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.

Toxoplasma gondii in an African crested porcupine (Hystrix cristata).

An adult female crested porcupine (Hystrix cristata) was evaluated for acute onset of neurologic signs including head tilt, circling, and ataxia. She was found dead in her holding area 2 days after initially exhibiting clinical signs. Necropsy was unremarkable. Histopathology of brain tissue revealed the presence of protozoal cysts associated with inflammation as the underlying cause of clinical signs and death. Immunohistochemical staining of brain tissue for Toxoplasma gondii was strongly positive. PCR on fresh brain confirmed T. gondii as the causative organism. An adult male in the same enclosure has demonstrated similar neurologic signs over the past 3 years and has failed to respond to various medical treatments. Clinical disease associated with T. gondii has not been previously reported in this porcupine species or any other Old World porcupines, although there are several reports of clinical toxoplasmosis involving New World porcupine species.

Toxoplasmosis in a free-ranging mink.

A free-ranging mink (Mustela vison), estimated to be 3 mo old, was found on the campus of Michigan State University, East Lansing, Michigan; it exhibited clinical signs of left hind limb lameness, ataxia, head tremors, and bilateral blindness. Histologically, the animal had a mild, nonsuppurative meningoencephalitis and severe chorioretinitis with intralesional bradyzoites and tachyzoites. Protozoal organisms were identified as Toxoplasma gondii based on histology, immunohistochemistry, and polymerase chain reaction. To the authors' knowledge, this is the first report of clinical toxoplasmosis in a free-ranging mink.

Seroprevalence of antibodies against Leishmania spp among dogs in the United States.

OBJECTIVE: To determine seroprevalence of antibodies against Leishmania spp among dogs other than Foxhounds in the United States. DESIGN: Cross-sectional study. SAMPLE POPULATION: 957 serum samples from dogs throughout the United States submitted between January 2000 and August 2001 to the Diagnostic Center for Population and Animal Health at Michigan State University for serologic testing for tick-borne diseases. PROCEDURE: Samples were tested for antibodies against Leishmania spp with an immunofluorescent antibody (IFA) assay. Samples with positive results were submitted to the Centers for Disease Control and Prevention for confirmatory testing. RESULTS: Results of the IFA assay were negative for 939 of 957 samples. For 16 samples, titers were from 1:16 to 1:64, and titers in these dogs were considered likely to be a result of cross-reactivity with antibodies directed against other organisms. For the remaining 2 samples, the titers were > or = 1:128. One of these samples was from a blood donor dog that had never had any clinical signs of leishmaniasis. Follow-up samples from both dogs also had Leishmania IFA titers > or = 1:128. Both dogs had antibodies against Trypanosoma cruzi, as determined with a radioimmunoprecipitation assay. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that the seroprevalence of antibodies against Leishmania spp in dogs in the United States was low. However, results further suggested that leishmaniasis may not be limited to Foxhounds in the United States.