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Salmonella sequestered in bovine peripheral lymph nodes may be a major source of ground beef contamination (Arthur et al. 2008, Gragg et al. 2013), and this infection is proposed to occur via transdermal introduction of the bacteria that is then shuttled to the nodes by lymphatic drainage (Edrington et al. 2013). Studies funded by the Beef Checkoﬀ highlighted a role for horn ﬂies as important reservoirs for, and mechanical vectors of, Salmonella (Olafson et al. 2014, Olafson et al. 2016). Interestingly, horn ﬂies are an intermediate host of Stephanoﬁlaria stilesi, a skin‐dwelling, ﬁlarial nematode that produces lesions on cattle resulting in dermatitis (bovine stephanoﬁlariasis). Nematodes can acquire bacterial pathogens from their environments, including Salmonella, and these pathogens can persist and be transmitted to subsequent nematode generations (Kenney et al. 2005, Lacharme‐Lora et al. 2009, Diaz and Restif 2014); acquisition of Salmonella by skin‐dwelling ﬁlarial nematodes via the hide or the intermediate arthropod host may provide an additional source of transdermally introduced pathogen. Several other ﬂy species are also known to vector arbonematodes that impact livestock, including Musca autumnalis (face ﬂy), Musca domestica (house ﬂy), and Stomoxys calcitrans (stable ﬂy). These non‐biting and biting dipteran species are frequently encountered on conventional and pasture settings, they exhibit seasonal variation in population density, and they have varied geographic distributions within the US. Collectively, these may account for the seasonal and regional diﬀerences observed in PLN prevalence. The frequency and diversity of skin‐dwelling ﬁlarial nematodes on cattle that inhabit conventional and pasture settings is not clearly documented in the US, nor is the prevalence of ﬁlariae harbored by ﬂies parasitizing these cattle and/or inhabiting these settings. Filling these gaps is critical to assessing an impact on Salmonella transmission due to interactions between the bovine host and these macroparasites.
The objective of the study was to screen for the presence of skin‐dwelling ﬁlarial nematode species from cattle hides and from ﬂy pests/ectoparasites inhabiting cattle production settings.
Horn, stable, and house ﬂy populations were collected on conventional and pasture settings from seven states (n=1,177) by either aerial sweep net or sticky traps (Fig. 1). Genomic DNA was isolated from individual ﬂies by maceration in a DNA Isolation buﬀer. These DNAs were used as template to detect nematode DNA using pan ﬁlarial primer pairs in PCR assays targeting nuclear rDNA. Reaction conditions consisted of optimized buﬀer (Advantage® HF2 PCR Buﬀer, Takara, Mountain View CA) for use with the Advantage® High Fidelity 2 Polymerase (Takara) system. Positive control genomic DNA templates from Diroﬁlaria immitis, Brugia malayi, and B. pahangi (BEI Resources, Manassas VA) and ‘no template’ controls were included in all experiments. Ampliﬁed products were puriﬁed and sequenced in both directions and resulting sequence data was compared to publicly available nematode sequences deposited in the Genbank database.
Less than 1% (n = 6) of the horn ﬂies screened were positive for nematode DNA using the nuclear rDNA assay (Table 1), and no products were ampliﬁed from house ﬂies or stable ﬂies. These indicate a low overall prevalence of nematode infection in ﬁlth ﬂy populations sampled in March – July. Biologically, stable ﬂies and house ﬂies do not associate with cattle for as long a period of time as horn ﬂies, and this may account for the diﬀerence in detection of ﬁlarial nematode DNA between the species. Two of the six sequenced nuclear rDNA products displayed similarity to publicly available ﬁlarial nematode sequences, while four were similar to non‐ﬁlarial species. Despite this, all six isolates remain ‘unidentiﬁed’ as they do not have high enough levels of sequence coverage to assign concrete identiﬁcations. This is, in part, due to a majority of the publicly available ﬁlarial nematode sequences being from the Onchocercidae, as they are associated with human diseases. Indeed, there are only a handful of sequences deposited for Filariidae nematodes, with no sequences deposited for S. stilesi, the nematode known to be vectored by horn ﬂies.
These data support molecular monitoring of ﬁlth ﬂy populations at livestock settings as an indicator for the prevalence of nematodes within cattle herds; however, limited nematode sequences in publicly available databases can preclude identiﬁcation. These data suggest that future sampling should be extended for the duration of the warmer season (June – October) when there is a reported increase in prevalence of Salmonella in peripheral lymph nodes, and it should focus on symbovine species, i.e. horn ﬂies.