A multiplex PCR to simultaneously distinguish Cryptosporidium species of veterinary and public health concern in cattle

Authors: Santin-Duran, Monica; Zarlenga, Dante

Submitted to: Veterinary Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/27/2009
Publication Date: 10/1/2009
Citation: Santin, M., Zarlenga, D.S. 2009. A multiplex PCR to simultaneously distinguish Cryptosporidium species of veterinary and public health concern in cattle. Veterinary Parasitology. 166(2009):32-37.

Interpretive Summary: Cryptosporidium is an enteric protozoan parasite that infects a wide range of vertebrate hosts. Cryptosporidial infection is known now as one of the most common causes of diarrhea in humans and livestock. Four species of Cryptosporidium are routinely found in cattle. It is crucial to know which species are actually present in Cryptosporidium -positive samples to determine the public health implications because only C. parvum is a human pathogen. PCR Identification of Cryptosporidium species currently relies on expensive molecular methods. Incorporation of these methods is expensive, requires highly trained personnel, and therefore is limited primarily to a few research and public health laboratories. To overcome this problem a new assay was developed for simultaneously detecting the 4 commonly identified species of Cryptosporidium that infect cattle using PCR only. This assay specifically identifies Cryptosporidium oocysts present in cattle feces, improves the detection of mixed infections and reduces the time and cost relative to current sequencing methods.

Technical Abstract: Four species of Cryptosporidium are routinely found in cattle: Cryptosporidium parvum, C. bovis, C. ryanae, and C. andersoni. It is important to determine the species of Cryptosporidium in infected cattle because C. parvum is the only serious pathogen, for humans as well as cattle. Identification of Cryptosporidium species and genotypes currently relies on molecular methods such as polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) or gene sequencing. Incorporation of these techniques in a routine veterinary diagnostic laboratory is cost prohibitive and their application is limited primarily to research and a few public health laboratories. To overcome this problem a multiplex PCR assay was developed for simultaneously detecting the 4 commonly identified species of Cryptosporidium that infect cattle using PCR only. This assay specifically identifies Cryptosporidium oocysts present in cattle feces, improves the detection of mixed infections, reduces the time and cost relative to current sequencing methods, and further demonstrates the shortcomings of sequencing as the definitive method for identification when analyzing samples obtained from mixed infections.