4. DISCUSSION
The role of cattle in SARS-CoV-2 transmission remains unclear. Although
cattle are potentially sensitive to SARS-CoV-2, this study aimed to
determine the susceptibility of cows to active SARS-CoV-2 infection at a
farm where there was a COVID-19 outbreak among the farmworkers.
SARS-CoV-2 and BCoV are both betacoronaviruses, and BCoV is very similar
to human coronavirus (HCoV) OC43 (Betacoronavirus 1) (Wensman &
Stokstad, 2020). Thus, after the samples tested positive for SARS-CoV-2,
they were also analysed for BCoV.
In addition to seroneutralisation, the other methods used for the
diagnosis of SARS-CoV-2 include PCR (to amplify and quantify nucleic
acids) and new ELISA tests (using serum from infected or vaccinated
animals), which allow the detection of antibodies in a wide range of
animal species or the discovery of reservoirs or intermediate hosts
(Wernike et al., 2021). In our study, we introduced chemiluminescence, a
methodology mainly used for qualitative and quantitative evaluation of
the antibody response in humans to infection with the wild strain of
SARS-CoV-2 and to the vaccine antigen, specifically anti-S
(quantitative). Using this test, our results showed that 15 of 24 cows
were positive for SARS-CoV-2, which was the only betacoronavirus
detected by serological tests.
The limited data on cattle, including our results, indicate that cattle
show low susceptibility to SARS-CoV-2 and probably do not function as
reservoirs. However, we suppose that in areas with large cattle
populations and a high prevalence of SARS-CoV-2 infection in humans,
close contact between livestock and farmworkers may cause reverse
zoonotic infections in cattle, as has already been described for highly
sensitive animal species, such as minks, cats, and dogs (Fenollar et
al., 2021; Decaro et al., 2021a; Clayton et al., 2022).
Interestingly, in our study, Pearson correlation analysis showed a
strongly significant relationship (P < 0.01) between anti-S
and anti-N antibodies, and a significant correlation (P <
0.05) between anti-S and neutralising antibodies against SARS-CoV-2 with
age. Particularly, of the 13 cows with neutralising antibodies to
SARS-CoV-2, only two were young animals (aged 1–5 years), whereas 11
were adults (aged 6–10 years); this significant difference is similar
to reports of SARS-CoV-2 infections in humans. In humans, children seem
to be less susceptible to SARS-CoV-2 than adults. This could be a result
of several factors, such as the decline of immune protection due to
aging, modulation of angiotensin converting enzyme 2 (ACE2) receptor
expression, and previous human-CoV infections (Felsenstein et al., 2020;
Wu et al., 2020; Dioguardi et al., 2021; Zhang et al., 2021).
Of the 13 cows with neutralising antibodies
against SARS-CoV-2, six were
pregnant. Further studies are required to evaluate this interesting
result. To date, there is very little data on COVID-19 during pregnancy
and vertical transmission in animals. For example, adult white-tailed
deer are highly susceptible to SARS-CoV-2 infection and can transmit the
virus vertically (Cool et al., 2022). Thus, there are concerns about the
risk of neonatal infections in the postpartum phases (Salma, 2021).
The data from the current study indicate that cattle do not seem to be
vehicles for the transmission of SARS-CoV-2. Indeed, after experimental
infection of a group of calves with SARS-CoV-2 by intranasal
inoculation, no intraspecies transmission of the virus to uninoculated
cattle that were in contact with inoculated cattle was detected at 24
hours post infection (Ulrich et
al., 2020). Therefore, based on the results of our study, we conclude
that there is no indication that cattle play a role in the SARS-CoV-2
human pandemic.
The susceptibility of various animal species to SARS-CoV-2 is of great
interest to the international scientific community, and it has been
hypothesised that the host range of SARS-CoV-2 may depend on the
interaction of the virus spike protein with host cell receptors. ACE2
plays a crucial role in host cell entry of the virus. Based on
phylogenetic and expression pattern analyses of ACE2, various mammals
may be susceptible to SARS-CoV-2. However, the amino acid sequence of
ACE2 is highly conserved in cattle (83% homology) (Lean et al., 2021)
and cattle share four of the five hotspot residues with humans,
suggesting a good probability of interaction between ACE2 and the spike
protein of SARS-CoV-2 (Liu et al., 2020). Other studies confirmed these
data (Bentum et al., 2022; Clayton et al., 2022; Lupala et al., 2022).
In humans, ACE2 is mainly expressed by the epithelial cells of the lung,
intestine, kidney, heart, and blood vessels (Liu et al., 2020). In
contrast, ACE2 receptor expression in cattle is only moderate in the
lungs but is higher in the liver and the kidneys (Sun et al., 2020).
Despite earlier reports on SARS-CoV-2 replication in respiratoryex vivo organ cultures of cattle (Di Teodoro et al., 2021) and
detection of low viral RNA levels after experimental intranasal
inoculation of SARS-CoV-2 in cattle (Ulrich et al., 2020), the nasal
swabs collected in our study were negative for SARS-CoV-2. A possible
explanation may involve the different distribution of ACE2 receptors in
cattle. Indeed, a recent investigation established that in cattle
(Bos taurus ), ACE2 was detected in the bronchiolar epithelium in
the lungs, but not in the nasal mucosa epithelium (Lean et al., 2021).
According to Lean et al. (2021), we hypothesise that this pattern of
distribution may explain the difference in the susceptibility of animals
to SARS-CoV-2.
Despite the negative results from swabs, the serological analyses in our
study confirmed infection of cattle with SARS-CoV-2 for the first time,
although no active viral replication was detected in colostrum-deprived
calves that were experimentally infected (Falkenberg et al., 2021). To
the best of our knowledge, no cases of natural SARS-CoV-2 infection in
cattle have been previously reported. As stated above, the serum samples
were negative for BCoV infection. Thus, we ruled out the possibility of
cross-reactivity due to other betacoronaviruses.
Finally, to avoid economic losses and threats to animal health,
biosecurity measures to control SARS-CoV-2 infections may be useful.
Conventional measures, including spray disinfection of each vehicle
entering the farm, ultraviolet light, and incoming and outgoing showers
for service personnel, as well as emerging technological measures, such
as electrostatic air filtration systems and heat treatments at high
temperatures for disinfection (Subedi et al., 2021), which have been
tested on chicken and pig farms in the USA, may be effective methods.
In conclusion, further studies are needed to validate the
chemiluminescence technique used to detect SARS-CoV-2 antibodies in
cattle, which appeared to seroconvert in the presence of circulating
virus. the advantages of the chemiluminescence techniques described
above are their speed, performance, and the fact that they are
inexpensive and can be carried out in BSL-2 labs.
To our knowledge, this is the first report of natural SARS-CoV-2
seroconversion in cattle. We hypothesise that this may represent a case
of reverse zoonosis. However, the role of cattle in SARS-CoV-2 infection
seems to be negligible. Further studies are needed to better define the
role of SARS-CoV-2 infection in cattle as well as its potential role in
the emergence of novel recombinant coronaviruses.