Shannon J. Hostetter

Effects of formalin-inactivated respiratory syncytial virus (FI-RSV) in the perinatal lamb model of RSV.

Respiratory syncytial virus (RSV) is the most frequent cause of bronchiolitis in infants and children worldwide. There are currently no licensed vaccines or effective antivirals. The lack of a vaccine is partly due to increased caution following the aftermath of a failed clinical trial of a formalin-inactivated RSV vaccine (FI-RSV) conducted in the 1960’s that led to enhanced disease, necessitating hospitalization of 80% of vaccine recipients and resulting in two fatalities. Perinatal lamb lungs are similar in size, structure and physiology to those of human infants and are susceptible to human strains of RSV that induce similar lesions as those observed in infected human infants. We sought to determine if perinatal lambs immunized with FI-RSV would develop key features of vaccine-enhanced disease. This was tested in colostrum-deprived lambs immunized at 3–5 days of age with FI-RSV followed two weeks later by RSV infection. The FI-RSV-vaccinated lambs exhibited several key features of RSV vaccine-enhanced disease, including reduced RSV titers in bronchoalveolar lavage fluid and lung, and increased infiltration of peribronchiolar and perivascular lymphocytes compared to lambs either undergoing an acute RSV infection or naïve controls; all features of RSV vaccine-enhanced disease. These results represent a first step proof-of-principle demonstration that the lamb can develop altered responses to RSV following FI-RSV vaccination. The lamb model may be useful for future mechanistic studies as well as the assessment of RSV vaccines designed for infants.

Increased concentration of iodide in airway secretions is associated with reduced respiratory syncytial virus disease severity.

Recent studies have revealed that the human and nonrodent mammalian airway mucosa contains an oxidative host defense system. This three-component system consists of the hydrogen peroxide (H2O2)-producing enzymes dual oxidase (Duox)1 and Duox2, thiocyanate (SCN(-)), and secreted lactoperoxidase (LPO). The LPO-catalyzed reaction between H2O2 and SCN(-) yields the bactericidal hypothiocyanite (OSCN(-)) in airway surface liquid (ASL). Although SCN(-) is the physiological substrate of LPO, the Duox/LPO/halide system can generate hypoiodous acid when the iodide (I(-)) concentration is elevated in ASL. Because hypoiodous acid, but not OSCN(-), inactivates respiratory syncytial virus (RSV) in cell culture, we used a lamb model of RSV to test whether potassium iodide (KI) could enhance this system in vivo. Newborn lambs received KI by intragastric gavage or were left untreated before intratracheal inoculation of RSV. KI treatment led to a 10-fold increase in ASL I(-) concentration, and this I(-) concentration was approximately 30-fold higher than that measured in the serum. Also, expiratory effort, gross lung lesions, and pulmonary expression of an RSV antigen and IL-8 were reduced in the KI-treated lambs as compared with nontreated control lambs. Inhibition of LPO activity significantly increased lesions, RSV mRNA, and antigen. Similar experiments in 3-week-old lambs demonstrated that KI administration was associated with reduced gross lesions, decreased RSV titers in bronchoalveolar lavage fluid, and reduced RSV antigen expression. Overall, these data indicate that high-dose KI supplementation can be used in vivo to lessen the severity of RSV infections, potentially through the augmentation of mucosal oxidative defenses.

Neutrophil Function in Small Animals

Neutrophils are highly mobile phagocytes that serve as the initial effectors against pathogens and are actively recruited to sites of inflammation. Chemoattractants guide them toward the inflammation, and their interaction with endothelial cells directs them through postcapillary venules and into the tissues. Once they have reached their destination, they can efficiently kill many microbes via phagocytosis, extracellular release of granule contents, and the formation of NETs. They also actively produce cytokines and other mediators to promote or suppress inflammation, repair tissues, and modulate the immune response. The importance of neutrophil function in host health is emphasized through discussion of inherited disorders of neutrophil function such as leukocyte adhesion deficiency and cyclic hematopoiesis.

Positive predictive value of albumin: globulin ratio for feline infectious peritonitis in a mid-western referral hospital population

Low albumin to globulin ratio has been found previously to have a high positive predictive value for feline infectious peritonitis (FIP) in cats with clinical signs highly suggestive of the disease. However, FIP can have a more vague clinical presentation. This retrospective study found that the positive predictive value of an albumin:globulin (A:G) ratio of <0.8 and <0.6 was only 12.5% and 25%, respectively, in a group of 100 cats with one or more clinical signs consistent with FIP. The negative predictive value was 100% and 99% for an A:G ratio of <0.8 and A:G<0.6%, respectively. Therefore, when the prevalence of FIP is low, the A:G ratio is useful to rule out FIP but is not helpful in making a positive diagnosis of FIP.

Sucrose stabilization of Respiratory Syncytial Virus (RSV) during nebulization and experimental infection

BackgroundRespiratory syncytial virus (RSV) is a common respiratory pathogen that can cause severe pneumonia. In vivo studies of RSV can be difficult due to variation in viral infection and disease severity in some animal models. Factors that may contribute to the variation are decreases in viral titer due to preparation and storage and method of virus administration. Nebulization is one method of RSV administration that provides even distribution of virus to all lung lobes; however, the exact quantity of the virus killed by nebulization is not defined. To test the hypothesis that sucrose enhances RSV stability and infectivity, a series of in vitro experiments were conducted with RSV strain Memphis 37 stored at varying concentrations (0%, 3%, 5%, 8%, 10%, 15%, and 20%) of sucrose as a possible cryo- and nebulization protectant. The optimal in vitro concentration was then assessed in vivo in a lamb model.MethodsPrior to titering the virus on HEp-2 cells, the various virus solutions were subjected to one freeze-thaw cycle and one nebulization cycle. Forty-eight hours after viral plating, infectious foci were detected and counted using immunofluorescent imaging. Titers were determined after freeze-thaw and after freeze-thaw followed by nebulization, then compared to the stock titers (before freezing) as well as to one another to determine the loss of infectivity. To further test this in vivo, lambs 2 to 3-days-old were infected via nebulization with RSV using inoculate containing either 20% sucrose or no sucrose followed by assessments of infection severity.ResultsNebulization of virus in 0% sucrose resulted in a 0.580 log reduction in infectivity while virus in 20% sucrose exhibited a 0.297 log reduction. In vivo studies demonstrated that 20% sucrose enhanced RSV lesions and antigen distribution.ConclusionsThe data suggests that both nebulization and freeze-thawing of RSV in the absence of sucrose cause unacceptable losses in viral infectivity and that sucrose acts as a RSV protectant in both regards.

Amyloid Deposition in 2 Feline Thymomas

Two cases of feline thymoma with amyloid deposition were encountered between 1982 and 2010. Neoplastic cells were separated by abundant, pale eosinophilic, homogeneous material that was congophilic and birefringent. Ultrastructurally, the neoplastic cells were connected by desmosomes, and the extracellular deposits were composed of nonbranching, hollow-cored fibrils, 8–10 nm in diameter. In the case with sufficient archived tissue for additional sections, the amyloid remained congophilic following potassium permanganate incubation, and the neoplastic cells were immunoreactive for pancytokeratin. The histologic, histochemical, ultrastructural, and immunohistochemical features of both neoplasms are consistent with epithelial-predominant thymoma with the unusual feature of intratumoral amyloid deposition. The affinity of the amyloid for Congo red following potassium permanganate incubation is consistent with non-AA amyloid. The ultrastructural findings were consistent with amyloid production by the neoplastic epithelial cells.

What is your diagnosis? Aspirate of a lung nodule in a dog

An 8-year-old male German Shepherd dog rescued by a Mower County humane society was presented for examination at the Cascade Animal Medical Center in Rochester, MN. The physical examination was unremarkable. No fever was detected and auscultation of the heart and lungs revealed no abnormalities. A heartworm antigen test (SNAP 4Dx, IDEXX, Westbrook, ME, USA) performed as part of a standard procedure for all stray intake animals was positive. A CBC revealed mild normocytic normochromic nonregenerative anemia with 0.6% reticulocytes. The only biochemical abnormality was mild hyperproteinemia with mild hyperglobulinemia. There were no abnormalities detected on urinalysis. Lateral and ventrodorsal thoracic radiographs to stage the heartworm disease revealed 4 nodular soft tissue opacities, each approximately the diameter of an intercostal space, visible in the right cranial, right middle, and left cranial lung lobes (Figure 1). Cardiac or pulmonary vascular changes of heartworm disease were not evident. Differential diagnoses for multiple pulmonary nodules included granulomas, abscesses, and neoplasia. Ultrasound-guided fine-needle aspirates of the lung nodules were collected, and slide preparations were submitted by mail to the Clinical Pathology Laboratory at Iowa State University for cytologic evaluation (Figure 2).

Age-related variation in the cellular composition of equine bronchoalveolar lavage fluid

BACKGROUND Previous reports reveal variation in the cellular composition of equine bronchoalveolar lavage fluid (BALF). OBJECTIVES The purpose of this study was to compare the profiles of BALF from horses to assess age-related differences. Serial BALF samples were collected from the same individuals over a one-year period to identify changes in individual animals as they aged. METHODS Collection of BALF was performed on horses aged one week and one, 2, 6, and 12 months. Total nucleated cell count (TNCC), protein concentration, and cytology were assessed. Longitudinal analysis was performed and compared to healthy adults. RESULTS Foals at one week and 6 months of age had significantly higher TNCC than adults (medians: 320/μL, 285/μL, and 90/μL, respectively); no differences in total protein were found. Foals at one month had the highest proportion of macrophages (median: 87.3%), differing significantly from both yearlings and adults (medians: 45.5% and 48.7%, respectively). Foals aged one week and one month had significantly lower proportions of lymphocytes than yearlings and adults (medians: 3.2% and 4.7% vs 43.2% and 45.8%, respectively). Eosinophil percentage was lowest in foals aged one week, one month, and 2 months (median: 0.0%) and highest in foals aged 6 months (median: 2.2%). Mast cell percentages were highest in yearlings and adults (medians: 2.2% and 3.3%, respectively) and neutrophil percentage was highest in foals aged one week (13.7%). CONCLUSIONS Cytologic profiles of BALF from foals and adult horses differed considerably. Significant changes in TNCC and percentages of lymphocytes, macrophages, and eosinophils occurred with age.

What is your diagnosis? Aspirate of a lung nodule in a dog. Paragonimiasis.

An 8-year-old male German Shepherd dog rescued by a Mower County humane society was presented for examination at the Cascade Animal Medical Center in Rochester, MN. The physical examination was unremarkable. No fever was detected and auscultation of the heart and lungs revealed no abnormalities. A heartworm antigen test (SNAP 4Dx, IDEXX, Westbrook, ME, USA) performed as part of a standard procedure for all stray intake animals was positive. A CBC revealed mild normocytic normochromic nonregenerative anemia with 0.6% reticulocytes. The only biochemical abnormality was mild hyperproteinemia with mild hyperglobulinemia. There were no abnormalities detected on urinalysis. Lateral and ventrodorsal thoracic radiographs to stage the heartworm disease revealed 4 nodular soft tissue opacities, each approximately the diameter of an intercostal space, visible in the right cranial, right middle, and left cranial lung lobes (Figure 1). Cardiac or pulmonary vascular changes of heartworm disease were not evident. Differential diagnoses for multiple pulmonary nodules included granulomas, abscesses, and neoplasia. Ultrasound-guided fine-needle aspirates of the lung nodules were collected, and slide preparations were submitted by mail to the Clinical Pathology Laboratory at Iowa State University for cytologic evaluation (Figure 2).

What is your diagnosis? Aspirate of a lung nodule in a dog: Lung nodule from a dog

An 8-year-old male German Shepherd dog rescued by a Mower County humane society was presented for examination at the Cascade Animal Medical Center in Rochester, MN. The physical examination was unremarkable. No fever was detected and auscultation of the heart and lungs revealed no abnormalities. A heartworm antigen test (SNAP 4Dx, IDEXX, Westbrook, ME, USA) performed as part of a standard procedure for all stray intake animals was positive. A CBC revealed mild normocytic normochromic nonregenerative anemia with 0.6% reticulocytes. The only biochemical abnormality was mild hyperproteinemia with mild hyperglobulinemia. There were no abnormalities detected on urinalysis. Lateral and ventrodorsal thoracic radiographs to stage the heartworm disease revealed 4 nodular soft tissue opacities, each approximately the diameter of an intercostal space, visible in the right cranial, right middle, and left cranial lung lobes (Figure 1). Cardiac or pulmonary vascular changes of heartworm disease were not evident. Differential diagnoses for multiple pulmonary nodules included granulomas, abscesses, and neoplasia. Ultrasound-guided fine-needle aspirates of the lung nodules were collected, and slide preparations were submitted by mail to the Clinical Pathology Laboratory at Iowa State University for cytologic evaluation (Figure 2).