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Parainfluenza virus 5 (PIV5), formerly known as simian virus 5 (SV5), is a paramyxovirus often referred to as canine parainfluenza virus (CPI) in the veterinary field. PIV5 is thought to be a contributing factor to kennel cough. Kennel cough vaccines containing live PIV5 have been used in dogs for many decades. PIV5 is not known to cause any diseases in humans or other animals. PIV5 has been used as a vector for vaccine development for humans and animals. One critical question concerning the use of PIV5 as a vector is whether prior exposure to PIV5 would prevent the use of PIV5-based vaccines. In this work, we have examined immunogenicity of a recombinant PIV5 expressing hemagglutinin (HA) of influenza A virus subtype 3 (rPIV5-H3) in dogs that were immunized against PIV5. We found that vaccination of the dogs containing neutralizing antibodies against PIV5 with rPIV5-H3 generated immunity against influenza A virus, indicting that PIV5-based vaccine is immunogenic in dogs with prior exposure. Furthermore, we have examined exposure of PIV5 in human populations. We have detected neutralizing antibody (nAb) against PIV5 in 13 out of 45 human serum samples (about 29 percent). The nAb titers in humans were lower than that in vaccinated dogs, suggesting that nAb in humans is unlikely to prevent PIV5 from being an efficacious vector in humans.

The experimental infection of dogs with TriGAS induced high levels of VNA in the serum, whereas wt RABV infection did not. Dogs infected with TriGAS developed antibodies against the virus including its glycoprotein, whereas dogs infected with DRV-NG11 only developed rabies antibodies that are presumably specific for the nucleoprotein, (N) and not the glycoprotein (G). We show that infection with TriGAS induces early activation of B cells in the draining lymph nodes and persistent activation of DCs and B cells in the blood. On the other hand, infection with DRV-NG11 fails to induce the activation of DCs and B cells and further reduces CD4 T cell production. Further, we show that intrathecal (IT) immunization of TriGAS not only induced high levels of VNA in the serum but also in the CSF while intramuscular (IM) immunization of TriGAS induced VNA only in the serum. In addition, high levels of total protein and WBC were detected in the CSF of IT immunized dogs, indicating the transient enhancement of blood-brain barrier (BBB) permeability, which is relevant to the passage of immune effectors from periphery into the CNS. IM infection of dogs with TriGAS induced the production of serum VNA whereas, IT immunization of TriGAS in dogs induces high levels of VNA in the periphery as well as in the CSF and transiently enhances BBB permeability. In contrast, infection with wt DRV-NG11 resulted in the production of RABV-reactive antibodies but VNA and antibodies specific for G were absent. As a consequence, all of the dogs infected with wt DRV-NG11 succumbed to rabies. Thus the failure to activate protective immunity is one of the important features of RABV pathogenesis in dogs.

Burkholderia mallei is a host-adapted bacterium that does not persist outside of its equine reservoir. The organism causes the zoonosis glanders, which is endemic in Asia, Africa, the Middle East and South America. Infection by B. mallei typically occurs via the respiratory or percutaneous route, and the most common manifestations are life-threatening pneumonia and bacteremia. Glanders is difficult to diagnose and requires prolonged antibiotic therapy with low success rates. There is no vaccine to protect against B. mallei and there is concern regarding its use as a biothreat agent. Thus, experiments were performed to establish a non-human primate model of intranasal infection to study the organism and develop countermeasures. Groups of marmosets (Callithrix jacchus) were inoculated intranasally with B. mallei strain ATCC 23344 and monitored for clinical signs of illness for up to 13 days. We discovered that 83% of marmosets inoculated with doses of 2.5 X 10(4) to 2.5 X 10(5) bacteria developed acute lethal infection within 3-4 days. Signs of disease were severe and included lethargy, inappetence, conjunctivitis, mucopurulent and hemorrhagic nasal discharges, and increased respiratory effort with abdominal lifts. Burkholderia mallei was cultured from the lungs, spleen and liver of these animals, and pathologic examination of tissues revealed lesions characteristic of glanders. Challenge experiments also revealed that 91% of animals infected with doses ranging from 25 to 2.5 X 10(3) bacteria exhibited mild non-specific signs of illness and were culture negative. One marmoset inoculated with 2.5 X 10(3) organisms developed moderate signs of disease and reached humane end-points 8 days post-infection. The liver and spleen of this animal were colonized with the agent and pathological analysis of tissues showed nasal, splenic and hepatic lesions. Taken together, these data indicate that the marmoset is a suitable model to study respiratory infection by B. mallei.

Rabies is traditionally considered a uniformly fatal disease after onset of clinical manifestations. However, increasing evidence indicates that non-lethal infection as well as recovery from flaccid paralysis and encephalitis occurs in laboratory animals as well as humans. Non-lethal rabies infection in dogs experimentally infected with wild type dog rabies virus (RABV, wt DRV-Mexico) correlates with the presence of high level of virus neutralizing antibodies (VNA) in the cerebral spinal fluid (CSF) and mild immune cell accumulation in the central nervous system (CNS). By contrast, dogs that succumbed to rabies showed only little or no VNA in the serum or in the CSF and severe inflammation in the CNS. Dogs vaccinated with a rabies vaccine showed no clinical signs of rabies and survived challenge with a lethal dose of wild-type DRV. VNA was detected in the serum, but not in the CSF of immunized dogs. Thus the presence of VNA is critical for inhibiting virus spread within the CNS and eventually clearing the virus from the CNS. Non-lethal infection with wt RABV correlates with the presence of VNA in the CNS. Therefore production of VNA within the CNS or invasion of VNA from the periphery into the CNS via compromised blood-brain barrier is important for clearing the virus infection from CNS, thereby preventing an otherwise lethal rabies virus infection.

The objective of this discovery-level investigation was to use mass spectrometry to identify low mass compounds in bronchoalveolar lavage fluid from lung transplant recipients that associate with bronchiolitis obliterans syndrome. Bronchoalveolar lavage fluid samples from lung transplant recipients were evaluated for small molecules using ESI-TOF mass spectrometry and correlated to the development of bronchiolitis obliterans syndrome. Peptides associated with samples from persons with bronchiolitis obliterans syndrome and controls were identified separately by MS/MS analysis. The average bronchoalveolar lavage fluid MS spectrum profile of individuals that developed bronchiolitis obliterans syndrome differed greatly compared to controls. Controls demonstrated close inter-sample correlation (R = 0.97+/-0.02, average+/-SD) while bronchiolitis obliterans syndrome showed greater heterogeneity (R = 0.86+/-0.09, average+/-SD). We identified 89 features that were predictive of developing BOS grade 1 and 66 features predictive of developing BOS grade 2 or higher. Fractions from MS analysis were pooled and evaluated for peptide content. Nearly 10-fold more peptides were found in bronchiolitis obliterans syndrome relative to controls. C-terminal residues suggested trypsin-like specificity among controls compared to elastase-type enzymes among those with bronchiolitis obliterans syndrome. Bronchoalveolar lavage fluid from individuals with bronchiolitis obliterans syndrome has an increase in low mass components detected by mass spectrometry. Many of these features were peptides that likely result from elevated neutrophil elastase activity.

Abdominal lavage is used in laboratory rodents for a variety of applications but carries an inherent risk of abdominal organ laceration; therefore, personnel carrying out this procedure must have considerable expertise. In this paper, the authors describe an improved method for delivering sterile media to and collecting peritoneal fluids from dark-clawed Mongolian gerbils ( Meriones unguiculatus ) that had been peritoneally infected with filarial nematode parasites (genus Brugia ). To carry out this gravity-assisted technique, the authors used a catheter to introduce sterile media into the peritoneal cavity of each gerbil and then to passively drain peritoneal fluid and larval worms for collection. Average fluid recovery was consistently greater when using this gravity-assisted method than when using aspiration. Larval parasites were recovered by both methods. To recover large volumes of fluid using the standard method of abdominal lavage, personnel typically must euthanize rodents. This gravity-assisted technique allows researchers to collect large numbers of parasite larvae without euthanizing gerbils.

Transtracheal access enables researchers to obtain diagnostic samples from or infuse materials into the lower respiratory tract. This column describes transtracheal aspiration and infusion of horses.Transtracheal access enables researchers to obtain diagnostic samples from or infuse materials into the lower respiratory tract. This column describes transtracheal aspiration and infusion of horses.

Confirmed reports of large domesticated cats becoming infected with highly pathogenic avian influenza (HPAI) H5N1 virus have raised questions about both the risk of infection for these animals, and their potential as vector or reservoir hosts in an influenza pandemic. With this in mind, we examined the immunogenicity of the hemagglutinin (HA) of H5N1 strain A/Vietnam/1203/04 using several different vaccination strategies. Data from ELISA assays showed that vaccination with a single dose of recombinant H5 HA protein induces a robust antibody response against both whole inactivated virus and recombinant HA antigen. Moreover, a single dose of the recombinant H5 HA protein induced hemagglutination inhibition titers ≥40, which is indicative of protective immunization. Cats receiving the IND H5N1 vaccine required two doses before similar H5 HA-specific antibody titers were observed, and despite boosting, these animals had HIA titers that were lower than or equivalent to those in the group receiving one injection of recombinant protein. In contrast, cats vaccinated with plasmid DNA encoding HA failed to develop HA-specific antibody responses above those seen in cohorts receiving an unrelated control plasmid. The results of this study indicate that recombinant H5 HA protein-based vaccines can rapidly induce high serum antibody titers, and may be more effective than either inactivated influenza virus or DNA vaccines in cats.