Espí Malillos, Alba

Listeria monocytogenes, a contaminant of raw milk, includes hypervirulent clonal complexes (CC) like CC1, CC4, and CC6, highly overrepresented in dairy products when compared to other food types. Whether their higher prevalence in dairy products is the consequence of a growth advantage in this food remains unknown. We examined growth kinetics of five L. monocytogenes isolates (CC1, CC4, CC6, CC9, and CC121) at 37 and 4 °C in ultra-high temperature (UHT) milk and raw milk. At 4 °C, hypovirulent CC9 and CC121 isolates exhibit better growth parameters in UHT milk compared to the hypervirulent CC1, CC4, and CC6 isolates. CC9 isolate in raw milk at 4 °C exhibited the fastest growth and the highest final concentrations. In contrast, hypervirulent isolates (CC1, CC4, and CC6) displayed better growth rates in UHT milk at 37 °C, the mammalian host temperature. Proteomic analysis of representative hyper- (CC1) and hypovirulent (CC9) isolates showed that they respond to milk cues differently with CC-specific traits. Proteins related to metabolism (such as LysA or different phosphotransferase systems), and stress response were upregulated in both isolates during growth in UHT milk. Our results show that there is a Listeria CC-specific and a Listeria CC-common response to the milk environment. These findings shed light on the overrepresentation of hypervirulent L. monocytogenes isolates in dairy products, suggesting that CC1 and CC4 overrepresentation in dairy products made of raw milk may arise from contamination during or after milking at the farm and discard an advantage of hypervirulent isolates in milk products when stored at refrigeration temperatures.

Introduction: Learning molecular genetics techniques is part of the program of the genetics course of the veterinary degree. It is essential to maintain motivation and interest in genetics practice by using a common thread that connects with the students’ interests. This work describes the design, implementation, and results of a gamification strategy developed during two practical sessions in which the student becomes a geneticist to identify which dog a faecal sample collected from the street belongs to, as some municipalities currently do to promote hygiene and public health in their streets. The aim was for students to understand basic concepts and techniques in molecular genetics during these two sessions. This included learning how to extract DNA from different types of samples, describing the amplification using Random Amplification Polymorphic DNA (RAPD), and getting a better understanding of molecular markers and the theory behind Polymerase Chain Reaction (PCR). Methodology: In the first practice, students extracted DNA from fresh dog faeces, while also discussing various DNA sources and extraction methods. Then they used the extracted DNA to create a simulated database of fictional dogs associated with students. They quantified the DNA, analysed its quality, and prepared a dilution to 10ng/μL. In the second practice, the students used RAPD to identify individuals by matching DNA from a simulated collected faeces sample to their fictional dogs DNA database. They performed amplification reactions with various primers pairs, followed by gel electrophoresis, to compare DNA band patterns and identify the dog and the fictional student associated with the uncollected dog faeces. The advantages and limitations of the RAPD technique were discussed, along with its potential applications in veterinary science and genetics. Results and discussion: The students were successful in extracting DNA with concentrations over 100 ng/μL in most cases as well as a good purity with respect to proteins. However, it was found that there was usually a low quality of DNA with respect to salts, although this did not influence the results of the second practice. They were able to generate reproducible RAPD profiles with all primer pairs. The unknown individual could be easily recognized within the database. Conclusions: It is concluded that this educational proposal is an effective option for teaching DNA extraction and the RAPD technique, as well as for many molecular genetics terms and concepts and contributes to the comprehensive training of future veterinary professionals. Additionally, the educational and social value of the practices are highlighted, as they promote interest in science, respect for the environment, and civic responsibility.