Amy L. Glaser

Detection of Antibodies to West Nile Virus in Equine Sera Using Microsphere Immunoassay

One hundred and ninety-one sera from horses that recently were exposed to West Nile virus (WNV) by either vaccination or natural infection or that were not vaccinated and remained free of infection were used to evaluate fluorescent microsphere immunoassays (MIAs) incorporating recombinant WNV envelope protein (rE) and recombinant nonstructural proteins (rNS1, rNS3, and rNS5) for detection of equine antibodies to WNV. The rE MIA had a diagnostic sensitivity and specificity, respectively, of 99.3% and 97.4% for detection of WNV antibodies in the serum of horses that were recently vaccinated or naturally infected with WNV, as compared to the plaque reduction neutralization test (PRNT). The positive rE MIA results were assumed to be WNV-specific because of the close agreement between this assay and the PRNT and the fact that unvaccinated control horses included in this study were confirmed to be free of exposure to the related St Louis encephalitis virus. The NS protein–based MIA were all less sensitive than either the rE MIA or PRNT (sensitivity 0–48.0), although the rNS1 MIA distinguished horses vaccinated with the recombinant WNV vaccine from those that were immunized with the inactivated WNV vaccine (P < 0.0001) or naturally infected with WNV (P < 0.0001). The rE MIA would appear to provide a rapid, convenient, inexpensive, and accurate test for the screening of equine sera for the presence of antibodies to WNV.

Prevalence of infectious diseases in cats and dogs rescued following Hurricane Katrina

OBJECTIVE To determine the prevalence of infectious diseases of animal and zoonotic importance in cats and dogs rescued and transferred from the Gulf Coast region following Hurricane Katrina. DESIGN Cross-sectional study. ANIMALS 414 dogs and 56 cats rescued and transferred from the Gulf Coast region within 4 months after the hurricane. PROCEDURES EDTA-anticoagulated blood and serum samples were tested via PCR and serologic assays for infectious diseases. RESULTS In dogs, prevalence was highest for anti-West Nile virus (WNV) antibodies (218/390 [55.9%]), Dirofilaria immitis antigen (195/400 [48.8%]), anti-Toxoplasma gondii antibodies (92/366 [25.1%]), and hemotropic mycoplasma DNA (40/345 [11.9%]). The DNA of Bartonella spp, Ehrlichia spp, or Babesia spp or anti-canine influenza virus antibodies were identified in < 2% of dogs. In cats, prevalence was highest for antibodies against Bartonella spp and DNA of Bartonella spp combined (49/55 [89.1 %]), anti-T gondii antibodies (13/55 [23.6%]), hemotropic mycoplasma DNA (5/47 [10.6%]), anti-WNV antibodies (5/48 [10.4%]), D immitis antigen (4/50 [8.0%]), and anti-FIV antibodies (4/56 [7.1%]). A total of 308 (74.4%) dogs and 52 (92.9%) cats had evidence of previous or current vector-borne infections. CONCLUSIONS AND CLINICAL RELEVANCE Cats and dogs rescued from the disaster region had evidence of multiple infectious diseases. The dispersal of potentially infectious animals to other regions of North America where some infections were not typically found could have contributed to new geographic ranges for these organisms or to underdiagnosis in affected animals because of a low index of suspicion in regions with low disease prevalence.

Pneumovirus in Dogs with Acute Respiratory Disease

To determine which respiratory viruses circulate among confined dogs, we analyzed nasal and pharyngeal swab specimens from shelter dogs with acute respiratory disease. An unknown virus was isolated. Monoclonal antibody testing indicated that it was probably a pneumovirus. PCR and sequence analysis indicated that it was closely related to murine pneumovirus.

Diagnosis of Borrelia‐associated uveitis in two horses

Borrelia burgdorferi, the etiologic agent of Lyme disease is a tick born spirochetal infection. Clinical signs of Lyme borreliosis are uncommon in horses, but when present they are often vague and nonspecific. In horses, Lyme borreliosis has been implicated in musculoskeletal, neurological, reproductive, and ocular disorders, including uveitis, but definitive diagnosis can be challenging as the causative agent is rarely isolated and serologic tests can be unreliable and do not confirm active disease. Here, we report two cases of equine uveitis associated with B. burgdorferi based on the identification of spirochetes within ocular fluids and confirmed with PCR testing. The two cases illustrate some of the challenges encountered in the recognition and diagnosis of equine Lyme borreliosis. Although only one of many possible causes of equine uveitis, Lyme disease should be considered a differential diagnosis, especially in endemic areas. Given the possibility for false negative results of serum tests during uveitis associated with B. burgdorferi and the failure of such tests to confirm active infection, a combination of cytologic assessment, antibody, and/or PCR testing of ocular fluids may be worthwhile if the clinical suspicion for Lyme uveitis is high.

Genomic analysis of a pneumovirus isolated from dogs with acute respiratory disease

A previously unrecognized virus belonging to the subfamily Pneumovirinae and most closely related to murine pneumovirus (MPV) was identified in domestic dogs in 2 related animal shelters. Additional diagnostic testing yielded 3 new viral isolates and identified 6 additional PCR positive dogs from other USA locations indicating that its distribution is not geographically limited. Nucleotide sequences encompassing 9 of the 10 genes were compared to the only 2 available MPV strains, 15 and J3666. Several features distinguished the canine pneumovirus (CnPnV) from the murine strains. Two regions of diversity were identified in the amino-proximal region of P and the overlapping P2 ORF was only 54 amino acids (aa) compared to 137aa in MPV. The G protein had an amino-terminal cytoplasmic tail 18aa longer than in the MPV strains. The CnPnV SH protein showed the highest divergence with only 90.2% aa identity when compared to MPV strain 15. Like strain 15, the CnPnV SH ORF coded for a protein of 92aa while J3666 has a 114aa variant. Comparison of CnPnV isolates at culture passages 4 and 17 revealed 7nt differences within the 8598nt sequenced. Of note was a substitution at nt 364 in G resulting in a termination codon that would produce a truncated G protein of 122aa. Analysis of early passage and ex vivo samples showed the termination codon in G to be predominant after 6 days in culture indicating rapid selection of the mutation in A72 cells.

Sentinel chickens as a surveillance tool for West Nile virus in New York City, 2000.

West Nile (WN) virus was first identified in the Western Hemisphere during an outbreak of encephalitis in New York City (NYC) in 1999. Prior to 1999, NYC had not had a locally acquired human arboviral infection since the 1800s (yellow fever), although eastern equine and St. Louis encephalitis have occurred in surrounding areas. There was also no existing surveillance system for arthropod-borne viruses and no citywide mosquito control program in NYC prior to 1999. The identification of this new agent prompted the NYC Department of Health to develop surveillance and control programs to prevent future outbreaks of WN and other arboviral diseases. Among the systems implemented for the 2000 mosquito season were sentinel chicken flocks. Experimental evidence has shown that domestic chickens are readily infected with isolates of NY-99 WN virus, shed virus for only a brief period of time, and experience a viremia high enough to infect mosquitoes for only about two days.1,2 The Centers for Disease Control and Prevention supported the use of sentinel chickens as a surveillance tool for the local presence and transmission of WN virus during the 2000 mosquito season.1

Clinical Findings, Lesions, and Viral Antigen Distribution in Great Gray Owls (Strix nebulosa) and Barred Owls (Strix varia) with Spontaneous West Nile Virus Infection

Abstract West Nile Virus (WNV) infection manifests itself clinically and pathologically differently in various species of birds. The clinicopathologic findings and WNV antigen tissue distribution of six great gray owls (Strix nebulosa) and two barred owls (Strix varia) with WNV infection are described in this report. Great gray owls usually live in northern Canada, whereas the phylogenetically related barred owls are native to the midwestern and eastern United States and southern Canada. Naturally acquired WNV infection caused death essentially without previous signs of disease in the six great gray owls during a mortality event. Lesions of WNV infection were dominated by hepatic and splenic necrosis, with evidence of disseminated intravascular coagulation in the great gray owls. WNV antigen was widely distributed in the organs of the great gray owls and appeared to target endothelial cells, macrophages, and hepatocytes. The barred owls represented two sporadic cases. They had neurologic disease with mental dullness that led to euthanasia. These birds had mild to moderate lymphoplasmacytic encephalitis with glial nodules and lymphoplasmacytic pectenitis. WNV antigen was sparse in barred owls and only present in a few brain neurons and renal tubular epithelial cells. The cause of the different manifestations of WNV disease in these fairly closely related owl species is uncertain.

Isolation of Salem virus, a novel equine paramyxovirus, and assessment of its etiologic role in a disease outbreak

Salem virus (SalV) is a recently identified equine virus belonging to the family Paramyxoviridae. The only known isolate was obtained from a horse that was involved in a disease outbreak of undetermined nature and the circumstances of its isolation suggested an etiologic role. However, the experimental infection of a colostrum-deprived foal failed to reproduce the disease; only mild neutropenia and temperature elevation were recorded. An additional attempt to establish an etiological relationship with the disease was made by conducting a retrospective evaluation of the serological profiles of animals involved in the outbreak. Animals reported as being affected by the disease according to a comprehensive United States Department of Agriculture (USDA) database were found to be 48% (n=27) positive for antibodies to SalV, but the percent positive for all horses, affected and unaffected, was actually higher at 56% (n=62). For 15 affected horses for which paired acute and convalescent serum specimens were available, no unequivocal seroconversions to SalV were identified. Furthermore, the horse from which SalV was isolated was not listed as one of the animals affected by the disease. In total, the evidence suggests that SalV was not the etiological agent of the disease and that its isolation was fortuitous.

Evaluation of a Mycobacterium avium subsp. paratuberculosis leuD Mutant as a Vaccine Candidate against Challenge in a Caprine Model

ABSTRACT Johnes disease (JD) is prevalent worldwide and has a significant impact on the global agricultural economy. In the present study, we evaluated the protective efficacy of a leuD (Δleud) mutant and gained insight into differential immune responses after challenge with virulent M. avium subsp. paratuberculosis in a caprine colonization model. The immune response and protective efficacy were compared with those of the killed vaccine Mycopar. In vitro stimulation of peripheral blood mononuclear cells with johnin purified protein derivative showed that Mycopar and ΔleuD generated similar levels of gamma interferon (IFN-γ) but significantly higher levels than unvaccinated and challenged phosphate-buffered saline controls. However, only with ΔleuD was the IFN-γ response maintained. Flow cytometric analysis showed that the increase in IFN-γ correlated with proliferation and activation (increased expression of CD25) of CD4, CD8, and γδT cells, but this response was significantly higher in ΔleuD-vaccinated animals at some time points after challenge. Both Mycopar and ΔleuD vaccines upregulated Th1/proinflammatory and Th17 cytokines and downregulated Th2/anti-inflammatory and regulatory cytokines at similar levels at almost all time points. However, significantly higher levels of IFN-γ (at weeks 26 and 30), interleukin-2 (IL-2; week 18), IL-1b (weeks 14 and 22), IL-17 (weeks 18 and 22), and IL-23 (week 18) and a significantly lower level of IL-10 (weeks 14 and 18) and transforming growth factor β (week 18) were detected in the ΔleuD-vaccinated group. Most importantly, ΔleuD elicited an immune response that significantly limited colonization of tissues compared to Mycopar upon challenge with wild-type M. avium subsp. paratuberculosis. In conclusion, the ΔleuD mutant is a promising vaccine candidate for development of a live attenuated vaccine for JD in ruminants.

Passive transfer of naturally acquired specific immunity against West Nile virus to foals in a semi-feral pony herd

Horses naturally exposed to West Nile Virus (WNV) or vaccinated against WNV develop humoral immunity thought to be protective against development of clinical disease in exposed or infected animals. No reports evaluate the efficacy of passive transfer of naturally acquired specific WNV humoral immunity from dam to foal. The purpose of this study was to investigate passive transfer of naturally acquired immunity to WNV to foals born in a herd of semi-feral ponies, not vaccinated against WNV, in an endemic area, with many dams having seroconverted because of natural exposure. Microwell serum neutralization titers against WNV were determined in all mares and foals. Serum IgG concentration was determined in foals by serial radial immunodiffusion. Differences in IgG concentration between seropositive and seronegative foals were examined by means of the Mann-Whitney U-test. Linear regression was used to evaluate the association between mare and foal titers. Seventeen mare-foal pairs were studied; 1 foal had inadequate IgG concentration. IgG concentration was not different between seronegative and seropositive foals (P = .24). Mare and foal titers were significantly correlated in foals with adequate passive transfer of immunity (Spearmans rho = .84; P < .001); >90% of the foals titer was explained by the mares titer (R2 = 0.91; P < .001). Passive transfer of specific immunity to WNV is present in pony foals with adequate passive transfer of immunity born to seroconverted mares.