Investigating genetic diversity within Cryptosporidium parvum outbreaks using multi-locus variable number tandem repeat analysis

<i>Cryptosporidium parvum</i> is a zoonotic protozoan parasite of human and veterinary public health concern that causes gastrointestinal disease. Animal contact is a major risk factor for <i>C. parvum</i> outbreaks which require thorough investigation through the use of molecular subtyping. Recently, a multi-locus variable-number tandem repeat analysis (MLVA) scheme was established for <i>C. parvum</i>, offering improved subtyping resolution compared to the commonly used single-locus 60 kDa glycoprotein gene (<i>gp60</i>) subtyping approach. Using the <i>C. parvum</i> MLVA scheme, the genetic diversity of known <i>gp60</i> subtyped faecal DNA extracts collected between April 1st 2023 and March 31st 2024 was explored. A representative group of a common Scottish <i>gp60</i> subtype (IIaA15G2R1, <i>n</i> = 28) was analysed by MLVA and found to consist of 8 distinct complete MLVA profiles, with 4-12-5-7-27-36-16 (<i>n</i> = 12) being the most common. Genetic diversity within samples involved in three historic animal contact outbreaks (Outbreaks A, B and C) was investigated. Outbreak A, involving a single <i>gp60</i> subtype (IIaA19G1R1), consisted of only one MLVA profile (4-12-5-8-27-15-17). Outbreak B was caused by two <i>gp60</i> subtypes (IIaA17G1R1 and IIaA15G2R1), which were further subdivided into four MLVA profiles, two per <i>gp60</i> subtype (4-14-4-7-27-37-15 and 4-14-5-7-27-27-15, and 4-13-4-8-27-31-17 and 4-12-5-7-27-42-16, respectively). Lastly, Outbreak C, thought to have two-point sources of infection, involved one <i>gp60</i> subtype (IIaA15G2R1), which was subdivided into four distinct MLVA profiles (4-12-5-7-27-36-16, 4-12-5-7-27-32-15, 4-12-5-7-27-30-15, and 4-14-5-7-36-33-15). Improved MLVA resolution allowed outbreak specimens with insufficient epidemiological data to be linked to a source through sharing a common MLVA profile.