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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 (Tm) 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. Tm 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 Tm. 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.

Background Zoonotic taeniid cestodes are amongst the most important food-borne parasites affecting human health worldwide. Contamination of fresh produce with the eggs of Echinococcus granulosus ( s.l. ), Echinococcus multilocularis , and some Taenia species pose a potential food safety risk. However, very few studies have attempted to investigate the potential contamination of fresh produce with taeniid eggs and the available methods are not standardized for this purpose. Established protocols do exist for testing leafy greens and berries for contamination with protozoan parasites and are used in national surveillance programmes. This methodology could be suitable for the detection of taeniids. The objective of this project was to develop and standardize a sensitive and reliable method to detect contamination of leafy greens and berries with eggs of zoonotic taeniids and to differentiate between E. multilocularis , E. granulosus ( s.l .) and Taenia spp. Methods We compared the efficacy of different wash solutions to remove Taenia spp. eggs from spiked produce, assessed two DNA extraction kits for their performance on Taenia spp. eggs, and adapted a published conventional multiplex PCR into a real-time PCR with fluorescence melting curve analysis (MCA) that was optimized for use on produce washes. Analytical specificity of this protocol was assessed using non-spiked produce washes as well as a variety of other potentially contaminating parasites. Results The protocol as established in this study had an analytical sensitivity of detecting five eggs per spiked sample for both romaine lettuce and strawberries. Unequivocal identification of E. multilocularis , E. granulosus ( s.l .) and Taenia spp. was possible through MCA. Amplicon sequencing allowed identification of Taenia to the species level. The real-time PCR also amplified DNA from Dicrocoelium sp., but with a clearly discernable melting curve profile. Conclusion The new protocol for screening produce for taeniid contamination was highly sensitive. Melting curve analysis and the possibility of amplicon sequencing made this assay very specific. Once further validated, this method could be employed for surveillance of produce for contamination with taeniid parasites to assess potential risks for consumers.

Milk depression is the major driver of economic loss due to mastitis in dairy animals. The aim of this study was to identify potential mediators of milk depression by investigating the local and systemic changes in gene expression or cytokine production during endotoxin challenge of the mammary gland in a mouse model. The left and right sides of the 4th pair of mouse mammary glands were alternatively injected with either lipopolysaccharide (LPS, 055: B5, 50 μL of 0.4 mg/mL) or sterile PBS through the teat meatus 3 days postpartum (n = 9). The 4th glands were individually collected 12 h after LPS injection and analyzed to identify gene expression changes by RNA sequencing and real-time PCR, and the plasma was collected before and after LPS challenge and analyzed to determine the levels of 32 cytokines. Transcriptome analysis showed that in addition to strong pro-inflammatory responses, which included granulocyte and monocyte migration and cytokine production and signaling, the LPS-treated glands exhibited strong ubiquitin-mediated and immune-mediated proteasome activation and an increase in nitric oxide-mediated oxidative stress. Furthermore, LPS induced a down-regulation in vesicle membrane, vesicle-mediated trafficking, and metabolic processes of amino acids and other organic molecules in the mammary gland. Of the 32 cytokines analyzed, the levels of 24 (mainly IL-6, G-CSF, MCP-1, RANTES, MIG, MIP-1b, KC, MIP-2, IP-10, and TNFα) were increased or tended to increase in the blood after LPS treatment, and only the levels of IL-9 were decreased. In the mammary gland after LPS challenge, the levels of IL-5, IL-6, IP-10, LIF, MCP-1, MIP-2, and TNFα were significantly increased, and the levels of INFΥ, IL-2, IL-4, IL-10, and IL-12 (p40) were decreased. These observations provide potential markers and targets for further studies on the prevention and treatment of gram-negative bacteria-induced mastitis.

Highly active antiretroviral therapy (HAART) is associated with improvement or resolution of several HIV-associated opportunistic infections. Although prophylaxis against disseminated Mycobacterium avium complex infection may be successfully discontinued after a favorable response to HAART, the 1999 guidelines from the U.S. Public Health Service/Infectious Diseases Society of America recommend continuing therapy for disseminated M. avium complex infection, regardless of the response to HAART. To examine the outcome among patients with disseminated M. avium complex infection whose antimycobacterial therapy was discontinued after a favorable response to HAART. Retrospective chart review between May 2000 and May 2001. 13 Canadian HIV clinics. 52 HIV-infected adults (43 men; mean age, 37.3 years) in whom successful antimycobacterial therapy for disseminated M. avium complex infection was discontinued after a favorable virologic response to HAART. Survival, survival free of disseminated M. avium complex infection, and CD4(+) cell count responses. At the time of diagnosis of disseminated M. avium complex infection, the median CD4(+) cell count was 0.016 x 10(9) cells/L, and the median plasma HIV RNA level was 90 000 copies/mL (plasma HIV RNA levels were available for only 21 patients). The patients received a median of 32 months of antimycobacterial therapy that included ethambutol plus either clarithromycin or azithromycin. When antimycobacterial therapy was discontinued, the median CD4(+) cell count was 0.23 x 10(9) cells/L and the median plasma HIV RNA level was less than 50 copies/mL. A median of 20 months after discontinuation of antimycobacterial therapy, only 1 patient had developed recurrent M. avium complex disease (37 months after stopping antimycobacterial therapy). This patient had stopped HAART 2 months earlier because of uncontrolled HIV viremia. Twenty months after stopping antimycobacterial therapy, the other 51 patients had a median CD4(+) cell count of 0.288 x 10(9) cells/L; 34 (67%) had undetectable plasma HIV RNA levels, and 8 (15%) had plasma HIV RNA levels of 50 to 1000 copies/mL. Discontinuation of successful disseminated M. avium complex therapy after a successful response to HAART is safe and reduces patients' pill burdens, potential drug adverse effects, drug interactions, and costs of therapy.