Resistance of colostrum-deprived domestic lambs to infection with deer adenovirus.

Seven colostrum-deprived, 3-4-wk-old Rambouillet-Hampshire lambs were inoculated via the mucous membranes with deer adenovirus (DAdV) and monitored for clinical signs for 21 d post-inoculation at which time animals were euthanized and postmortem examinations were performed. Pre-inoculation and post-inoculation serum samples were tested for antibodies to DAdV, ovine adenovirus 7, bovine adenovirus 7, and goat adenovirus 1. Evidence for DAdV infection was determined by virus isolation, PCR tests, and histopathology with immunohistochemistry tests for DAdV. No clinical signs or lesions consistent with adenoviral hemorrhagic disease (AHD) in deer were seen in the lambs, and the lambs did not seroconvert to DAdV. DAdV was not detected by PCR, virus isolation, or immunohistochemistry in any of the samples tested from the lambs. A positive control deer similarly inoculated with DAdV developed fatal AHD 1 wk post-inoculation. Our colostrum-deprived lambs did not become infected when inoculated with DAdV.

Differential expression of cytokine transcripts in neonatal and adult ovine alveolar macrophages in response to respiratory syncytial virus or toll-like receptor ligation.

Alveolar macrophages (AMvarphis) secrete regulatory molecules that are believed to be critical in maintaining normal lung homeostasis. However, in response to activating signals, AMvarphis have been shown to become highly phagocytic cells capable of secreting significant levels of pro-inflammatory cytokines. There is evidence to suggest that susceptibility of Mvarphi subpopulations to viral infection, and their subsequent cytokine/chemokine response, is dependent on age of the host. In the present study, we compared bovine respiratory syncytial virus (BRSV) replication and induction of cytokine responses in neonatal ovine AMvarphis to those cells isolated from adult animals. While neonatal AMvarphis could be infected with BRSV, viral replication was limited as previously shown for AMvarphis from mature animals. Interestingly, following BRSV infection, peak mRNA levels of IL-1beta and IL-8 in neonatal AMvarphi were several fold higher than levels induced in adult AMvarphis. In addition, peak mRNA expression for the cytokines examined occurred at earlier time points in neonatal AMvarphis compared to adult AMvarphis. However, the data indicated that viral replication was not required for the induction of specific cytokines in either neonatal or adult AMvarphis. TLR3 and TLR4 agonists induced significantly higher levels of cytokine transcripts than BRSV in both neonatal and adult AMvarphis. It was recently proposed that immaturity of the neonatal immune system extends from production of pro-inflammatory cytokines to regulation of such responses. Differential regulation of cytokines in neonatal AMvarphis compared to adult AMvarphis in response to RSV could be a contributory factor to more severe clinical episodes seen in neonates.

Minimum intravenous infectious dose of ovine progressive pneumonia virus (OPPV).

The minimum intravenous infectious dose for ovine progressive pneumonia virus (OPPV) WLC1 was determined using twenty-four 6month-old lambs. Twelve groups of two 6month-old lambs were inoculated intravenously (i.v.) with tissue culture fluid containing ovine progressive pneumonia virus (OPPV) WLC1 titers ranging from 10(7.6) TCID(50)/lamb down to 10(-3.4) TCID(50)/lamb and were monitored for seroconversion using the OPPV agar gel immunodiffusion assay (AGID). Fifteen of the 16 lambs given equal or greater than 10(0.6) TCID(50) seroconverted, and virus could be isolated from peripheral blood leukocytes in 13 out of the 15 of these lambs. None of the eight lambs receiving less than 10(0.6) TCID(50) seroconverted during the 12months. The results of this study indicated that 10(0.6) or 4 TCID(50)/lamb given i.v. was capable of establishing infection.

Serologic and hexon phylogenetic analysis of ruminant adenoviruses.

The objectives of this study were to determine the antigenic relationship among ruminant adenoviruses and determine their phylogenetic relationship based on the deduced hexon gene amino acid sequence. Results of reciprocal cross-neutralization tests demonstrated antigenic relationships in either one or both directions among bovine adenovirus type 6 (BAdV-6), BAdV-7, ovine adenovirus type 7 (OAdV-7), caprine adenovirus type 1 (GAdV-1), and deer adenovirus (Odocoileus adenovirus 1, OdAdV-1). No antigenic cross-reactivity was observed among BAdV-1 through -5, and -8 and the other putative ruminant adenoviruses. Two PCR primer sets, one for mastadenovirus and atadenovirus that amplified an approximately 2,700-bp region in the hexon genes were used for comparative studies. Phylogenetic analysis of the deduced hexon amino acid sequences clustered the ruminant adenoviruses on the Mastadenovirus and Atadenovirus genus branches of the Adenoviridae tree. The recent classification of BAdV-6, and -7 as members of the genus Atadenovirus was supported by phylogenetic distance matrix analysis of their deduced hexon amino acid sequences. Further, we propose that BAdV-6 and -7 be recognized as members of new Atadenovirus species, Bovine adenovirus E and Bovine adenovirus F, respectively. Phylogenetic analysis of OdAdV-1 places this virus in the genus Atadenovirus with a proposed new species Odocoileus adenovirus A. OAdV-6 and GAdV-2 are proposed as members of new Mastadenovirus species Ovine adenovirus C and Goat adenovirus A, respectively.

Evaluation of the pathogenic potential of cervid adenovirus in calves.

Four 3-month-old Jersey calves and three 3-month-old Holstein calves were inoculated with cervid adenovirus and monitored for clinical signs until necropsied between 10 and 42 days postinoculation. The neonatal Jersey calves had received colostrum, and the Holstein calves were colostrum deprived. Preinoculation and postinoculation serum samples were tested for antibodies to the cervid adenovirus, bovine adenovirus type 6, bovine adenovirus type 7, and goat adenovirus type 1. Virus isolation was performed on kidney, nasal secretion, and/or lung homogenates in fetal white-tailed deer lung cells. Negatively stained preparations of feces from Jersey calves were examined weekly using an electron microscope, and weekly blood samples were collected for complete blood counts. Full necropsies were performed on all calves. A complete selection of tissues was evaluated for microscopic changes, and immunohistochemistry was performed on all tissues using a polyclonal antibody to deer adenovirus. No clinical signs were observed in the calves during the study period. Following inoculation, colostrum-deprived calves developed low antibody titers to deer adenovirus, while the Jersey calves that received colostrum did not. Calves that received colostrum had high antibody titers to bovine adenovirus type 7 and goat adenovirus type 1. No consistent gross or microscopic lesions were seen. Adenovirus was not observed in negatively stained preparations of feces. Immunohistochemistry results did not demonstrate virus in all tissues examined microscopically, and virus was not isolated from lungs, nasal secretions, and kidneys.

Neonatal ovine pulmonary dendritic cells support bovine respiratory syncytial virus replication with enhanced interleukin (IL)-4 And IL-10 gene transcripts.

The lung microenvironment is constantly exposed to microorganisms and particulate matter. Lung dendritic cells (DCs) play a crucial role in the uptake and processing of antigens found within the respiratory tract. Respiratory syncytial virus (RSV) is a common respiratory tract pathogen in children that induces an influx of DCs to the mucosal surfaces of the lung. Using a neonatal lamb model, we examined the in vivo permissiveness of DCs to RSV infection, as well as overall cell surface changes and cytokine responses of isolated lung DCs after bovine RSV (BRSV) infection. We report that isolated lung DCs and alveolar macrophages support BRSV replication. Isolated lung DCs were determined to be susceptible to BRSV infection as demonstrated by quantification of BRSV non-structural protein 2 mRNA. BRSV infection induced an initial upregulation of CD14 expression on lung DCs, but by 5 d postinfection expression was similar to that on control cells. No significant changes in CD80/86 or MHC class I expression were seen on lung DCs after BRSV infection. Low to moderate expression of MHC class II and DEC-205 was detected by day 5 postinfection. Initially, on day 3 postinfection, lung DCs from BRSV-infected lambs had decreased endocytosis of fluorescein isothiocyanate (FITC)-ovalbumin (OVA). The amount of FITC-OVA endocytosed by lung DCs isolated on day 5 postinfection was similar to that of controls. The most interesting observation was the induction of immunomodulatory interleukin (IL)-4 and IL-10 cytokine gene transcription in lung DCs and alveolar macrophages after in vivo infection with BRSV. Overall, these findings are the first to demonstrate that neonatal lung DCs support in vivo BRSV replication and produce type II cytokines after viral infection.

Pretreatment with recombinant human vascular endothelial growth factor reduces virus replication and inflammation in a perinatal lamb model of respiratory syncytial virus infection.

Vascular endothelial growth factor (VEGF) is increasingly recognized as a perinatal regulator of lung maturation and surfactant protein expression. Preterm and young infants are at increased risk for pulmonary immaturity characterized by insufficient surfactant production as well as increased risk for severe manifestations of respiratory syncytial virus (RSV) infection. Innate immune components including surfactant proteins A and D, and beta-defensins have putative antimicrobial activity against pulmonary pathogens including RSV. Our hypothesis was that recombinant human VEGF (rhVEGF) pretreatment therapy would decrease RSV disease in the perinatal lamb RSV model. Newborn lambs were pretreated with rhVEGF, betamethasone, or saline and then inoculated with bovine RSV or sterile medium. Tissues were collected 5 d postinoculation, corresponding to the initiation of severe lesions and peak viral replication. In RSV-infected lambs, rhVEGF therapy increased the mean daily body temperature, decreased airway neutrophil exudate, and reduced RSV replication compared with betamethasone or saline pretreatment. Furthermore, rhVEGF therapy significantly mitigated the RSV-induced increase in surfactant protein A mRNA expression and decrease in surfactant protein D mRNA expression. In control (non-RSV-infected) lambs, pretreatment with rhVEGF increased sheep beta-defensin-1 (SBD1) mRNA expression, but no alteration in surfactant proteins A and D was detected. This novel study demonstrates that rhVEGF pretreatment mitigates RSV disease and, in addition, rhVEGF regulation of innate immune genes is dependent on RSV infection status.

Differential expression of ovine innate immune genes by preterm and neonatal lung epithelia infected with respiratory syncytial virus.

Preterm infants have increased susceptibility to severe manifestations of respiratory syncytial virus (RSV) infection. The cause(s) for this age-dependent vulnerability is/are not well-defined, but alterations in innate immune products have been implicated. In sheep, RSV disease severity has similar age-dependent characteristics and sheep have several related innate molecules for study during pulmonary infection including surfactant protein A (SP-A), surfactant protein D (SP-D), sheep beta defensin 1 (SBD1), monocyte chemotactic protein 1 (MCP1), and Toll-like receptor 4 (TLR4). However, the in vivo cellular gene expression as a response to RSV infection is poorly understood. In this study, the effect of RSV infection on expression of these innate immune genes was determined for bovine RSV-infected (bRSV+ fluorescence) epithelial cells, adjacent cells lacking bRSV antigen (adjoining cells lacking fluorescence), and control cells from non-infected lung using laser capture microdissection (LCM) and real-time RT-PCR. Control lambs had increased expression of innate immune molecules in full term (term) compared to preterm epithelia with statistical significance in SBD1, SP-D, and TLR4 mRNA. Infected cells (bRSV+ fluorescent cells) had consistently higher mRNA levels of SP-A (preterm and term), MCP1 (preterm and term), and SP-D (preterm). Interestingly, bRSV- cells of infected term lambs had significantly reduced SP-D mRNA expression compared to bRSV+ and control epithelia, suggesting that RSV infected cells may regulate the adjacent epithelial SP-D expression. This study defines specific innate immune components (e.g., SBD1, SP-D, and TLR4) that have differential age-dependent expression in the airway epithelia. Furthermore, cellular bRSV infection enhanced certain innate immune components while suppressing adjacent cellular SP-D expression in term animals. These in vivo gene expression results provide a framework for future studies on age-dependent susceptibility to RSV and RSV pathogenesis.

Pulmonary dendritic cells isolated from neonatal and adult ovine lung tissue.

Lung dendritic cells (DCs) are potent antigen presenting cells (APCs) that initiate and modulate the adaptive immune response upon microbial infection within the pulmonary environment. For the first time, neonatal and adult lung DCs in a large animal model were compared in these studies. Here, we isolated and identified lung DCs in both neonatal and adult sheep, a valuable experimental animal utilized in pulmonary studies of naturally occurring respiratory diseases. Neonatal lung DCs exhibited characteristic dendrites and morphology when observed by transmission electron microscopy and expressed low to moderate DEC-205, CD80/86, MHC class II and CD 14. Regardless of age, lung DCs were functionally able to endocytose FITC conjugated ovalbumin but to a lesser degree than monocyte-derived DCs. In addition, neonatal lung DCs were demonstrated to be potent stimulators of allogeneic T cell proliferation. Together, these results demonstrate that neonatal and adult lung DCs are functionally similar. It is apparent from the data presented that neonatal pulmonary DCs do not exhibit an intrinsic functional defect that would impair their ability to take up antigen and stimulate naïve T cells. These data support growing evidence that neonatal immune responses may differ from adults due to different microenvironmental influences rather than differences in dendritic cell maturation states.

Reduced clearance of respiratory syncytial virus infection in a preterm lamb model.

Respiratory syncytial virus (RSV) causes significant respiratory disease in children worldwide. For the study of severe RSV disease seen in preterm infants, a suitable animal model is lacking. The novel hypothesis of this study was that preterm lambs are susceptible to bovine RSV (bRSV) infection, an analogous pneumovirus with ruminant host specificity, and that there would be age-dependent differences in select RSV disease parameters. During RSV infection, preterm lambs had elevated temperatures and respiration rates with mild anorexia and cough compared to controls. Gross lesions included multifocal consolidation and atelectasis with foci of hyperinflation. Microscopic lesions included multifocal alveolar septal thickening and bronchiolitis. Immunohistochemistry localized the RSV antigen to all layers of bronchiolar epithelium from a few basal cells to numerous sloughing epithelia. A few mononuclear cells were also immunoreactive. To assess for age-dependent differences in RSV infection, neonatal lambs were infected similarly to the preterm lambs or with a high-titer viral inoculum. Using morphometry at day 7 of infection, preterm lambs had significantly more cellular immunoreactivity for RSV antigen (P <0.05) and syncytial cell formation (P <0.05) than either group of neonatal lambs. This work suggests that perinatal RSV clearance is age-dependent, which may explain the severity of RSV infection in preterm infants. The preterm lamb model is useful for assessing age-dependent mechanisms of severe RSV infection.

Isolation of an adenovirus and an adeno-associated virus from goat kids with enteritis.

A dairy goat operation in Minnesota experienced a sudden, markedly increased mortality among its neonatal goats. Approximately 60 of 130 kids (46%) died. The animals had diarrhea and dyspnea of 1-2 days duration before death. Necropsy of 4 goat kids revealed marked, acute, catarrhal enteritis and fibrinous pleuropneumonia. Mannheimia haemolytica was isolated from the lungs. Basophilic inclusion bodies filling the entire nucleus were present in enterocytes of the ileum of 3 goats. Adenoviral particles were detected in the feces by electron microscopy and adenovirus was subsequently isolated from the intestinal content together with a parvo-like virus (dependovirus). Morphology, physicochemical characteristics, and neutralization tests indicated that the adenovirus resembled ovine adenovirus-2 (OAdV-2). However, the PstI restriction endonuclease pattern produced by the goat adenovirus was distinct from that of OAdV-2. This is the first report of enteritis in goats with an adenovirus antigenically related to OAdV-2 and with a parvo-like dependovirus.

Adenovirus-mediated gene therapy enhances parainfluenza virus 3 infection in neonatal lambs.

Parainfluenza viruses are a common cause of seasonal respiratory disease, but in high-risk individuals (e.g., young children) these viruses can cause severe clinical manifestations that require hospitalization. Beta-defensins are a subclass of antimicrobial peptides with antiviral activity. Use of adenovirus-mediated beta-defensin gene expression has been proposed as therapy for chronic bacterial infections commonly seen in cystic fibrosis patients; however, its use during parainfluenza virus 3 (PIV3) infection has not been evaluated. The hypothesis in this experiment was that adenovirus expression of human beta-defensin 6 (HBD6) would diminish concurrent PIV3 infection in neonatal lambs. The group infected with adenovirus HBD6 and PIV3 had increased levels of pulmonary neutrophil recruitment compared to those for the group infected with PIV3 or PIV3 and adenovirus, with an increased respiration rate and body temperature late in the course of the PIV3-adenovirus HBD6 infection. Interestingly, the adenovirus-treated groups had higher levels of immunohistochemical staining for PIV3 and syncytial cell formation than the group infected with PIV3, suggesting that treatment with the adenovirus vector, regardless of whether it was carrying a target gene, exacerbated the PIV3 infection. The levels of expression of mRNA for antimicrobial surfactant proteins A and D and sheep beta-defensin 1 were increased by PIV3 and adenovirus treatment, and the increased levels of expression roughly corresponded to the degree of inflammation. While pulmonary administration of a high-dose adenovirus vector has been associated with undesirable inflammation, this is the first study to show that it can exacerbate concurrent viral infection, a concern that needs to be addressed for future studies of adenovirus in the lung. Additionally, this study showed that adenovirus-mediated HBD6 expression increases neutrophil recruitment, a recently described attribute of beta-defensins, with mild accentuation of PIV3 activity and inflammation.

Enhanced surfactant protein and defensin mRNA levels and reduced viral replication during parainfluenza virus type 3 pneumonia in neonatal lambs.

Defensins and surfactant protein A (SP-A) and SP-D are antimicrobial components of the pulmonary innate immune system. The purpose of this study was to determine the extent to which parainfluenza type 3 virus infection in neonatal lambs alters expression of sheep beta-defensin 1 (SBD-1), SP-A, and SP-D, all of which are constitutively transcribed by respiratory epithelia. Parainfluenza type 3 viral antigen was detected by immunohistochemistry (IHC) in the bronchioles of all infected lambs 3 days postinoculation and at diminished levels 6 days postinoculation, but it was absent 17 days postinoculation. At all times postinoculation, lung homogenates from parainfluenza type 3 virus-inoculated animals had increased SBD-1, SP-A, and SP-D mRNA levels as detected by fluorogenic real-time reverse transcriptase PCR. Protein levels of SP-A in lung homogenates detected by quantitative-competitive enzyme-linked immunosorbent assay and protein antigen of SP-A detected by IHC were not altered. These studies demonstrate that parainfluenza type 3 virus infection results in enhanced expression of constitutively transcribed innate immune factors expressed by respiratory epithelia and that this increased expression occurs concurrently with decreased viral replication.

Adenoviral infection in captive moose (Alces alces) in Canada.

Adenoviral infection was associated with hemorrhagic enteritis, serosal hemorrhages, and severe pulmonary edema in six captive moose (Alces alces) in Toronto, Ontario, Canada: an adult female moose and three calves in 1985 and two calves in 1998. Adenoviral disease was suspected based on histological findings of systemic vasculitis and widespread thrombosis associated with amphophilic intranuclear inclusions in endothelial cells. Diagnosis was confirmed by immunohistochemistry using antiserum to bovine adenovirus type 5, transmission electron microscopic identification of viral particles consistent in morphology with adenovirus within nuclei of pulmonary endothelial cells in an affected calf, and virus isolation. The restriction pattern of virus isolated from the lung of one of the calves indicated that the virus was identical to a recently characterized adenovirus in black-tailed deer (Odocoileus hemionus) in California. The moose adenovirus reported here may have been endemic in the captive moose herd, or infection may have resulted from either direct or indirect contact with other species of captive or wild cervids. This is the first report of adenoviral infection in moose and of the presence of adenoviral disease in a cervid in Canada.

Bovine adenovirus serotype 7 infections in postweaning calves.

OBJECTIVE: To detect bovine adenovirus serotype 7 (BAV-7) infections in calves by use of viral isolation and serologic testing. ANIMALS: 205 postweaning calves. PROCEDURE: 121 calves were assembled by an order buyer through auction markets in eastern Tennessee and transported to New Mexico where they were commingled with 84 healthy ranch-reared calves. Tests included viral isolation in cell culture from peripheral blood leukocytes (PBL) and detection of serum BAV-7 antibodies by use of microtitration viral neutralization. RESULTS: BAV-7 was isolated from PBL of 8 calves and seroconversion to BAV-7 was detected for 38 of 199 (19.1%) calves. Concurrent bovine viral diarrhea virus infections were detected in most calves from which BAV-7 was isolated. CONCLUSIONS AND CLINICAL RELEVANCE: Results of our study indicate that BAV-7 infections can be found in postweaning commingled calves and may develop more commonly in calves with concurrent infections with viruses such as bovine viral diarrhea virus (BVDV).