Discussions
Our aim was twofold: (1) Develop and validate a SARS-CoV-2 crude lysate ELISA that could be routinely applied for surveying the presence of SARS-CoV-2 like antibodies in wild and domestic animals (2) utilize the validated SARS-CoV-2 crude lysate ELISA to analyses antibody responses in camels, cats and dogs from geographically widely separated areas in Kenya.
The SARS-CoV-2 crude lysate ELISA that we developed represent a major advance, particularly with respect to the ability to conduct broad analyses of SARS-CoV-2 like antibody responses in livestock and wildlife. To validate our SARS-CoV-2 crude lysate ELISA, we modified a commercial (PISHTAZ TEB) kit coated with Spike specific -RBD antigen and used an ELISA protocol developed by Amanat et al., (2020 to detect SARS-CoV-2 antibodies in camels, cats and dogs (Fig. 1, 2 & 3). By modification of the commercial kit by replacing anti-human IgG-HRP with anti-Llama IgG HRP for camel samples and dogs samples (with anti-human IgG-HRP) we were able to positively detect SARS CoV-2 like antibodies in those animal species. The modified commercial kit gave 83% exposure for camel samples, at the 10% PP cutoff recommended by the manufacturers for human samples. Using the more stringent 20% PP cut off we were still able to record a 57% exposure and the negative controls were convincing. As already mentioned, these high titers diminish concerns that positive samples could largely be accounted for by antigenic cross reactions and not actual SARS-CoV-2 viral exposures.
Our comparative validation analysis revealed that the SARS-CoV-2 crude lysate ELISA corroborated the results obtained using the commercial kit based on the Spike specific RBD antigen (Fig. 9 and 10), albeit with a few more positive samples. This however is not surprising and one plausible explanation could be that SARS-CoV-2, like many pathogens induce antibody responses to multiple antigens. In this respect, it is important to recognize that there could be additional epitopes in the crude lysate that are potentially identified by the antibodies (whole spike S1 and the RBD, nucleocapsid protein and the membrane protein). Although, the spike S1 and RBD portions could be the most immunogenic (Meyer et al., 2014; Tai et al., 2020), it is conceivable that in the crude lysate, the other two antigens present could help in boosting the overall antibodies titers and hence increase the chance of positivity. On the basis of these findings, we suggest that the crude lysate can be used for initial screening as it avoids false negatives while simultaneously taking into account potential background cross-reactivity.
The data presented here, based both on our SARS-CoV-2 crude lysate ELISA and the modified commercial kit, indicate that SARS-CoV-2 antibodies or antibodies cross-reacting with the SARS-CoV-2 virus, are present in camels, cats and dogs in Kenya. An obvious question arising from these findings on the presence of SARS-CoV-2 in camel, cats and dogs in Kenya concerns how these infections relates to the human SARS-CoV-2 that has triggered an ongoing crises of public health, and economic wellbeing worldwide, including Kenya and continue to cause deaths daily. It is important to emphasize here that some of the camel samples that tested positive had been collected prior to the first reported human case in Kenya. Although this observation in itself does not suffice to rule out the possibility that the suggested exposure might have been a case of human to animal transmission, it suggests that this is an issue worthy of further investigation. This is because one possibility suggested by these results is that, the virus or a closely related virus had been in existence in Kenya before the pandemic. If that were to be the case, it would mean that the animals and or human have been previously exposed and so could reasonably be expected to have developed a certain level of protection against the SARS CoV-2 virus. If this were the case, understanding the mechanism leading to such protection would be the next logical step and the findings could have far reaching implications.
It is now known that the origin of SARS-CoV was in bats and that it was transmitted to humans via civet cats (Wang and Eaton 2007) while MERS virus is known to have originated in camels (Dudas et al., 2018). As already mentioned, the early evidences had suggested that pangolins were the mammals originating the pandemic in humans; possibly after a failed attempt to sneak Malaysian pangolin into China and subsequent single cell sequencing suggesting that SARS-CoV-2 could infect pangolin cell types (Chen et al., 2020). The probable cause of the infections in their case could have been from human transmissions during the shipment or rather from the other animals being traded together at the illegal market. More recently, through experimental studies cats (Shi et al., 2020; Chen et al., 2020), ferrets (Kim et al., 2020), dogs (Shi et al., 2020), hamsters (Chan et al., 2020; Osterrieder et al., 2020), bats (Schlottau, et al., 2020) and non-human primates (Lu et al., 2020; Denis, et al., 2020; Hartmann et al., 2020; Munster, et al 2020) can be infected with SARS-CoV-2 and transmission can occur (Damas et al., 2020). Dogs appear to have limited susceptibility to SARS-CoV-2, while other domestic species including pigs and poultry do not appear susceptible (Shi et al., 2020; Suarez et al., 2020). While most of these studies have relied on experimental infection, natural SARS-CoV-2 infections in animals haven’t been confirmed. There are a few cases of 14 pets, 8 captive big cats and farmed mink thought to have been positive for SARS-CoV-2 (Hobbs & Reids 2020). Thus our data is a valuable addition to the efforts aimed at ultimately confirming possible viral exposure in domesticated animals (Camel, Cats and the Dogs) (Fig 3,5,6,7 8 &).
Though the camels showed no clinical symptoms of COVID-19, cats and dogs were brought for treatments and thus it might have been possible that they were somehow symptomatic. Furthermore, infections in dogs are typically asymptomatic (Hobbs and Reids 2020) and hence it might have been hard to suspect SARS-CoV-2. Whether the seropositivity were as a result of natural exposure or human to animal transmission is still unclear, though the animal numbers 18, 19, 20, 21 and 22 have been traced to owners who were exposed to COVID-19, while dog number 22 and 23 came from owners with confirmed SARS-CoV-2 (Fig. 8). Furthermore like in the case of the camel samples, the fact that the commercial antigen based on spike RBD was used to screen the sample and the result gave an exposure level of 83% is reinforces the possibility that some of the animals testing positive are as a result of SARS-CoV-2 exposure rather than non- specific cross-reactions. It’s important to note that some of the samples (n=23) which tested positive (Fig.9) had been collected 1 week before the first confirmed human case in Kenya. This might confirm the fact that the disease could have been in the country much earlier or overlaps from other coronaviruses such as MERS COV, SARS COV-2 or HKU8 is a possibility. SARS COV-2 antibodies have been shown to be present in cat samples, taken after the SARS-CoV-2 outbreak in Wuhan China (Qiang et al., 2020) with titers ranging 1/20 to 1/1080.
The presence of SARS COV-2 antibodies in animal might not be limited to Kenya alone, in the UK they have shown the presence of the antibodies in sera collected during the second wave, 1.4% (n=4) of dogs and 2.2% (n=2) cats tested positive for neutralizing antibodies (Smith et al., 2021). There was a strong reaction and high titers in camel samples, while dogs and cats showed lower titers. The high titers and strong reaction in camels might be due to either co-infection with MERS CoV or HKU8, as these viruses have been shown serologically to co-infect the dromedary camels in the same region where we collected the samples (Wei Zhang et al 2019) and therefore their cross reaction especially with the crude lysate ELISA (Fig 6) cannot be ruled out. The fact that both MERS COV and HKU8 which is a bat Coronaviruses has been previously found in camel and also the fact that coronaviruses have been shown to undergo recombination could elicit questions relating to whether that recombination has led to a more human infective SARS-CoV-2 . This data should be supported by molecular screening of the camel, cat, dog and bat samples for possible presence of SARS-CoV-2 viruses. Bat samples need to be tested, especially those totally in the wild. Bats are suspected as the key reservoir of the viruses and show close similarity with SARS-CoV-2 with as much as 96.2% identical in genome sequencing of SARS-CoV-2 with bat CoV RaTG13 (Zhou et al.,2020).
In summary the present study demonstrated for the first time the presence of SARS-CoV-2 antibodies in camels, cats, bats and dogs. Studies aimed at validating these findings will most likely benefit from the array of molecular tools for virus characterization. Serological detection of other coronaviruses in the same blood samples is also a priority area for further research. More broadly, the implication of our data on the current pandemic cannot be understated as the following key question need to be addressed: (1) If the present results are attributable to specific cross-reaction with other corona viruses such as MERS COV or HKU8, then would the antibodies neutralize, block or even improves immunity against SARS-CoV-2 and (2) If indeed the animals examined were SARS-CoV-2 positive then did the viruses exist in the country before the pandemic or is it the case of human to animal transmissions. If it’s the latter, an additional question related to the duration for which the virus has been in humans in Kenya prior to detection and what the correlates of immunity in such a symptomatic human hosts might be important to scrutinize.