The source(s) of phages as well as their dissemination routes should be identified in a cheese manufacturing plant in order to implement long-term corrective actions to limit phage propagation and improve overall product quality (13
). Contamination sources and dissemination routes are not easy to identify, as several control points need to be checked. Because virulent dairy phages have a narrow host range (12
), it is not practicable to use culture assays to detect them in various dairy environments, including air samples. Additionally, phages can deteriorate and lose their infectivity during sampling and sample processing (40
). Molecular biology methods independent of phage infectivity and of bacterial hosts can facilitate the analysis of the viral content of environmental samples (40
). In this study, quantitative PCR using SYBR green fluorescence and primers specific to conserved regions was successfully performed to detect the two main lactococcal phage groups in various samples obtained from a cheese manufacturing plant, including air samples.
Only a few studies to date have shown that airborne phages can be detected in industrial cheese manufacturing plant settings (30
). We also had some anecdotal evidences that ventilation breakdowns lead to increased phage contamination. Our analyses of air samples confirmed that lactococcal phages can be disseminated through the airborne route, since up to 2.7 × 104
and 6.6 × 104
lactococcal phage genomes per m3
of air were detected using the BioSampler (Table ). Considering that it takes only a few infectious phages to infect phage-sensitive L. lactis
cells and start the phage lytic cycle, cheese factories should possess adequate ventilation and control the airflow to minimize phage dissemination as much as possible.
Our study also clearly demonstrates that phage genomes can be found on various surfaces, including floors, walls, cleaning materials, pipes, door handles, and office tables. While it is not known if these viral nucleic acids are still part of infectious phages, it is safe to assume that they were at some point. These findings underscore the need to train workers regarding the importance of surfaces as sources of phage contaminations. They also suggest that the use of appropriate cleaning procedures and effective sanitizers needs to be carefully evaluated to reduce the risks of phage problems.
Although we successfully detected phages in aerosols using different air samplers, not all samplers showed the same level of efficiency. The air sampling devices used in earlier studies on airborne phages in cheese manufacturing plants (30
) were based on gelatin membrane filtration and on impaction on agar. Although the devices were able to detect phages, they have practical drawbacks. For example, gelatin membrane filtration can dry and break during prolonged sampling, or it can dissolve if liquid droplets are sampled. In contrast, impaction on agar relies on plaque assays, which is difficult to apply when a variety of bacterial strains are employed daily. Besides the concern about drying during prolonged sampling with the latter system, only large aerosol particles can be sampled. In fact, there is no standard and approved protocol to detect viral aerosols, let alone phages.
The NIOSH sampler gave the most reliable results in this study. It had the highest proportion of positive samples, low between-sample variability, and the lowest detection limit. The two stages of the NIOSH sampler were analyzed separately to take advantage of the aerodynamic size separation that took place with this sampler. At a sampling rate of 10 liters/min, the 50% cutoff is 2.1 μm for the first stage and 0.41 μm for the second stage, while the remainder of the aerosol is captured by the third stage. The second stage of this sampler detected the lowest airborne concentrations of 936-like phages, while the first stage detected the highest concentration. This suggests that most airborne 936-like phages where bound to larger particles. However, for the c2-like phages, there was no difference in the concentrations detected in the two stages of the sampler, indicating that these phages were present at similar concentrations on the smaller and the larger particles. It is not clear at present why this difference was observed. Both phage groups belong to the Siphoviridae
family but have somewhat different morphology. Phages belonging to the 936 group have an isometric capsid that is approximately 60 nm in diameter and a long noncontractile tail ranging from 140 nm to 200 nm (morphotype B1), whereas c2-like phages have a prolate capsid (60 nm by 40 nm) and a 100-nm-long noncontractile tail (morphotype B2) (26
Of all the samplers tested, the BioSampler allowed the highest recovery of airborne phage concentrations. However, given its very high detection limit and the few positive samples collected, this sampler was less suitable for the determination of the airborne phage concentrations.
The Coriolis sampler has the advantage of collecting a large volume of air in a very short time period. However, with its very high flow rate (300 liters/min), this sampler can draw in particles with greater inertia (greater aerodynamic size) than the filters (2 liters/min), the NIOSH sampler (10 liters/min), or the BioSampler (12.5 liters/min). Considering that the mass of a particle is proportional to the cubic value of the radius, a few large particles can drastically raise the concentration of airborne viruses detected. The presence of these larger aggregated particles in the liquid sample can also cause large differences in viral concentrations between aliquots (Table , c2-like phages). Our attempts to reduce these variations consisted of purifying the viral DNA with commercial kits and exposing the samples to sonication (data not shown). However, viral DNA purification lowered the concentrations of DNA and led to the underestimation of the viral load in the air sample, while sonication had no effect on the sample variability (data not shown). This inherent variability may be due to the variation of the concentrations of airborne viruses over the course of a day, reflected by the short sampling period of the Coriolis sampler. A sampler that slowly collects its sample, like the NIOSH sampler or PC and PTFE filters, likely provides a more representative evaluation of the airborne viral concentration over the course of a day.
In conclusion, various types of samplers were successfully used to collect airborne viruses, but the NIOSH sampler was the most efficient. Most samplers detected concentrations of at least 103
of air for both the lactococcal 936 and c2 phage groups. The NIOSH sampler results indicate that a significant portion of the airborne phages was bound to small particles (<2.1 μm). Since these smaller particles can remain airborne for longer periods of time and are influenced by air movements, it is likely that they can be carried far away from their aerosolization source. Although the dynamics of airborne viral transmission are poorly understood (29
), appropriate ventilation practices should reduce airborne dissemination. Finally, a qPCR protocol was effectively adapted to detect lactococcal phages. However, it is not known whether these phages were active or inactive or whether they were inactivated by the sampling/elution procedure. The detection level and the limit of detection are the most important characteristics to consider when choosing a sampler for field studies.