , Masculino , Dados de Sequência Molecular , Testículo/virologia , Viremia/virologiaRESUMO
Beef cattle from a herd in north Alabama were examined because of an outbreak of nonfatal skin disease characterized by discrete circumscribed areas of inflammation that developed on the skin from the neck to the hips. Areas of inflammation, which tended to be superficial, underwent necrosis and scabbed over. The scabs eventually dropped off leaving discrete, round, whitish, hairless lesions that were 1.2 to 2.5 cm diameter. Because clinical signs were consistent with those expected with pseudo-lumpy skin disease (PLSD) caused by bovine herpesvirus type 2 (BHV-2), samples from 16 representative animals were submitted for BHV-2 testing. All 16 animals were seropositive for BHV-2, but the virus could not be isolated from skin biopsy specimens or buffy coat samples. Results of a polymerase chain reaction assay incorporating primers designed to amplify 2 DNA sequences from BHV-2 were positive for 3 of the 10 cattle, suggesting that skin lesions in these cattle were a result of PLSD. Our findings suggest that PLSD may be more common and widespread in the United States than suggested by the frequency with which BHV-2 has been isolated from cattle with PLSD-like skin lesions.
Assuntos
Doenças dos Bovinos/diagnóstico , DNA Viral/análise , Herpes Simples/veterinária , Herpesvirus Bovino 2/isolamento & purificação , Reação em Cadeia da Polimerase/veterinária , Alabama/epidemiologia , Animais , Sequência de Bases , Bovinos , Doenças dos Bovinos/epidemiologia , Surtos de Doenças/veterinária , Feminino , Herpes Simples/diagnóstico , Herpes Simples/epidemiologia , Herpesvirus Bovino 2/genética , Doença Nodular Cutânea/diagnóstico , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Pele/patologiaRESUMO
The complex and unique nature of bovine viral diarrhea virus(BVDV) continues to present challenges to infectious disease re-searchers, veterinarians, and the cattle industry. In addition, the BVDV pathogen will undoubtedly continue to change and present itself in many different configurations.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Vírus da Diarreia Viral Bovina/patogenicidade , Animais , BovinosRESUMO
Several key factors influence the success of bovine viral diarrhea virus (BVDV) prevention and control measures. Current knowledge and an understanding of the problem and the impact of BVDV-associated disease by the livestock industry will facilitate the organization and tremendous effort required to control BVDV successfully.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/prevenção & controle , Vírus da Diarreia Viral Bovina/imunologia , Vacinas Virais/imunologia , Animais , Doença das Mucosas por Vírus da Diarreia Viral Bovina/diagnóstico , Doença das Mucosas por Vírus da Diarreia Viral Bovina/economia , Bovinos , Análise Custo-Benefício , Diagnóstico Diferencial , Resultado do TratamentoRESUMO
The objective was to compare the mRNA expression of pro-inflammatory (TNF-α, IL-1ß, IFN-γ, IL-2, IL-12, IL-15) and anti-inflammatory (IL-4, IL-10, TGF-ß) cytokines, after experimental infection with low or high virulence noncytopathic (ncp) bovine viral diarrhea virus (BVDV). Thirty BVDV-naïve, beef calves were intranasally inoculated with low (LV; n=10, SD-1) or high (HV; n=10, 1373) virulence ncp BVDV or with BVDV-free cell culture medium (Control, n=10). Calves were euthanized on day 5 post-inoculation, and tracheo-bronchial lymph node and spleen samples were collected for mRNA expression through quantitative-RT-PCR. mRNA levels of pro-inflammatory (TNF-α, IL-1ß, IL-2, IFN-γ) and anti-inflammatory (IL-4 and IL-10) cytokines were up-regulated in tracheo-bronchial lymph nodes of HV, but not in LV, compared to the control group (P<0.05). IL-12 mRNA level was up-regulated in tracheo-bronchial lymph nodes of both LV and HV groups (P ≤ 0.05). A significant up-regulation of IL-15 mRNA was observed in tracheo-bronchial lymph nodes for LV calves (P<0.002), but not for HV calves. Experimental inoculation with BVDV-2 1373 stimulated significant mRNA expression of pro-inflammatory and anti-inflammatory cytokines. In contrast, inoculation with BVDV-1a SD-1 only resulted in up-regulation of IL-12 and IL-15 mRNA, which is associated with activation of macrophages and NK cells during innate immune response.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/imunologia , Citocinas/genética , Animais , Bovinos , Interleucina-1beta/genética , Interleucina-2/genética , RNA Mensageiro/análise , Fator de Necrose Tumoral alfa/genéticaRESUMO
OBJECTIVE: To determine the effects of constant exposure to cattle persistently infected (PI) with bovine viral diarrhea virus (BVDV) on health and performance of feedlot cattle. DESIGN: 3 controlled trials. ANIMALS: Crossbred feedlot cattle (trial 1, n = 184; trial 2, 138; trial 3, 138). PROCEDURES: Weaned calves were or were not vaccinated against BVDV at feedlot arrival (trial 1) or 2 (trial 2) or 3 (trial 3) weeks before feedlot arrival. During trial 1, half of the calves were commingled with PI cattle throughout the feeding period. During trial 2, 63 calves were exposed to PI cattle before weaning and all calves were exposed to PI cattle throughout the feeding period. During trial 3, all study calves were exposed to PI cattle throughout the feeding period. Morbidity and mortality rates and average daily gain (ADG) data were analyzed. RESULTS: During trial 1, calves maintained with PI cattle had a higher morbidity rate regardless of BVDV vaccination than did calves not exposed to PI cattle; however, for calves maintained with PI cattle, the morbidity rate for those vaccinated against BVDV was less than that for those not vaccinated against BVDV. During trial 2, calves exposed to PI cattle before weaning or vaccinated against BVDV had lower morbidity and mortality rates and increased ADG, compared with those for calves not exposed to PI cattle before weaning or vaccinated against BVDV. During trial 3, health and performance did not vary between calves that were and were not vaccinated against BVDV. CONCLUSIONS AND CLINICAL RELEVANCE: Exposure of cattle to BVDV naturally or through vaccination before or at feedlot arrival mitigated the negative effects of constant exposure to PI cattle.