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\begin{document}

\title{First detection of SARS CoV-2 like antibodies in domestic and wild
animals in Kenya}



\author[1]{Tindih Sheltone Heshborne}%
\author[2]{Isaiah Obara}%
\author[3]{Marcello Alessandro}%
\author[4]{Akinyi Matindi Mercy}%
\author[5]{John Nyambega}%
\author[4]{Adinoh Omondi George}%
\affil[1]{Machakos University}%
\affil[2]{Freie Universitat Berlin Fachbereich Veterinarmedizin}%
\affil[3]{International Centre for Genetic Engineering and Biotechnology}%
\affil[4]{Institute of Primate Research}%
\affil[5]{Directorate of Research Kenyatta University Teaching and Referral Hospital}%


\vspace{-1em}



  
  \date{June 18, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
The severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2),
etiological agent of the novel coronavirus disease 2019 (COVID-19), has
spread since December 2019, resulting in massive health and economic
crisis worldwide. While efforts to stop the pandemic are crucial,
collecting epidemiological data to help manage current and future
pandemics will be important. In addition to humans, serological and
molecular based studies have demonstrated SARS CoV-2 exposure in several
wild, domestic and farmed animals. For examples Shriner and the team
showed serologically an exposure of 40\% to the white deer living in
close proximity to urban centers. Additional reports have also emerged
of susceptibility of animal's species like cats, ferrets, raccoon dogs,
cynomolgus macaques, rhesus macaques, white-tailed deer, rabbits,
Egyptian fruit bats, and Syrian hamsters to SARS-CoV-2 infection.. It's
worth emphasizing that these reports are based on experimental data
mostly derived from Europe, USA, South America and parts of Asia. In
limited instances natural infections of SARS-CoV-2 have been reported in
pet dogs, cats, tigers, lions, snow leopards, pumas, gorillas at zoos
and farmed mink and ferrets. The presence of the virus in animal species
and an understanding of whether these are natural or recent human to
animal transmissions is important. It's possible that such transmission
could passage the virus or subject the virus to a different
immunological pressure thereby helping with the development of viral
variants in addition to being a host for future reservoirs of the virus.
In Kenya SARS-CoV-2 was first detected on March 12 \textsuperscript{th}
2020 from imported human cases of persons who had travelled from the
United States. This was followed by detection of imported cases majorly
from China, Sweden and United Kingdom. Later infections were confirmed
in Nairobi and Mombasa suggesting further cases of disease importations
through the major ports of entry. However, no comparable data on animal
exposure have hitherto been generated in Kenya. To address this key
concern, we focused on three objectives; 1) development of a robust
antibody ELISA based on crude SARS-CoV-2 lysate. 2) SARS-CoV-2 serology
of domestic animals in Kenya. 3) Corroboration of the crude lysate based
seroprevalence data and a commercial ELISA kit based on the Spike
receptor binding domain (RBD) antigen. Our sample set included camel
sera (both pre- \& post outbreak sera), as well as sera from cats and
dogs collected at the peak of the pandemic. Our results using the ELISA
based on crude SARS-CoV-2 lysate indicated SARS-CoV-2 antibodies in
camels (71\%, N=145), cats 11\% (N=16) and dogs (81\%, N=36) with
varying titer levels.%
\end{abstract}%



\sloppy


\textbf{First detection of SARS CoV-2 like antibodies in domestic and
wild animals in Kenya}

\textbf{Tindih Sheltone Heshborne\textsuperscript{1**}, Isaiah
Obara\textsuperscript{5*}, Marcello
Alessandro\textsuperscript{4}\textsubscript{,} Akinyi Matindi
Mercy\textsuperscript{2}, John Nyambega\textsuperscript{3}, Adinoh
Omondi George\textsuperscript{2}}

\begin{enumerate}
\tightlist
\item
  \textbf{Department of Biological Sciences, Machakos University,
  Kenya;}
\item
  \textbf{Kenya snake venom Research Centre, Institute of Primate
  Research, Karen Kenya;}
\item
  \textbf{Directorate of Research, Kenyatta University Teaching and
  Referral Hospital;}
\item
  \textbf{Laboratory of Molecular Virology, International Centre for
  Genetic Engineering and Biotechnology (ICGEB)}
\item
  \textbf{Institute for Parasitology and Tropical Veterinary Medicine,
  Freie Universitaet Berlin}
\end{enumerate}

**Corresponding author: tindih.heshborne@mksu.ac.ke

\textbf{*} Current Affiliations: I\textbf{nstitute for Parasitology and
Tropical Veterinary Medicine, Freie Universitaet Berlin}

NB: The order of the authors does not indicate the level of
contributions.

\textbf{First detection of SARS CoV-2 like antibodies in domestic and
wild animals in Kenya}

\textbf{KEYWORDS: Animals, Detection, SARS CoV-2,}

\textbf{Abstract/Summary}

The severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2),
etiological agent of the novel coronavirus disease 2019 (COVID-19), has
spread since December 2019, resulting in massive health and economic
crisis worldwide. While efforts to stop the pandemic are crucial,
collecting epidemiological data to help manage current and future
pandemics will be important.

In addition to humans, serological and molecular based studies have
demonstrated SARS CoV-2 exposure in several wild, domestic and farmed
animals. For examples Shriner and the team showed serologically an
exposure of 40\% to the white deer living in close proximity to urban
centers. Additional reports have also emerged of susceptibility of
animal's species like cats, ferrets, raccoon dogs, cynomolgus macaques,
rhesus macaques, white-tailed deer, rabbits, Egyptian fruit bats, and
Syrian hamsters to SARS-CoV-2 infection.. It's worth emphasizing that
these reports are based on experimental data mostly derived from Europe,
USA, South America and parts of Asia. In limited instances natural
infections of SARS-CoV-2 have been reported in pet dogs, cats, tigers,
lions, snow leopards, pumas, gorillas at zoos and farmed mink and
ferrets. The presence of the virus in animal species and an
understanding of whether these are natural or recent human to animal
transmissions is important. It's possible that such transmission could
passage the virus or subject the virus to a different immunological
pressure thereby helping with the development of viral variants in
addition to being a host for future reservoirs of the virus.

In Kenya SARS-CoV-2 was first detected on March
12\textsuperscript{th}2020 from imported human cases of persons who had
travelled from the United States. This was followed by detection of
imported cases majorly from China, Sweden and United Kingdom. Later
infections were confirmed in Nairobi and Mombasa suggesting further
cases of disease importations through the major ports of entry. However,
no comparable data on animal exposure have hitherto been generated in
Kenya. To address this key concern, we focused on three objectives; 1)
development of a robust antibody ELISA based on crude SARS-CoV-2 lysate.
2) SARS-CoV-2 serology of domestic animals in Kenya. 3) Corroboration of
the crude lysate based seroprevalence data and a commercial ELISA kit
based on the Spike receptor binding domain (RBD) antigen.

Our sample set included camel sera (both pre- \& post outbreak sera), as
well as sera from cats and dogs collected at the peak of the pandemic.
Our results using the ELISA based on crude SARS-CoV-2 lysate indicated
SARS-CoV-2 antibodies in camels (71\%, N=145), cats 11\% (N=16) and dogs
(81\%, N=36) with varying titer levels. These findings were comparable
to those obtained using the commercial ELISA kit based on the spike RBD
antigens.

In summary, the data warrants two key conclusions: (i) we have
demonstrated that the crude lysate ELISA allows for SARS-CoV-2 antibody
detection, and given its potential to offer robust detection could be
applied for initial mass screening (ii) although the current study
cannot disentangle the relative contributions of antigenic
cross-reactivity, pre-pandemic exposure to SARS-CoV-2 or human-animal
transmission, it nonetheless demonstrates for the first time the
prevalence of SARS-CoV-2 like antibodies in domestic and wild animals in
Kenya. Our findings set the scene for further research into the
prevalence of SARS-CoV-2 in domestic and wild animals to understand
their potential epidemiological implications.



\textbf{Introduction and Background}

Initial investigations into the origin of the SARS-CoV-2 virus pointed
to the animals sold at the Wuhan seafood market (Huang et al, 2020). In
particular, the virus is believed to have originated from either bats,
pangolins or a recombination between the virus found in bat and
pangolins sold in the market (Zhou et al., 2020; Liu et al 2020). Though
it is now highly doubtful whether pangolins are the original source, it
is important to note that genetic comparisons have shown that the genome
of the coronaviruses found in pangolins and thatof the SARS CoV-2
exhibit a genome sequence similarity of 99\% (Cyranoski D 2020). By
contrast, it is now highly suspected that SARS CoV-2 has its ecological
reservoirs in bats. This is based on similarities in the biological
features and the sequence identity between bat-nCoV and SARS-CoV-2 (Chan
et al 2013; Nguyen et al, 2020: Ren et al., 2020).

SARS-Cov-2 virus is highly contagious and is transmitted between humans
via respiratory droplets or close contact (Li et al., 2020). One
important aspect of SARS-CoV-2 epidemiology that has received limited
attention, particularly in Africa, is the transmission of the virus
between humans and animals. This is important because SARS-CoV-2 is
known to infect animals (Prince et al., 2021) and human to animal
transmissions have been demonstrated. In addition, SARS-CoV-2 infections
have been documented in cats and dogs in the UK (Smith et al., 2021),
Belgium and the USA {[}Garigliany et al., 2020; Newman et al., 2020; Sit
et al., 2020). The detection of SARS-CoV-2 infections in animals in the
studies outlined above were based on RT-PCR or virus isolation {[}Barrs
et al., 2020; Decaro et al., 2020; Hamers et al., 2021; Ruis-Arrondo et
al., 2020 \& Sailleau et al., 2020).

The use of sero-detection of the anti-SARS CoV-2 antibodies has also
been deployed in a number of countries with the aim of providing
evidence of exposure of cats and dogs to SARS CoV-2. Independent studies
in Italy have also shown evidence of exposure to SARS-CoV-2 in cats and
dogs from households (Patterson et al (2020)), while in France, Frits et
al (2020) showed a high prevalence of SARS-CoV-2 antibodies in pets from
COVID-19 positive households (Fritzs et al., 2020). In Germany, Croatia
and China the presence of anti-SARS-CoV-2 antibodies have also been
shown in domestic cats and other pet populations. Collectively, these
studies have demonstrated that serosurveys can provide reliable evidence
of animal infections (Michelitsch et al., 2020; Stevanovic et al., 2020;
Zhang et al., 2020).

A close examination of these studies reveal that most of the animals in
which SARS-CoV-2 were detected were either companion or domesticated
animals. This evidence has been interpreted to suggest that such
domestic and companion animals could reliably predict human to animal
transmissions especially post the pandemic outbreak. Additional data in
support of this conclusion has also recently become available. For
example, cases of experimentally induced infections have recently been
documented in cats (Halfmann et al., 2020; Bosco-Lauth et al., 2020) and
dogs have also been shown to be susceptible to SARS-CoV-2.

As for the epidemiological significance of these infections, cats have
been shown to shed off the virus and able to transmit the virus to other
cats {[}Shi et al., 2020; Bosco Lauth et al 2020) and cases of possible
natural infections and transmissions have been detected in farmed minks
that have been in contact with cats and dogs (Oude Munnink et al., 2020;
van Aart et al., 2020). This data therefore suggest the possibility of
human to animal transmission and vice versa. While in the studies
especially in cats and dogs there were no clinical symptoms, the studies
in farmed Minks whose transmissions were from infected human contact
respiratory and gastrointestinal signs, and also sudden death after
infection (Molenaar et al., 2020). However there were cases of
nondomestic felids at the Bronx Zoo and pet cats in New York and Europe,
which showed respiratory disease and/or conjunctivitis.

Although this data by itself already suggests a complex persistent
zoonosis, it can be further complicated by the presence of wild animals
that interface with domestic animals and humans as is the case in most
parts of Africa. In other parts of the world with a domesticated animal-
wild animal interface, new evidence of infection of some of these
animals has recently been documented. These include: Croatia where
fifteen out of 533 (2.8\%) positive ELISA results has been detected in
wild boars (3.9\%), red foxes (2.9\%) and jackals (4.6\%) (Jemer\selectlanguage{polish}š\selectlanguage{english}i\selectlanguage{polish}ć, \selectlanguage{english}et
al., 2021). Given these findings, it's only a matter of time before the
virus start spreading in the wild. These facts together with the
evidences that a certain wild animals like the lion are susceptible to
SARS CoV-2 (Koeppel et al., 2022) and develop pneumonia, could mean
maintenance of the virus in the wild, with potentially negative
consequences on wild populations and biodiversity. These therefore
underscore the importance of extending surveillance to animals in the
wild in future..

Herein, in pursuit of the broad objective of SARS-CoV-2 surveillance in
domestic animals in Africa, we have developed a robust ELISA diagnostic
assay based on a crude viral lysate and used it to detect antibodies to
SARS-CoV-2 in cats, dogs and camels in Kenya. Our specific goal was to
develop a robust ELISA diagnostics assay for SARS-CoV-2 in order to
understand the seroprevalence of SARS-CoV-2 in domestic animals.



\textbf{Methodology}

\textbf{Ethical approval, Consent and Research License}

This research was approval by the Institutional Research and Ethics
Committee of the Institute of Primate Research, Karen Kenya reference
number (ISERC/01/2021).

\textbf{Samples}

Two sets of samples from three geographically distinct sites in Isiolo,
Nairobi and Machakos that are 500 km apart were utilized in the present
study. These included: (i) randomly collected camel samples from Isiolo
and from Athi -- River, Machakos County, and (ii) randomly collected
animal samples from the veterinary clinics around Nairobi (cats, \&
dogs) and one veterinary clinic in Syokimau-Machakos County.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1 Sampling sites in Isiolo (camels), Nairobi (cats and
dogs) \& Machakos (camels) are indicated by the red dots.}

Camels were sampled randomly as follows: 144 fecal specimens, 144 nasal
swabs and 144 blood samples collected into serum vacutainers. The camels
were identified through their owners whose consent was initially sought;
the animals were restrained using trained animal handlers and samples
taken. The blood samples were processed for serum the same day, labeled,
aliquoted into two portions and stored at -20\textsuperscript{0}C. The
duplicate samples were then transported to the Machakos University,
Department of Biological sciences laboratory and the other part
transported to the institute of Primate research, Kenya-snake bite
research center, Karen Kenya.

Samples from cats (16 samples) and dogs (36 samples) were collected at
the veterinary clinics on different dates. The consent was sought from
their owners before drawing of the blood. 6 ml of blood was drawn from
each animal onto a serum vacutainer and stored at 4\textsuperscript{0}C
until they were collected and transported to the Institute of primate
Research. The blood sera were transported in vacutainer tubes to the
Institute of Primate Research where the sera separation was done. The
sera were aliquoted and stored at -20\textsuperscript{0}C till use.

In order to ascertain that the samples were suitable for the ELISA
assay, we tested a selected number of samples with a commercial kit
based on purified SARS-CoV-2 receptor domain binding site specific ELISA
(PISHTAZ Teb) based ELISA. The kit has been optimized for human samples,
in our test we modified the kit to be able to detect camel, cats and
dogs samples by replacing the kit's anti-Human IgG-HRP with either
anti-Llama IgG-HRP (for camels and dogs or Invitrogen anti-human IgG-HRP
(for cats samples). To ensure that our modified kits was working well we
used the kits confirmed human positive controls to ascertain that the
kit was also working on camel, cat and dog samples. The rest of the
procedure and reagents used was as described in the kits manual. We
eventually compared the results from the commercial kit to those
obtained using our crude lysate ELISA designed as described below.

\textbf{Development of a robust in-house ELISA for SARS-CoV-2 antibodies
in cats, dogs and camels}

The SARS CoV-2 virus was isolated from a COVID-19 patient via
nasopharyngeal swab. The swab was then inoculated onto vero cells
cultured in DMEM media with 15\%FBS \& Pen/Strep and allowed to infect
the and grow in the cells. The presence of SARS-CoV-2 was confirmed with
RT-PCR reactions. The virus infected vero cells were then cultured in
DMEM media with 15\%FBS \& Pen/Strep. The cells were monitored and
harvested at 80\% CPE. The harvested infected cells were broken down by
freeze thawing and under 5\% SDS detergent to inactivate them and viral
particles was separated on sucrose gradient sedimentation solution and
resuspended in 100\selectlanguage{greek}µ\selectlanguage{english}l of DMEM media. The harvested virus was further
inactivated by three cycles of freeze thawing and then at 55 degree
Celsius for 1 hour and then stored at -20\textsuperscript{0}C. The crude
viral lysate was re- suspended in 400 \selectlanguage{greek}µ\selectlanguage{english}l ml of PBS and taken as the neat
lysate. The viral antigen titration curve was done by diluting the viral
lysate in the range of 5 x 10\textsuperscript{-1} to 5 x
10\textsuperscript{-10} with PBS. The 50 \selectlanguage{greek}µ\selectlanguage{english}l of titrated viral lysate was
used to coat the plates overnight at 4\textsuperscript{0}C for the
titration and determination of the appropriate dilution assay. Finally a
dilution of of 5 x 10\textsuperscript{-7} was adopted and used for
subsequent plate coating and further assays. Wells without coating (No
coats) were also included to compensate for nonspecific interactions of
the serum with other factors other than the antigen. Before addition of
the sera the excess antigen flicked out and the plate tapped upside down
on the soft tissue to remove the excess of the antigen.

In developing the assay further ELISA protocol developed byAmanat et
al., 2020 was adopted with a few modifications as described below.
First, in order to identify the positive and negative camel control sera
a aselected pre-COVID-19 and post COVID-19 camel samples were screened
using PISHTAZ Teb commercial kit. The camel sera was selected, titrated
in the range of 1/100, 1/500, 1/1000, 1/5000, 1/10 000 and 1/50 000, and
finally 1/1000 was adopted. Cat \& dogs sera was titrated in the 1/5,
1/10, 1/50, 1/100, 1/500 and 1/1000 and finally 1/50 was adopted. Wells
with antigen coating but no sera (No Sera) were included to check on
nonspecific cross-reactivities. Positive controls sera from camel (C0874
\& C1191), cats (CAT 2 \& 4) and dogs (DOG 1 \& 2) were identified and
used to check if the serum is binding on anything other than the
antigens. Serum dilutions were done in PBS /1\% skimmed milk/0.1\% SDS
and incubation was done for 1hr at 37\textsuperscript{0}C. For the
safety of technologist the animal samples/sera were inactivated at
55\textsuperscript{0}C and stored at -20\textsuperscript{0}C till used
for the ELISA assay.

The secondary step antibody used was either a reacting anti-Human IgG
HRP for Cats sera or anti-Llama IgG --HRP for camel and dogs samples,
the conjugate incubation was done for 1hr at 37\textsuperscript{0}C.
Washing of the wells was done five times with 200\selectlanguage{greek}µ\selectlanguage{english}l per well of
PBS/0.1\% SDS. 100\selectlanguage{greek}µ\selectlanguage{english}l of substrate was used to develop the assay and
incubated for 30 minutes at 37\textsuperscript{0}C after which the
reaction was stopped with sulphuric acid and the plate read at 450nm.

To test if our crude lysate ELISA assay could be used for surveillance,
we collected a pool of randomly sampled animal sera from three different
animal species (Camel, n=144, cats, n= 16 and dogs, n=36) and three
different locations (Fig.1). The samples were screened with our crude
lysate ELISA assay for the presence of cross reacting antibodies and
compared with a commercial kits based on the purified recombinant spike
RBD antigen. The camel sera samples were used at 1/1000, while the cats
and dogs sera samples were used at 1/50.

\textbf{Results}

\textbf{Development of a robust ELISA for SARS-CoV-2 antibodies in cats,
dogs and camels.}

\textbf{Titrating the crude lysate}

The crude lysate used to coat the ELISA plates was titrated in the range
of 5x10\textsuperscript{-2} and 5x10\textsuperscript{-10} of the viral
lysate of 80\% CPE viral harvest. Wells without coating (No coats) were
included and a positive serum (C0874) was used to check if the serum is
binding on anything other than the antigen. First pre-COVID-19 and post
COVID-19 camel sera were screened using a commercial kit to identify
reacting samples. The positively reacting camel sera was selected and
used at a dilution of 1/1000, cat \& dogs sera was used at 1/50. Wells
with antigen coating but no sera (No Sera) were included to check for
nonspecific cross-reactions.

The crude lysate antigen reactivity with camel, cats and dogs sera was
optimal at a dilution range of 5x10\textsuperscript{-7} and
5x10\textsuperscript{-8} of the lysate of 80\% CPE viral harvest. Beyond
5x10\textsuperscript{-8} dilution, the lysate couldn't pick or
distinguish between a SARS CoV-2 negative from a positive sera (Fig.2).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Figure 2: Titration of Crude SARS COV-2 Lysate antigen. Shown on the
y-axis is the ELISA OD scores, while the X-axis shows the crude lysate
titrations. The serum for the o titration gradient was used at 1/10 for
Cat, 1/10 for Dogs sera and 1/1000 for the Camel sera. The camel and
cats titres were higher than those of the dogs.

\textbf{b) Screening of positive and negative controls against best
performing Secondary HRP conjugated antibodies}To check if anti-Llama
IgG conjugated with HRP could also cross react with dogs and cats sera
and also if anti-human IgG conjugated with --HRP could cross react with
camel, cats and dogs sera, we coated the ELISA plates with the crude
lysate. The wells were then probed with sera from camel, cats and dogs.
The sera were then detected with both Anti-Llama IgG-HRP and anti-Human
IgG-HRP. The rest of the conditions were as described in Amanat et al.,
2020. Anti-Llama IgG Conjugated -- HRP detects /cross reacts with dogs
antibodies whereas Anti-human IgG Conjugated -- HRP with dogs antibodies
do not. Using crude lysate ELISA, it's possible to discriminate positive
\& negatives on dog samples: D1, D2, D3 and D5 dogs' samples are
positive (figure 3). Crude lysate ELISA can pick positive \& negatives
camel sera: Camel D8-874 react positively with both anti-human IgG-HRP
and anti-Llama IgG- HRP-conjugated antibody while the negative camel
sera (D0) 0852 remains negative.

Figure 3: Bar graph showing positive cat, dogs and camel Sera detected
either with Secondary second step anti-human IgG HRP or anti-Llama IgG
HRP conjugated antibodies. The selected anti-human IgG conjugated with
HRP can discriminate between positive sera (CAT1 \& 2) and negative cat
sera (CAT3, 4 \&5) samples, while anti-Llama IgG-conjugated HRP cannot
discriminate between positives and negative cats sera. cat sera dogs
sera (D1, 2, 3 \& 5). Anti-human IgG conjugated HRP does not detect
Camel sera therefore doesn't distinguish between positive and negatives
(C0852-Antihuman IgG HRP, C874 anti-Human) while anti-Llama IgG HRP does
detect camel sera and can discriminate between a SARS CoV-2 positive
sera (C874 --Anti-Llama IgG-HRP from the negative sera(C0852-Anti-Llama
IgG HRP)

\textbf{c) Titration of serum samples (camel, dogs and cats) against
Crude SARS-CoV-2 lysate antigen}

To determine the right sera dilution to use for assay and to help avoid
non-specific antibody-antigen interactions, we titrated the three serum
samples (camel, cats and dogs sera). Camel sera was titrated in the
range of 1/100, 1/500, 1/1000, 1/5000, 1/10 000 and 1/50 000, cats and
dogs sera was titrated in the 1/5, 1/10, 1/50, 1/100, 1/500 and 1/1000.
The diluted sera was then used to perform the ELISA assay and detection
of the antibodies was achieved by either Anti-Llama IgG --HRP for Camels
and dogs samples while the cats antibodies were detected using
anti-Human IgG--HRP.

The camel sera C1191 and C0874 had high titers such that even at a
higher dilution series they were still positive for SARS-CoV-2 and it
titrated out confirming the positive detection of SARS COV-2, while
C0852 remained constantly low confirming its negativity (figure 4a)..
The positive SARS CoV-2 cats samples (CAT2, \& CAT4) also titrated out
while the negative cat samples remained constant low suggesting that the
test is capable of discriminating between cats SARS CoV-2 positive and
negative sera (figure 4b). The same dynamics was seen with the dogs
samples where DOG 1 \& 2 shown positive titration trend in detection of
SARS COV-2 while DOG4 sera remained negative suggesting the capability
of the system to positively detect dogs samples (figure 4c) .\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

Figure 4: Titrating Serum against Crude SARS COV-2 Lysate antigen.
Samples in panel A: C1191, C0874 and C0852 are Camel samples titrated
from 1/100 to 1/50,000. Samples in panel B: CAT1, CAT2, CAT3 and CAT4
are CAT samples titrated from 1/5 to 1/1000 and Samples in panel C:
DOG1, Dog2 and Dog4 are selected dogs sera titrated from 1/5 to 1/1000.

\textbf{2) Assessment of the SARCoV-2 sero-status of domestic and wild
animals in Kenya.}

Using the validated crude lysate ELISA, we found that 71\% of camel
samples, 11\% cat samples and 83\% of dog samples were positive.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

Figure 5: Distribution of SARS COV-2 antibodies among cats, dogs and
camels in Kenya as assessed using the crude lysate ELISA developed
herein. Center lines show the medians; box limits indicate the 25th and
75th percentiles as determined by R software; whiskers extend 1.5 times
the interquartile range from the 25th and 75th percentiles, outliers are
represented by dots; crosses represent sample means; bars indicate 95\%
confidence intervals of the means; data points are plotted as open
circles. Results are given as a percentage of the positive control serum
(percent positivity, PP). The data interpretation was based on the
stringent cut-offs (20\% PP) for negative or positive ELISA results.

SAR CoV-2 positive Camel samples are strongly reacting to SARS CoV-2\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

Figure 6: Screening of randomly selected Camel Sera for the detection of
SARS-CoV -- 2 like antibodies. Despite the high background at 1/100 Sera
dilution, at 41\% PP cut -- off the exposure is 71\% (comparable to the
83 \% on the commercial Kit.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

Figure 7: Screening of randomly selected Cats Sera for the Detection of
SARS-CoV -- 2 like antibodies. 1/50 sera dilution

15\% PP cutoff: 89 \% exposure. 20\% PP cut off: 11 \% exposure\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

Figure 8: Screening of randomly selected Dogs Sera for the detection of
SARS-CoV -- 2 like antibodies. Sera dilution 1/50, (antigen at 5 x
10\^{}-6). 15\% PP cutoff: 83\% exposure; 20\%, cutoff point: 81\%
exposure to COVID-like antibodies

\textbf{3) Corroboration of results using a commercial kit ELISA based
on the spike RBD antigen s}

This finding of the presence of SARS COV-2 like antibodies in Camels in
Kenya based on our crude lysate ELISA, was corroborated by the results
obtained using the commercial kit based on the Spike specific RBD
antigen (Figure 9 \& 10). One potential explanation for the generally
higher OD scores in crude lysate ELISA relative to the commercial kit is
that the crude lysate ELISA could include up to 4 (NP, S1 \& S2 and M)
major antigens as opposed to the single antigen (Spike RBD) in the
commercial kit.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image9/image9}

\end{center}
\end{figure}

Figure 9: ELISA detection of SARS-CoV -- 2 like antibodies in animal
samples using modified human Diagnostics Commercial Kit. The assay is a
modification of the ELISA test described by Amanat et al, (2020) by
replacing the anti-human IgG conjugate antibody with (anti-Llama IgG ).
D0 -- samples collected before COVID-19 outbreak announcement in Kenya
while D8 samples were collected 8 days after the COVID-19 outbreak
announcement in the country. The 10\% PP cut off: 83 \% exposed / cross
reacting, 20\% PP cut off: 57\% exposed

The mean OD absorbance values generated in Figure 9 above were used to
calculate the percent positivity scores relative to the absorbance
values for the positive control serum. On the basis of the stringent pp
threshold of 20\%, we found evidence of SARS COV-2 like antibodies in
all the three species studied.

The plot in figure 10 enables clearer visual comparisons to be drawn
between the results of the analysis of SARS COV-2 like antibody titers
based on the crude lysate ELISA and the commercial kit based on the
Spike specific RBD antigen. In the figure, the individual camel antibody
titers are shown as small white lines in a one-dimensional scatter plot.
The estimated density of the distributions of the percent positivity
values is visible as a polygon and the average is also shown as a black
line. It is clear that the results based on the crude lysate are
corroborated by commercial kit ELISA and as already mentioned, the
higher pp scores in the crude lysate ELISA relative to the commercial
kit could be due to the additional antigens present in the crude lysate.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image10/image10}

\end{center}
\end{figure}

Figure 10: A comparison of the distribution of SARS CoV-2 like
antibodies in camel sera determined both by a commercial kit (n=18) and
by the crude lysate ELISA (n=32), and expressed as a percentage of the
positive control serum (percent positivity). Black lines show the means;
white lines represent individual data points; polygons represent the
density of the data. The plot was generated using the R package beanplot
(Kampastra 2009).



\textbf{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.

\textbf{Acknowledgements}

We would like to acknowledge and appreciate the funding through
International Centre for genetic Engineering and Biotechnology (ICGEB)
which allowed us to perform this work. Machakos University management
for the provision of the humble working environment and the laboratory
space and the personnel resources that enable us to undertake this work.
Ms. Sandra Ngelel, Alphas Amollo and Faith Ndunda for their technical
support in carrying out some of the experiments. Special thanks go to
the technical staffs at the Kenyasnake Venon Research Centre (KSRIC) who
participated in sample collection and helping with the analysis of the
samples.

\textbf{Declarations of conflicts of interest:}

\textbf{None}



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