Inducing Long Lasting B Cell and T Cell Immunity Against Multiple Variants of SARS-CoV-2 Through Mutant Bacteriophage Qß-Receptor Binding Domain Conjugate.

More than 3 years into the global pandemic, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a significant threat to public health. Immunities acquired from infection or current vaccines fail to provide long term protection against subsequent infections, mainly due to their fast-waning nature and the emergence of variants of concerns (VOCs) such as Omicron. To overcome these limitations, SARS-CoV-2 Spike protein receptor binding domain (RBD)-based epitopes are investigated as conjugates with a powerful carrier, the mutant bacteriophage Qß (mQß). The epitope design is critical to eliciting potent antibody responses with the full length RBD being superior to peptide and glycopeptide antigens. The full length RBD conjugated with mQß activates both humoral and cellular immune systems in vivo, inducing broad spectrum, persistent, and comprehensive immune responses effective against multiple VOCs including Delta and Omicron variants, rendering it a promising vaccine candidate.

Shape Anisotropy-Governed High-Performance Nanomagnetosol for In Vivo Magnetic Particle Imaging of Lungs.

Caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), coronavirus disease 2019 (COVID-19) has shown extensive lung manifestations in vulnerable individuals, putting lung imaging and monitoring at the forefront of early detection and treatment. Magnetic particle imaging (MPI) is an imaging modality, which can bring excellent contrast, sensitivity, and signal-to-noise ratios to lung imaging for the development of new theranostic approaches for respiratory diseases. Advances in MPI tracers would offer additional improvements and increase the potential for clinical translation of MPI. Here, a high-performance nanotracer based on shape anisotropy of magnetic nanoparticles is developed and its use in MPI imaging of the lung is demonstrated. Shape anisotropy proves to be a critical parameter for increasing signal intensity and resolution and exceeding those properties of conventional spherical nanoparticles. The 0D nanoparticles exhibit a 2-fold increase, while the 1D nanorods have a > 5-fold increase in signal intensity when compared to VivoTrax. Newly designed 1D nanorods displayed high signal intensities and excellent resolution in lung images. A spatiotemporal lung imaging study in mice revealed that this tracer offers new opportunities for monitoring disease and guiding intervention.

Fatal Sarcocystis calchasi-associated meningoencephalitis in 2 captive vulturine guineafowl.

Two captive vulturine guineafowl (Acryllium vulturinum) were presented with lethargy, hyporexia, weight loss, and progressive neurologic signs. One of the guineafowl was seropositive for Sarcocystis falcatula (1:50 dilution). Both guineafowl died within 5 d of presentation. Histologic examination revealed nonsuppurative meningoencephalitis with gliosis, associated with occasional schizonts in the neuropil. Using fresh-frozen brain tissue, PCR was performed to amplify the ITS1 RNA region and portions of the 18S ribosomal RNA gene (18S gene) and the 28S ribosomal RNA gene (28S gene). Analysis of nucleic acid sequences from the resulting amplicons indicated that Sarcocystis calchasi was the likely cause of disease. To our knowledge, S. calchasi-associated disease has not been reported previously in the order Galliformes.

Eastern Equine Encephalomyelitis in Michigan: Historical Review of Equine, Human, and Wildlife Involvement, Epidemiology, Vector Associations, and Factors Contributing to Endemicity.

Eastern equine encephalomyelitis (EEE) is a mosquito-borne viral disease that is an emerging public health concern in the state of Michigan. Although Michigan has one of the highest incidence rates of EEE in the United States, much of the information known about cases in humans, equines, and other animals residing in Michigan is unpublished. This article summarizes such information and explores spatial trends in the historic distribution of EEE in Michigan. Outbreaks in Michigan have occurred over an 80-yr interval, involving only horses in 1942-1943 and 1973-1976, and then episodically from 1980 to 2020, and involving horses, humans, and wild and domestic animals. An estimated 1,036 equine cases (confirmed and suspected) and 36 confirmed human cases have occurred, including 10 in 2019 (6 deaths) and 4 in 2020 (2 deaths). Human cases ranged in age from 1 to 81 yr; 70% were male, and fatality rate of 34.3%. Equine and human cases occurred from July to October, peaked in August, and cluster in space in southwestern and southeastern lower Michigan. Cases occurred in glacial outwash and ice-contact landscapes in glacial interlobate zones. EEE virus (EEEV) was recovered from Culiseta melanura, Coquillettidia perturbans, five species of Aedes, and other mosquito species near horse and human case sites. Virus isolations or presence of neutralizing antibodies in several passerine species of birds suggest broad EEEV-bird associations. White-tailed deer and other wildlife were also affected. Geographic spread to northern areas of the state suggests expansion of this disease system into new and unsuspected foci.

Detection of pathogens in blood or feces of adult horses with enteric disease and association with outcome of colitis.

BACKGROUND: Rates of detecting ≥1 potential enteric pathogens (PEP) or toxins (PEP-T) in feces, blood, or both of horses ≥6 months of age with enteric disease and impact of multiple detections on outcome of horses with colitis has not been reported. OBJECTIVE: To determine detection rates of PEP/PEP-T in feces, blood, or both of horses with enteric disease and effect of detecting multiple agents on outcome of horses with colitis. ANIMALS: Thirty-seven hundred fifty-three fecal samples submitted to IDEXX Laboratories and 239 fecal and blood samples submitted to Michigan State University's Veterinary Diagnostic Laboratory (MSUVDL). METHODS: Retrospective evaluation of PEP/PEP-T testing results was performed to determine rates of detection of 1 or more PEP/PEP-T. Impact of detecting multiple agents on outcome was assessed in 239 horses hospitalized for colitis. RESULTS: One or more PEP/PEP-T was detected in 1175/3753 (31.3%) and 145/239 (60.7%) of samples submitted to IDEXX Laboratories and MSUVDL, respectively. In a hospitalized cohort, survival to discharge was lower (76%) in horses with 1 agent, compared to horses with either no (88%) or multiple (89%) agents. There was no difference (P = .78) in days of hospitalization between horses with 0 (1-17), 1 (1-33), and > 1 positive (1-20) result. There was no difference in cost of hospitalization (P = .25) between horses with 0 ($2357, $1110-15 553), 1 ($2742, $788-11 005), and >1 positive ($2560, $1091-10 895) result. CONCLUSIONS AND CLINICAL IMPORTANCE: Detection rates of PEP/PEP-T in horses with colitis vary with cohorts and tests performed. Detection of more than 1 PEP or PEP-T did not affect outcome.

High SARS-CoV-2 Viral Load in Urine Sediment Correlates with Acute Kidney Injury and Poor COVID-19 Outcome.

BACKGROUND: AKI is a complication of coronavirus disease 2019 (COVID-19) that is associated with high mortality. Despite documented kidney tropism of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there are no consistent reports of viral detection in urine or correlation with AKI or COVID-19 severity. Here, we hypothesize that quantification of the viral load of SARS-CoV-2 in urine sediment from patients with COVID-19 correlates with occurrence of AKI and mortality. METHODS: The viral load of SARS-CoV-2 in urine sediments (U-viral load) was quantified by qRT-PCR in 52 patients with PCR-confirmed COVID-19 diagnosis, who were hospitalized between March 15 and June 8, 2020. Immunolabeling of SARS-CoV-2 proteins Spike and Nucleocapsid was performed in two COVID-19 kidney biopsy specimens and urine sediments. Viral infectivity assays were performed from 32 urine sediments. RESULTS: A total of 20 patients with COVID-19 (39%) had detectable SARS-CoV-2 U-viral load, of which 17 (85%) developed AKI with an average U-viral load four-times higher than patients with COVID-19 who did not have AKI. U-viral load was highest (7.7-fold) within 2 weeks after AKI diagnosis. A higher U-viral load correlated with mortality but not with albuminuria or AKI stage. SARS-CoV-2 proteins partially colocalized with the viral receptor ACE2 in kidney biopsy specimens in tubules and parietal cells, and in urine sediment cells. Infective SARS-CoV-2 was not detected in urine sediments. CONCLUSION: Our results further support SARS-CoV-2 kidney tropism. A higher SARS-CoV-2 viral load in urine sediments from patients with COVID-19 correlated with increased incidence of AKI and mortality. Urinary viral detection could inform the medical care of patients with COVID-19 and kidney injury to improve prognosis.

Eastern Equine Encephalitis Virus in Visibly Affected Ruffed Grouse (Bonasa umbellus) in Michigan, Minnesota, and Wisconsin, USA.

Eastern equine encephalitis virus (EEEV) infects many avian species but has rarely been described in Ruffed Grouse (Bonasa umbellus). Between September and December 2019, 40 Ruffed Grouse, most in poor physical condition, were submitted to the Michigan, Wisconsin, and Minnesota (US) Departments of Natural Resources; eight were positive for EEEV.

Evaluation of Real-Time Quaking-Induced Conversion, ELISA, and Immunohistochemistry for Chronic Wasting Disease Diagnosis.

Chronic wasting disease (CWD) is a transmissible prion disorder, primarily affecting free-ranging and captive cervids in North America (United States and Canada), South Korea, and Europe (Finland, Norway, and Sweden). Current diagnostic methods used in the United States for detection of CWD in hunter harvested deer involve demonstration of the causal misfolded prion protein (PrPCWD) in the obex or retropharyngeal lymph nodes (RLNs) using an antigen detection ELISA as a screening tool, followed by a confirmation by the gold standard method, immunohistochemistry (IHC). Real-time quaking-induced conversion (RT-QuIC) assay is a newer approach that amplifies misfolded CWD prions in vitro and has facilitated CWD prion detection in a variety of tissues, body fluids, and excreta. The current study was undertaken to compare ELISA, IHC, and RT-QuIC on RLNs (n = 1,300 animals) from white-tailed deer (WTD) in Michigan. In addition, prescapular, prefemoral and popliteal lymph nodes collected from a small subset (n = 7) of animals were tested. Lastly, the location of the positive samples within Michigan was documented and the percentage of CWD positive RLNs was calculated by sex and age. ELISA and RT-QuIC detected PrPCWD in 184 and 178 out of 1,300 RLNs, respectively. Of the 184 ELISA positive samples, 176 were also IHC positive for CWD. There were seven discordant results when comparing IHC and ELISA. RT-QuIC revealed that six of the seven samples matched the IHC outcomes. One RLN was negative by IHC, but positive by ELISA and RT-QuIC. RT-QuIC, IHC, and ELISA also detected PrPCWD in prescapular, prefemoral and popliteal lymph nodes. CWD infection heterogeneities were observed in different age and sex groups, with young males having higher CWD prevalence. All, except one, CWD positive RLNs analyzed were from ten Counties geographically located in the West Michigan region of the Lower Peninsula. Taken together, we show evidence that the RT-QuIC assay is comparable to ELISA and IHC and could be helpful for routine CWD detection in surveillance programs. RT-QuIC also demonstrated that CWD prions are distributed across lymph nodes in a variety of anatomic locations. A multi-laboratory validation on blinded sample panels is underway and is likely to help to provide insight into the variability (lab-to-lab), analytical sensitivity, and specificity of gold standard diagnostics vs. RT-QuIC assay.

Suggested guidelines for validation of real-time PCR assays in veterinary diagnostic laboratories.

This consensus document presents the suggested guidelines developed by the Laboratory Technology Committee (LTC) of the American Association of Veterinary Laboratory Diagnosticians (AAVLD) for development, validation, and modification (methods comparability) of real-time PCR (rtPCR) assays. These suggested guidelines are presented with reference to the World Organisation for Animal Health (OIE) guidelines for validation of nucleic acid detection assays used in veterinary diagnostic laboratories. Additionally, our proposed practices are compared to the guidelines from the Foods Program Regulatory Subdivision of the U.S. Food and Drug Administration (FDA) and from the American Society for Veterinary Clinical Pathology (ASVCP). The LTC suggestions are closely aligned with those from the OIE and comply with version 2021-01 of the AAVLD Requirements for an Accredited Veterinary Medical Diagnostic Laboratory, although some LTC recommendations are more stringent and extend beyond the AAVLD requirements. LTC suggested guidelines are substantially different than the guidelines recently published by the U.S. FDA for validation and modification of regulated tests used for detection of pathogens in pet food and animal-derived products, such as dairy. Veterinary diagnostic laboratories that perform assays from the FDA Bacteriological Analytical Method (BAM) manual must be aware of the different standard.

Survival of Mycobacterium bovis during forage ensiling.

OBJECTIVE To determine whether Mycobacterium bovis remains viable in ensiled forages. SAMPLE Alfalfa, mixed mostly grass, and corn silages. PROCEDURES For each of 10 sampling days, six 250-g replicate samples of each feedstuff were created and placed in a film pouch that could be vacuum sealed to simulate the ensiling process. Within each set of replicate samples, 4 were inoculated with 10 mL of mycobacterial liquid culture medium containing viable M bovis and 2 were inoculated with 10 mL of sterile mycobacterial liquid culture medium (controls) on day 0. Pouches were vacuum sealed and stored in the dark at room temperature. On the designated sampling day, 1 control pouch was submitted for forage analysis, and the other pouches were opened, and forage samples were obtained for M bovis culture and analysis with a PCR assay immediately and 24 hours later. RESULTS None of the control samples had positive M bovis culture or PCR assay results. Among M bovis-inoculated samples, the organism was not cultured from alfalfa and corn silage for > 2 days but was cultured from mixed mostly grass silage for 28 days after inoculation and ensiling initiation. Mycobacterium bovis DNA was detected by PCR assay in samples of all 3 feedstuffs throughout the 112-day observation period. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that properly ensiled forages would be an unlikely source for M bovis transmission to cattle. Further research is necessary to determine whether ensiling kills M bovis or forces it to become dormant and, if the latter, elucidate the conditions that cause it to revert to an infectious state.

Dietary Fatty Acids Differentially Regulate Secretion of Adiponectin and Interleukin-6 in Primary Canine Adipose Tissue Culture.

The aim of this study was to determine the effect of n3 polyunsaturated fatty acids (PUFA) on canine adipose tissue secretion of adiponectin, interleukin-6 (IL6), and tumor necrosis factor-α (TNFα). Subcutaneous and omental visceral adipose tissue samples were collected from 16 healthy intact female dogs. Concentrations of adiponectin were measured in mature adipocyte cultures, and concentrations of IL6 and TNFα were measured in undifferentiated stromovascular cell (SVC) cultures following treatment with eicosapentaenic acid (EPA, 20:5n-3), arachidonic acid (ARA, 20:4n-6), or palmitic acid (PAM, 16:0) at 25, 50, or 100 µM. Secretion of adiponectin from mature adipocytes was higher (p < 0.001) following EPA treatment at 50 µM compared to control in subcutaneous tissue, and higher following EPA treatment compared to PAM treatment at 25 µM in both subcutaneous (p < 0.001) and visceral tissues (p = 0.010). Secretion of IL6 from SVC derived from subcutaneous tissue was lower following EPA treatment and higher following PAM treatment compared to control both at 50 µM (p = 0.001 and p = 0.041, respectively) and 100 µM (p = 0.013 and p < 0.001, respectively). These findings of stimulation of adiponectin secretion and inhibition of IL6 secretion by EPA, and stimulation of IL6 secretion by PAM, are consistent with findings of increased circulating concentrations of adiponectin and decreased circulating concentration of IL6 in dogs supplemented with dietary fish oil, and show that the effect of fish oil on circulating concentrations of adiponectin and IL6 is, at least partially, the result of local effects of EPA and PAM on adipose tissue.

Equine multinodular pulmonary fibrosis in southern Brazil: pathology and differential diagnosis / Fibrose pulmonar multinodular em equinos no Sul do Rio Grande do Sul: patologia e diagnóstico diferencial

Equine multinodular pulmonary fibrosis (EMPF) diagnosed at the Laboratório Regional de Diagnóstico of Faculdade de Medicina Veterinária, Universidade Federal de Pelotas (LRD/UFPel), is described. Differential aspects of other pulmonary diseases in horses with pneumonia and interstitial fibrosis were discussed. The disease occurred in a 15-year-old equine that presented with clinical signs of respiratory distress, intermittent fever, anorexia, and dyspnea. Macroscopically, there was enlargement of the lungs with whitish, pale, firm and well-delimited nodules, approximately 7-10 cm in diameter, distributed throughout the parenchyma. Histologically, the lung nodules had alveolar spaces with walls covered by cuboidal epithelium containing macrophages, neutrophils, lymphocytes, hyperplasia of type II pneumocytes and, eventually, multinucleated giant cells. The interstitium was markedly thickened by mature fibrous connective tissue and collagen. There were intranuclear inclusion bodies in the macrophages. The PCR technique for detecting the EHV-5 DNA was positive. In a retrospective study of pneumonia cases in horses with interstitial fibrosis diagnosed in the LRD/UFPel, two animals had macroscopic and histological lesions similar to those with EMPF, but they were negative for EHV-5 in PCR. Four cases diagnosed with pneumonia and interstitial tissue fibrosis had a histological pattern that was different from that observed in the EMPF animal, thus eliminating the possibility of EMPF. It is concluded that EMPF is a sporadic disease that should be considered in cases of respiratory disease in horses. Reports of such cases are important to alert technicians about the occurrence of rare diseases in Brazil. It is also necessary to establish the true role of EHV-5 in the pathogenesis of EMPF. Cases of pulmonary fibrosis such as EMPF, in which the virus is not present, should be studied to establish whether it could be an idiopathic form of the disease.(AU)

Descreve-se a fibrose multinodular pulmonar equina (EMPF) diagnosticado no Laboratório Regional de Diagnóstico da Faculdade de Veterinária da Universidade Federal de Pelotas. Foram discutidos a patologia da doença e os aspectos diferenciais de outras enfermidades pulmonares de equinos que cursam com pneumonia e fibrose intersticial. A doença ocorreu em um equino sem raça definida de 15 anos de idade que apresentou sinais clínicos de dificuldade respiratória febre intermitente, anorexia e dispneia, com evolução de aproximadamente 10 dias. Macroscopicamente havia aumento de volume dos pulmões e nódulos esbranquiçados, pálidos, firmes e bem delimitados, de aproximadamente 7-10 cm de diâmetro, distribuídos pelo parênquima. Histologicamente, o tecido pulmonar apresentava nódulos caracterizados pela presença de espaços alveolares, com paredes revestidas por epitélio cuboidal achatado, contendo macrófagos e neutrófilos e havia, também, linfócitos e hiperplasia de pneumócitos tipo II e eventualmente células gigantes multinuacleadas. O interstício estava acentuadamente espessado por tecido conjuntivo fibroso maduro e por colágeno. Havia corpúsculos de inclusão intranucleares em macrófagos. A técnica de PCR para detecção do DNA de herpes vírus equino-5 (EHV-5) resultou positiva. Em um estudo retrospectivo de casos de pneumonia com fibrose intersticial diagnosticados no LRD entre 2000 e 2015, dois equinos apresentaram lesões macroscópicas e histológicas similares às de EMPF, porém resultaram negativos na PCR para detecção de EHV-5. Quatro casos de pneumonia com fibrose do tecido intersticial apresentaram padrão histológico diverso da EMPF descartando-se a possibilidade de tratar-se da doença. Conclui-se que EMPF é uma enfermidade esporádica, no entanto deve ser levada em consideração em casos de doença respiratória em equinos. A descrição dos casos diagnosticados é importante para alertar técnicos sobre a ocorrência da mesma no Brasil. É necessário estabelecer o real papel do EHV-5 na patogenia da doença. Casos de fibrose pulmonar semelhantes à EMPF em que não esteja presente o vírus, devem ser estudados a fim de ficar estabelecido se poderia ser uma forma idiopática da mesma doença.(AU)

Use of whole-genome sequencing and evaluation of the apparent sensitivity and specificity of antemortem tuberculosis tests in the investigation of an unusual outbreak of Mycobacterium bovis infection in a Michigan dairy herd.

OBJECTIVE To describe use of whole-genome sequencing (WGS) and evaluate the apparent sensitivity and specificity of antemortem tuberculosis tests during investigation of an unusual outbreak of Mycobacterium bovis infection in a Michigan dairy herd. DESIGN Bovine tuberculosis (bTB) outbreak investigation. ANIMALS Cattle, cats, dog, and wildlife. PROCEDURES All cattle in the index dairy herd were screened for bTB with the caudal fold test (CFT), and cattle ≥ 6 months old were also screened with a γ-interferon (γIFN) assay. The index herd was depopulated along with all barn cats and a dog that were fed unpasteurized milk from the herd. Select isolates from M bovis-infected animals from the index herd and other bTB-affected herds underwent WGS. Wildlife around all affected premises was examined for bTB. RESULTS No evidence of bTB was found in any wildlife examined. Within the index herd, 53 of 451 (11.8%) cattle and 12 of 21 (57%) cats were confirmed to be infected with M bovis. Prevalence of M bovis-infected cattle was greatest among 4- to 7-month-old calves (16/49 [33%]) followed by adult cows (36/203 [18%]). The apparent sensitivity and specificity were 86.8% and 92.7% for the CFT and 80.4% and 96.5% for the γIFN assay when results for those tests were interpreted separately and 96.1% and 91.7% when results were interpreted in parallel. Results of WGS revealed that M bovis-infected barn cats and cattle from the index herd and 6 beef operations were infected with the same strain of M bovis. Of the 6 bTB-affected beef operations identified during the investigation, 3 were linked to the index herd only by WGS results; there was no record of movement of livestock or waste milk from the index herd to those operations. CONCLUSIONS AND CLINICAL RELEVANCE Whole-genome sequencing enhanced the epidemiological investigation and should be used in all disease investigations. Performing the CFT and γIFN assay in parallel improved the antemortem ability to detect M bovis-infected animals. Contact with M bovis-infected cattle and contaminated milk were major risk factors for transmission of bTB within and between herds of this outbreak.

Development and application of a quantitative PCR assay to study equine herpesvirus 5 invasion and replication in equine tissues in vitro and in vivo.

Equine herpesvirus 5 (EHV-5) infection is associated with pulmonary fibrosis in horses, but further studies on EHV-5 persistence in equine cells are needed to fully understand viral and host contributions to disease pathogenesis. Our aim was to develop a quantitative PCR (qPCR) assay to measure EHV-5 viral copy number in equine cell cultures, blood lymphocytes, and nasal swabs of horses. Furthermore, we used a recently developed equine primary respiratory cell culture system to study EHV-5 pathogenesis at the respiratory tract. PCR primers and a probe were designed to target gene E11 of the EHV-5 genome. Sensitivity and repeatability were established, and specificity was verified by testing multiple isolates of EHV-5, as well as DNA from other equine herpesviruses. Four-week old fully differentiated (mature), newly seeded (immature) primary equine respiratory epithelial cell (ERECs), and equine dermal cell cultures were inoculated with EHV-5 and the cells and supernatants collected daily for 14days. Blood lymphocytes and nasal swabs were collected from horses experimentally infected with equine herpesvirus 1 (EHV-1). The qPCR assay detected EHV-5 at stable concentrations throughout 14days in inoculated mature EREC and equine dermal cell cultures (peaking at 202 and 5861 viral genomes per 106 cellular ß actin, respectively). EHV-5 copies detected in the immature EREC cultures increased over 14days and reached levels greater than 10,000 viral genomes per 106 cellular ß actin. Moreover, EHV-5 was detected in the lymphocytes of 76% of horses and in the nasal swabs of 84% of horses experimentally infected with EHV-1 pre-inoculation with EHV-1. Post-inoculation with EHV-1, EHV-5 was detected in lymphocytes of 52% of horses while EHV-5 levels in nasal swabs were not significantly different from pre-inoculation levels. In conclusion, qPCR was a reliable technique to investigate viral load in in vivo and in vitro samples, and EHV-5 replication in equine epithelial cells may be influenced by cellular stages of differentiation.

PREVALENCE AND DISTRIBUTION OF PSEUDOGYMNOASCUS DESTRUCTANS IN MICHIGAN BATS SUBMITTED FOR RABIES SURVEILLANCE.

Since 2006, bat populations in North America have suffered devastating mortality from an emerging disease known as white-nose syndrome (WNS). The causal agent of WNS is the fungus Pseudogymnoascus destructans. In April 2014, WNS was discovered in little brown bats ( Myotis lucifugus ) in Michigan, US, and has since been documented in 12 counties. Because current surveillance for WNS focuses primarily on mine-hibernating species in winter, it is subject to geographic, species, and seasonal bias. To investigate species affected and potential associations of gender, seasonal life cycle, and region with P. destructans prevalence, 1,040 rabies-negative bats were sampled from May 2014 to May 2015 from animals submitted as part of statewide rabies surveillance. The vast majority (96%) of the sample population consisted of big brown bats ( Eptesicus fuscus ), a noncavernicolous species. Two methods were used to detect P. destructans: fluorescence of the muzzle, wing, and tail membranes under ultraviolet light and PCR targeting genomic DNA on wing samples. Only five bats (0.5%), all M. lucifugus , were confirmed positive after nucleic acid sequencing of PCR amplicons. No other species were infected. All infected bats were collected from April to May, coinciding with their emergence from hibernation. As P. destructans and WNS spread westward, novel surveillance streams may provide a useful tool for wildlife management agencies seeking to detect the fungus where winter hibernacula such as caves and mines are absent or otherwise inaccessible.

Eastern equine encephalomyelitis virus infection in six captive southern cassowaries (Casuarius casuarius).

CASE DESCRIPTION Within a 2-week period, 4 southern cassowaries (Casuarius casuarius) at an exhibit at a Virginia zoo died acutely subsequent to eastern equine encephalitis virus (EEEV) infection. This prompted a search for other EEEV outbreaks in cassowaries, which resulted in the identification of 2 additional cassowaries that died of EEEV infection at a conservation center in Florida. CLINICAL FINDINGS Both juvenile and adult birds were affected. Three of the 6 birds died acutely with no premonitory signs. Clinical disease in the other 3 birds was characterized by lethargy and ataxia. Clinicopathologic findings typically included leukocytosis, hyperuricemia, abnormally high liver enzyme activities, and hyper-ß globulinemia, which was indicative of acute inflammation. TREATMENT AND OUTCOME The 3 birds with clinical disease died despite supportive treatment. Gross abnormalities commonly observed during necropsy included coelomitis and evidence of diarrhea. Frequently observed histologic abnormalities were encephalitis, vasculitis, hepatitis, nephritis, and splenitis. The diagnosis of EEEV infection was confirmed by detection of serum anti-EEEV antibodies or detection of viral RNA in brain tissue by use of a reverse-transcriptase PCR assay. CLINICAL RELEVANCE Findings suggested that EEEV can cause high morbidity and mortality rates in southern cassowaries. Clinical disease might be reduced or prevented by vaccination, isolation of ill birds, and mosquito control strategies.

ACUTE CLINICAL LEPTOSPIROSIS (GRIPPOTYPHOSA SEROVAR) IN AN ADULT DROMEDARY CAMEL (CAMELUS DROMEDARIUS).

A 9-yr-old castrated male dromedary camel (Camelus dromedarius) presented with lethargy and partial anorexia. A diagnostic examination revealed fever, and further workup revealed a neutrophilia, hyperfibrinogenemia, renal azotemia, and a rapid onset of a high Leptospira antibody titer during the acute clinical period (Grippotyphosa serovar). The camel responded clinically to antimicrobial treatment with ceftiofur crystalline free acid injections, but renal azotemia persisted, presumably secondary to chronic renal damage. Subsequent Leptospira polymerase chain reaction testing on urine samples obtained over the following 4 mo revealed no evidence of urinary shedding, so a persistent infection was unlikely. Although often mentioned as a potential cause of reproductive loss, well-documented case reports of clinical leptospirosis in camelids are very rare. In this case, native wildlife contamination of a small watering hole is suspected to have been the source of infection. In response to this experience, the camel and two conspecifics were prescribed a vaccination regimen using an inactivated pentavalent Leptospira vaccine licensed for cattle.

Prevalence and diagnosis of hemotrophic mycoplasma infection in research sheep and its effects on hematology variables and erythrocyte membrane fragility.

Hemotrophic mycoplasma (hemoplasma) infection in research sheep can confound experimental results and contribute to morbidity and mortality. Prevalence and clinicopathologic studies historically relied on blood-smear diagnosis, but systematic studies using current molecular techniques are warranted. Here we sought to report the prevalence of subclinical infection in our study population, compare diagnostic sensitivity and specificity between blood smears and a PCR assay, and determine the effects of infection on CBC variables and erythrocyte membrane fragility. We collected whole-blood samples from 111 convenience-sampled research sheep. All samples were tested for hemoplasmas by using a PCR assay, blood smears were evaluated for visual presence of hemoplasmas, and CBC and osmotic fragility assays were performed. Subclinical prevalence, according to PCR diagnosis, was 14.1% (14 of 99) in our study population. Relative to the PCR assay, blood-smear diagnosis was 8.3% sensitive and 100% specific for hemoplasma detection. Subclinical infection was associated with changes in MCV, MCHC, RBC distribution width, and absolute monocyte count. Acute infection was associated with changes in RBC mass, Hgb concentration, MCV, MCH, MCHC, and absolute lymphocyte and monocyte counts. Acute infection was associated with increased mean erythrocyte fragility compared with that in uninfected control and treated sheep. We demonstrated that hemoplasma infection is common in our study population, blood-smear evaluation is insensitive at detecting infection, and infection is associated with changes in CBC variables and increased erythrocyte membrane fragility. These findings raise concerns regarding the suitability of hemoplasma-infected sheep for biomedical research.

Comparison between a conductometric biosensor and ELISA in the evaluation of Johne's disease.

Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), is an important gastrointestinal disease of cattle worldwide because of the economic losses encountered in JD-affected herds. These losses include reduction in milk yield in cows, premature culling and reduced carcass weight of culled diseased animals. In the U.S. dairy industry, economic losses from reduced productivity associated with JD are estimated to cost between $200 and $250 million annually. The development of non-laboratory-based assays would support more frequent testing of animals for JD and could improve its control. Conductometric biosensors combine immunomigration technology with electronic signal detection and have been adapted for the detection of IgG antibody against MAP. In the present study, a capture membrane with limited variability in the immunomigration channel and an optimal concentration of the secondary anti-bovine antibody used in a previously developed conductometric biosensor were compared with a commercially available antibody detection ELISA in their evaluation of JD, using samples of serum from cattle whose JD status where unknown. There was a moderate strength of agreement (kappa = 0.41) between the two assays. Findings from this preliminary study support the continued development of conductometric biosensors for use in the diagnosis of JD.