Noninvasive Detection of Equid Herpesviruses in Fecal Samples

Abstract

Equid herpesviruses (EHVs) are pathogens of equid and nonequid hosts that can cause disease and fatalities in captivity and in the wild. EHVs establish latent infections but can reactivate, and most EHVs are shed via the nasal passage. Therefore, nasal swabs are generally used for EHV monitoring. However, invasive sampling of wild equids is difficult. While feces is a commonly used substrate for detecting other pathogens, to our knowledge, EHVs have never been detected in feces of naturally infected equids. We systematically tested zebra feces for EHV presence by (i) establishing nested PCR conditions for fecal DNA extracts, (ii) controlling for environmental EHV contamination, and (iii) large-scale testing on a free-ranging zebra population. A dilution minimizing inhibition while maximizing viral DNA concentrations was determined in captive Grévy’s zebra (Equus grevyi) fecal samples from individuals shedding EHV nasally. Sixteen of 42 fecal samples (38%) were EHV positive. To demonstrate that the EHV positivity was not a result of environmental contamination, rectal swabs of wild zebras were screened (n = 18 [EquusquaggaandE. zebra]), and 50% were EHV positive, indicating that the source of EHV in feces is likely the intestinal mucosa and not postdefecation contamination. Out of 270 fecal samples of wild zebras, 26% were EHV positive. Quantitative PCRs showed that the amount of virus DNA in feces was not significantly smaller than that in other samples. In summary, fecal sampling facilitates large-scale screening and may be useful to noninvasively investigate phylogenetic EHV diversity in wild and domestic equids.IMPORTANCEEquid herpesviruses (EHVs) establish latent infections, and many EHVs are shed and transmitted via nasal discharge primarily through droplet and aerosol infection. Obtaining nasal swabs and other invasive samples from wildlife is often not possible without capture and physical restraint of individuals, which are resource intensive and a health risk for the captured animals. Fecal EHV shedding has never been demonstrated for naturally infected equids. We established the conditions for fecal EHV screening, and our results suggest that testing fecal samples is an effective noninvasive approach for monitoring acute EHV shedding in equids.

Immune differences in captive and free-ranging zebras (Equus zebra and E. quagga)

“…Equine herpesviruses are endemic in equid populations throughout the world, with prevalences of over 90%, depending on the EHV strain (Ataseven et al 2009; Barnard and Paweska 1993;Borchers and Frölich 1997;Ehlers et al 1999;Guevara et al 2018;Seeber et al 2019). Contrary to our expectations, lytic EHV infection was not a significant predictor for general immune function and, thus, might not function as a proxy measure of general immune function in zebras.…”

Terrestrial mammal surveillance using hybridization capture of environmental DNA from African waterholes

“…The compiled mitochondrial sequences were submitted to Arbor Biosciences for custom design of 80 bp baits. In total, 19,496 unique baits were produced; the bait set was made available in an online repository (https://doi.org/10.17632/zsd3w5vttk.1; Seeber et al, ). Due to the expected degree of target DNA degradation in environmental samples (Pilliod, Goldberg, Arkle, & Waits, ), baits were tiled threefold to allow for overlap and ensure sufficient coverage.…”

“…Seeber et al, 2019). Due to the expected degree of target DNA degradation in environmental samples(Pilliod, Goldberg, Arkle, & Waits, 2014), baits were tiled threefold to allow for overlap and ensure sufficient coverage.For each hybridization capture, we pooled two genomic libraries of approximately equal DNA concentration.…”

Environmental Detection and Potential Transmission of Equine Herpesviruses

“…The use of feces for EHV monitoring can facilitate sampling at larger scales and would be useful for screening active virus shedding in wild and domestic equids. In free-ranging and captive zebras, various EHV species were found to occur in feces, with EHV-5 and asinine herpesvirus 5 being most common [ 17 ]. However, EHV species detected in feces also included viruses that have been reported relatively rarely in previous studies (e.g., EHV-7, Wild Ass herpesvirus, and Equus zebra herpesvirus) [ 31 ].…”

“…In the case of nasal shedding, the host’s gut passage time must be considered, for example, EHVs stemming from swallowed nasal exudate would be expected to occur in feces only 1–2 days after the start of nasal virus shedding [ 32 ]. Compared to nasal swabbing, fecal EHV screening produces a higher rate of false-negative results [ 17 ], which, apart from presumably lower abundance of EHV in feces, may also be an effect of PCR-inhibiting substances common in feces (e.g., bile salts, plant secondary metabolites, and complex sugars) [ 17 ]. Thus, EHV screening of fecal samples may need to be optimized regarding the dilution of PCR template in order to obtain the optimal ratio of target DNA to PCR inhibitors.…”