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.