abstract Rapid and reliable detection and identification of coccidian oocysts are essential for animal health and foodborne disease outbreak investigations. Traditional microscopy and morphological techniques can identify large and unique oocysts, but they are often subjective and require parasitological expertise. The objective of this study was to develop a real-time quantitative PCR (qPCR) assay using melting curve analysis (MCA) to detect, differentiate, and identify DNA from coccidian species of animal health, zoonotic, and food safety concern. A universal coccidia primer cocktail was designed and employed to amplify DNA from Cryptosporidium parvum , Toxoplasma gondii , Cyclospora cayetanensis , and several species of Eimeria , Sarcocystis , and Isospora using qPCR with SYBR Green detection. MCA was performed following amplification, and melting temperatures (T m ) were determined for each species based on multiple replicates. A standard curve was constructed from DNA of serial dilutions of T. gondii oocysts to estimate assay sensitivity. The qPCR assay consistently detected DNA from as few as 10 T. gondii oocysts. T m data analysis showed that C. cayetanensis , C. parvum , Cryptosporidium muris , T. gondii , Eimeria bovis , Eimeria acervulina , Isospora suis , and Sarcocystis cruzi could each be identified by unique melting curves and could be differentiated based on T m . DNA of coccidian oocysts in fecal, food, or clinical diagnostic samples could be sensitively detected, reliably differentiated, and identified using qPCR with MCA. This assay may also be used to detect other life-cycle stages of coccidia in tissues, fluids, and other matrices. MCA studies on multiple isolates of each species will further validate the assay and support its application as a routine parasitology screening tool.

Two enzyme-linked immunosorbent assay (ELISA) systems, one using natural excretory–secretory (ES) antigens and the other a synthetic glycan antigen (3,6-dideoxy- d -arabinohexose [tyvelose, TY]), were evaluated for the serological diagnosis of trichinellosis in swine. Sensitivity was estimated using samples (n = 113) collected 3–21 wk PI from 15 experimentally infected pigs, and specificity was estimated using samples (n = 397) from a population of Trichinella spp.–free pigs. Results were analyzed using 2 cutoff values recommended in international guidelines (Office Internationale des Epizooties [OIE]) and by the optimal cutoff level as determined by receiver–operator characteristic (ROC) analysis. The ROC-optimized TY-ELISA consistently performed better than all other combinations. None of the combinations of test and cut-off detected infected pigs sooner than 35 days; however, the ROC-optimized TY-ELISA identified 8 of 15 pigs earlier than the ES-ELISA and detected 2 pigs missed by all other tests. At 49 days PI the sensitivity and specificity of the ROC-optimized TY-ELISA were 94.3 and 96.7%, respectively, as compared with the ROC-optimized ES-ELISA at 84.9 and 96.0%, respectively. The ROC-optimized TY-ELISA was 100% specific at OIE-recommended cut-offs. This study indicates that the TY-ELISA is as good or better than the ES-ELISA for the detection of trichinellosis in swine.

A study was undertaken to determine whether domestic goats can serve as patent hosts of Elaphostrongylus cervi under natural or experimental conditions. Three-hundred and two fecal samples from 124 domestic goats raised outdoors in New Zealand, where E. cervi is enzootic, were tested for nematode larvae by the Baermann method. All samples were negative for E. cervi dorsal-spined larvae. Twenty juvenile male Nubian and Saanen goats obtained locally were assigned randomly to 5 dosage groups and were orally administered 5, 15, 35, 65, or 125 third-stage larvae of E. cervi , respectively. Two yearling female red deer ( Cervus elaphus elaphus ) each received 35 or 65 third-stage larvae as positive controls, and 2 uninoculated juvenile male goats served as negative controls. Fecal Baermann testing of pooled samples from the inoculated goats was conducted weekly for the first 80 days postinoculation (DPI) and daily thereafter until 250 DPI. No dorsal-spined larvae were recovered. One goat that had received 15 third-stage larvae displayed a mild transient posterior ataxia suggestive of cerebrospinal elaphostrongylosis. Gross postmortem examination did not reveal any direct evidence of nematodes in any of the goats, and only a few minor lesions were present. Histologically, these lesions were consistent with a parasite etiology. Histological evaluation of grossly normal lumbar and sacral spinal cord from 2 goats that had each received 125 third-stage larvae revealed eosinophilic meningoencephalitis and leukomyelitis, respectively, suggestive of the presence of parasites in the central nervous system. The 2 positive control red deer became patent with dorsal-spined larvae consistent with E. cervi at 131 DPI. These findings suggest that goats, at least those breeds utilized in this study, are not suitable patent hosts for E. cervi .