Discussion
The concept of FMD as a fulminant, outbreak-driven animal emergency is
appropriate for considering the disease within FMD-free countries.
Useful as it is, the concept has subsequently been “imported” into
countries where FMDV is widespread and endemic, and where virus presence
is known to exist without ongoing clinical outbreaks (Farooq et al.,
2018; de Carvalho Ferreira et al. 2017). Although outbreaks occur
frequently in many endemic regions, they are not sufficient to explain
the high seroprevalence in many regions where no recent clinical
outbreak has been reported (van Andel, 2020b).
We found that 2.3% of healthy, commercially-slaughtered large ruminants
from an endemic region had detectable FMDV RNA within the oral cavity,
and that 1.5% had FMD RNA in the nasal cavity. The results were similar
for both Laos and Myanmar, suggesting this might be a pattern which is
common throughout the region. This is the first study to sample the oral
and nasal mucosal surface of a healthy population of large ruminants
within an endemic region, and adds to a small but growing literature
examining the state of FMDV infection in animals from endemic regions.
By comparison, in naïve cattle, oral and nasal shedding following
experimental infection sharply declines over 21 days (Parthiban et al.
2015) with extinction of virus in the oral or nasal mucosa regardless of
whether animals clear infection or become chronic carriers (Stenfeldt et
al., 2016). By contrast, the presence of FMDV RNA within the dorsal
nasopharynx, has been well-established by multiple other studies
(Stenfeldt et al., 2016; Barend et al 2016; among others) which have
defined the role of that region in the chronic carrier state. Although
FMDV detection in the nasopharynx is typically demonstrated by
collection of oropharyngeal fluid from live animals with a probang cup,
the current study demonstrates the utility of detection by swabbing the
nasopharynx in the post-mortem context.
The animals we sampled were chosen by convenience, and nothing was known
about their previous exposure to FMDV except that they originated from
an FMD-endemic region. None of the animals in this study had visible
scars, lesions or other clinical signs (e.g. salivation, lameness)
suggestive of current or recent FMD. We used NSP ELISA positivity as an
imperfect proxy for prior exposure, as a way of judging whether any
viral excretion might be related to pre-clinical acute FMD infection.
Vaccination with whole vaccines (which could cause NSP-positive
reactors) is thought to be low in both Laos and Myanmar, and was not
considered likely to influence NSP ELISA results. Animals with
detectable mucosal viral RNA at any site were significantly more likely
to be seropositive than the sampled population, but not all RNA-positive
animals had antibodies. Since development of antibodies postdates
initial infection and early replication, we hypothesized that animals
with detectable FMDV RNA but no antibodies might have been in the early
(neoteric) stages of infection (Stenfeldt and Arzt, 2020). Six
seropositive animals had detectable viral RNA in the oral and nasal
cavity. Since NSP serology is not serotype specific, one possible reason
for shedding is that these animals did not have protective antibodies to
the same virus that was detected by RT-PCR.
One benefit of slaughterhouses is that they aggregate animals and
present a single site for sampling many animals with minimal handling
complications compared to field sampling. Others have used
slaughterhouse sampling as a way of accessing FMDV samples, including in
Pakistan and Kenya: Navid et al (2019) found that 11% of buffalo tested
at Pakistani slaughterhouses carried detectable FMDV antigen in their
probang sample. Anderson et al. (1974) found that over 3% of Kenyan
cattle at slaughter had viable virus in pharyngeal scrapings. However,
animals sent for slaughter might not be indicative of a population at
large, and more work remains to be done before it is clear whether our
results can be extrapolated to the general large ruminant population of
Laos and Myanmar. In addition, animals transported for slaughter are
more likely to have undergone stress and mixing, with potential new
infection or reactivation of viral activity. The potential role of
stress in FMDV reactivation has been an area of interest, although
experiments using immunomodulatory drugs have been unable to effect
active shedding of virus in chronic carriers (reviewed more thoroughly
in Stenfeldt and Arzt, 2020).
Outbreaks of active FMD in Myanmar and Laos have been attributed to
incursions of new strains of FMDV (Bo et al, 2019; Khounsey et al.
2008). Our results suggest the interesting possibility that in addition
to these virus “incursions”, the epidemiology of FMDV spread within
endemic contexts could include subclinical viral infection and shedding
within populations of apparently healthy animals. Dissecting the
pathophysiology of infection in each animal in our study was not
attempted, since too much was unknown about previous exposure and
infection. Our intent was to evaluate a cross-section of healthy animals
for a possible role in viral shedding, to inform a regional
epidemiological understanding. Descriptions of viral location in chronic
carrier cattle and buffalo focus heavily on nasopharyngeal virus
detection (Stenfeldt et al., 2016; Moonen et al. 2004; de Carvalho
Ferreira et al. 2017). Our results suggest that perhaps more attention
should be paid to other mucosal surfaces, and that the relationship
between FMDV and large ruminants in endemic settings may differ from
that predicted experimentally. If small numbers of cattle and buffalo
contain viable virus on environmentally-accessible mucosal surfaces such
as the nose and mouth, there is the potential for low-level transmission
of virus on a regular basis between animals in close contact. This might
help explain the phenomena seen in FMDV-enzootic contexts, where
seroprevalence rises without disease (Bertram et al, 2018), and where
outbreak reporting imperfectly predicts seroprevalence (van Andel
2020b). The observation that healthy dairy cattle and buffalo shed virus
in their milk (Nawaz, 2019; Ahmed 2017), raises the possibility that in
endemic countries, some cattle might be exposed from birth to both virus
and protective maternal antibody. Additionally, intensive sampling of
buffalo in a dairy colony in Pakistan demonstrated that within one year
numerous serotypes and strains could move through a herd without any
clinical signs of infection (DOI: 10.1111/tbed.12963).
A limitation of this study is that we cannot be sure the presence of
viral RNA is indicative of the presence of viable FMDV. In addition,
although environmental control swabs were negative, the possibility of
cross contamination within different anatomical sites or between animals
cannot be discounted. Positive results however were widespread
throughout the sampling period and locations, suggesting contamination
was not a prominent cause of positivity. Dry swabs were employed for
sampling rather than metal cuvettes used at slaughter by Anderson et al.
(1974); this was to prevent possible contamination between animals.
Swabs were also considered an affordable, useful surveillance tool that
might enable use of the technique by agencies seeking to increase their
PCP-FMD surveillance options. Because we chose not to obtain probang
samples prior to slaughter in order to prevent possible contamination of
sites, our swab-based dorsal nasopharyngeal results are not comparable
with results from other studies. Future studies should make use of virus
isolation to determine presence of viable virus in animals with
detectable FMDV RNA, and should work to establish the relative
sensitivity of the post-mortem swab-based technique with more
established techniques such as the probang.
In summary, we found that 5.3% of healthy, asymptomatic cattle and
buffalo sampled during slaughter in Laos and Myanmar had detectable FMDV
RNA in their dorsal nasopharynx, and 3.4% had detectable FMDV RNA in
their oral and/or nasal cavities. Although the viability of this viral
RNA is unclear, our results suggest the possibility that FMDV present on
the oral and nasal mucosa of asymptomatic large ruminants could play a
role in the epidemiology of FMD in Southeast Asia. It is known that
surveillance approaches focussing solely on clinical outbreaks of FMD
may not be sufficient to detect all circulating virus in populations of
large ruminants. Yet, field sampling of live animals is highly
labor-intensive and often logistically prohibitive. The current study
demonstrates the utility of detection of FMDV RNA by swabbing of the
nasopharyngeal, oral and nasal mucosa in a post-mortem context, and may
constitute a useful complementary tool for surveillance in endemic
regions.