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A canine model of experimental infection with Cryptosporidium canis
Accepted Manuscript A canine model of experimental infection with Cryptosporidium canis Zhaohui Cui, Heping Dong, Rongjun Wang, Fuchun Jian, Sumei Zhang, Changshen Ning, Longxian Zhang PII:
S0014-4894(18)30008-0
DOI:
10.1016/j.exppara.2018.09.019
Reference:
YEXPR 7617
To appear in:
Experimental Parasitology
Received Date: 9 January 2018 Revised Date:
22 June 2018
Accepted Date: 23 September 2018
Please cite this article as: Cui, Z., Dong, H., Wang, R., Jian, F., Zhang, S., Ning, C., Zhang, L., A canine model of experimental infection with Cryptosporidium canis, Experimental Parasitology (2018), doi: https://doi.org/10.1016/j.exppara.2018.09.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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A canine model of experimental infection with Cryptosporidium canis
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Zhaohui Cuia,b#, Heping Donga,b#, Rongjun Wanga,b, Fuchun Jiana,b, Sumei Zhanga,b,
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Changshen Ninga,b, Longxian Zhanga,b*
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a
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Zhengzhou 450002, China
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b
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China
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International Joint Research Laboratory for Zoonotic Diseases of Henan, Zhengzhou,
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These authors contributed equally to this work.
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College of Animal Science and Veterinary Medicine, Henan Agricultural University,
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*Corresponding author. Longxian Zhang, College of Animal Science and Veterinary
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Medicine, Henan Agricultural University, No. 15 University Distract, Zhengdong Newly-
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developed Area, Zhengzhou 450046, China.
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Tel.: 86-371-56990163; Fax: 86-371-56990163;
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E-mail: [email protected]; [email protected].
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ABSTRACT
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Cryptosporidium is a genus of protozoal parasites that affects the gastrointestinal
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epithelium of a variety of hosts. Several models of experimental infection have been
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described to study the susceptibility, infectivity and pathogenicity among different
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Cryptosporidium species and isolates. This study aimed to establish an experimental
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infection of Cryptodporidium canis in canids. Infectivity and pathogenicity have been
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measured by evaluating the clinical status, pattern of oocyst excretion and histological
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examination. Results showed that C. canis was not infective for immunocompetent dogs
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or mice with severe combined immunodeficiency syndrome (SCID). Oocysts were first
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detected in the feces of immunosuppressed dogs on day 3 post-infection (p.i.), with levels
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peaking twice on days 10 and 17 p.i. during the patent period. cryptosporidial
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developmental stages were found in the duodenum and jejunum of dogs in histological
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sections stained with hematoxylin and eosin (H & E) and using scanning electron
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microscopy (SEM). Histopathological changes in the intestinal tract of infected dogs
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were characterized by epithelial metaplasia and dilatation; the integrity of intestinal
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mucosal epithelial cells was distinctly damaged with whole sheets of cilia sloughed away.
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Ultrastructural observation data were consistent with histological observations. Based on
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these findings, the canine model described in this work will be useful to evaluate clinical,
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parasitological and histological aspects of C. canis infection and will be useful for the
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further understanding of cryptosporidiosis, drug development, and vaccine development.
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Keywords: Cryptosporidium canis; Canine model, Experimental infection
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1. Introduction Cryptosporidium is a protozoal parasite that infects a variety of hosts, including humans, domestic and wild animals, worldwide (Ryan et al., 2016). Cryptosporidiosis
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characteristically results in watery diarrhea, but clinical symptoms can vary from
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asymptomatic shedding of oocysts to severe and life-threatening disease, depending on
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the immune status of the host (Bouzid et al., 2013). Immunocompetent individuals
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typically experience self-limiting illness (up to 2 to 3 weeks in duration) and recover
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without treatment. However, in immunocompromised patients, cryptosporidiosis can
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result in intractable diarrhea and may even be fatal in some cases (Chalmers and Davies,
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2010). An effective vaccine or drug treatment for cryptosporidiosis is not yet available.
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Dogs have been considered to be faithful friends and shared a close relationship with
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humans for over 10,000 years. Pet ownership has been recognizably proven to exert
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beneficial effects on human health, by improving physical condition as well as mental
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and emotional well-being (Hodgson et al., 2015). Consequently, pets are being
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increasingly used as a therapeutic option in healthcare facilities (Wells, 2007). However,
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dogs may not only act as intimate companions of humans but also as natural reservoirs of
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a large number of zoonotic pathogens, including Cryptosporidium spp. (Chomel, 2014;
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Esch and Petersen, 2013).
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Cryptosporidiosis has been reported in dogs worldwide (Jian et al., 2014). Currently,
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four species of Cryptosporidium, namely C. canis, C. parvum, C. muris, and C.
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meleagridis, have been detected in dogs (Lupo et al., 2008; Yoshiuchi et al., 2010; Scorza 3
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et al., 2011). Cryptosporidium canis causes the vast majority of infections in dogs (Ryan
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et al., 2014) and is considered to be potentially zoonotic due to its presence in human
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patients in a number of countries (Fayer, 2010; Gatei et al., 2008; Elwin K et al., 2012).
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Over the decades, different models of experimental infection have been described to study the susceptibility, infectivity and pathogenicity of Cryptosporidium (Ayinmode et
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al., 2017; Kvac et al., 2013; Modry et al., 2012; Masuno et al., 2014; Petermann et al.,
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2014). Nevertheless, information on cryptosporidiosis caused by C. canis infection in an
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experimental host has yet been reported.
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In this study, we developed a canine model of cryptosporidiosis caused by experimental infection with C. canis parasites. We analyzed the infectivity of C. canis
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using severe combined immunodeficiency (SCID) mice and immunosuppressed dogs,
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and described the clinical, parasitological, and histological aspects of experimental
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infection in immunosuppressed dogs, which provide a good model for the future testing
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of novel drugs or vaccines against cryptosporidisis.
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2. Materials and methods
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2.1. Ethics approval and consent to participate
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This study was conducted in accordance with the Chinese Laboratory Animal
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Administration Act (1988). The experimental protocol was approved by the Institutional
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Animal Care and Use Committee of Henan Agricultural University (authorization number
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IACUC-henau-20070503). 4
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2.2. Inoculum Cryptosporidium oocysts were obtained from a dog located in a pet hospital at the province of Henan, China. Oocysts were concentrated using the water ether technique
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(Bukhari and Smith, 1995), and purified by discontinuous sucrose density centrifugation
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(Heyman et al., 1986). Oocysts were counted using a Neubauer hemocytometer. A
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combination of streptomycin and penicillin was added and this oocyst suspension was
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kept at 4°C. C. canis oocysts for morphometry analyses were determined using digital
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analysis of images (Motic Images Plus 2.0 soft-ware). Length and width of oocysts (n =
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50) were measured under bright-field microscopy at 1000-fold magnification, and these
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were used to calculate the length-to-width ratio of each oocyst.
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Total DNA was extracted using an E.Z.N.A.® Stool DNA Kit (OMEGA Biotek Inc.,
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Norcross, GA, USA). To determine the species of Cryptosporidium for the isolate used in
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this study, nested PCR protocols were used to amplify the SSU rRNA gene, actin gene
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and HSP70 gene according to the previously-reported studies (Xiao et al.,1999; Sulaiman
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et al., 2002; Sulaiman et al., 2000). DNA sequencing indicated that the SSU rRNA and
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HSP70 nucleotide sequences had a 100% similarity with C. canis (GenBank accession
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number AB210854 and AY120920, respectively), while the actin nucleotide sequence had
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a 99% similarity with C. canis (GenBank accession number AY120927). The nucleotide
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sequences obtained in this study has been deposited in the GenBank database under
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accession numbers: EU754826, EU754835 and EU754843. 5
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2.23. Experimental animals Six 8-week old SCID mice were obtained from Shanghai SLAC Laboratory Animal
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Co., Ltd., Shanghai, China. All mice were housed individually in plastic cages with wire
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mesh tops, under pathogen-free conditions, and kept on daily 12 h cycles of light and
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dark. Mice received sterilized food and water, and were randomly divided into control and
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test groups with 3 mice per group. Each mouse in the test group was inoculated orally by
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a stomach tube with a dose of 1×106 oocysts.
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Nine beagle dogs, 7 weeks of age, were obtained from the Animal Lab Center of Henan Agricultural University. All dogs were confirmed to be free of C. canis infection
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by microscopic examination of feces. During the adaptation period, dogs were vaccinated
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against regional infectious diseases (Canine distemper, Parvovirus, Canine hepatitis,
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Leptospirosis, and Rabies). Animals received commercial dog food, according to their
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physiologic development, and water ad libitum.
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Experimental dogs (n = 3) were immunosuppressed according to the protocol as
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described previously (Young et al., 2002). Dogs were fed 6 mg of dexamethasone acetate
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per day for 5 days before infection. At day 8 of immunosuppression, each dog was
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inoculated orally by a stomach tube with a dose of 1×106 oocysts. The following two
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control groups were also included: immunocompetent dogs inoculated orally with 1×106
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oocysts of C. canis (n = 3), and immunocompetent, non-inoculated dogs (n = 3). At days
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12 and 28 post infection (p.i.), one dog per group was euthanized by an intravenous
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injection of a lethal dose of pentobarbital 100 mg/kg and their organs were removed.
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2.34 Clinical status
Dogs were examined daily for clinical signs including rectal temperature, respiratory
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rate, and heart rate. Additionally, the presence of diarrhea and any abnormal behaviours
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were observed.
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2.45. Oocyst excretion
To determine the prepatent period, daily fecal samples were collected from each mouse and dog from day 1 to 28 p.i. Briefly, 1g of the feces from each animal was
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examined using the Sheather’s sugar floatation method and counted using hemocytometer
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slide under bright-field microscopy at 400-fold magnification. Repeat the count three
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times and calculate the average. Infection intensity was determined by the number of
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oocysts per gram (OPG). The OPG was estimated on the basis of number of oocysts
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counted.
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2.56. Histological examination
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For histological analysis, tissue samples harvested from duodenum, jejunum, ileum,
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cecum, stomach, lungs, liver, spleen and kidney were fixed in 10% buffered formalin for
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24 h, dehydrated in absolute ethanol, cleared in xylene, and embedded in paraffin. Tissue 7
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sections were stained with hematoxylin and eosin (H & E), and observed microscopically
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at 400x and 1000x magnification. Tissue samples harvested from duodenum, jejunum, ileum and cecum were also
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prepared for scanning electron microscopy (SEM). Samples were fixed in 2.5%
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glutaraldehyde for one week at 4 , and then washed with 0.1 mol/L phosphoric acid
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buffer (pH = 7.4) three times for 10 min each. The dehydration procedure followed
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conventional methods in a graded ethanol series of 30%, 50%, 70%, 90%, 100%, and two
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more changes of 100%, each for 5 min followed by 50% isoamyl acetate solution (v/v,
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isoamyl acetate: ethanol = 1:1) and 100% isoamyl acetate solution for 10 min,
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respectively. After specimens were critically point-dried using CO2 and coated with gold,
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observations were made by an S-3400 SEM (HITACHI).
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3. Results
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3.1. Clinical status
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The appetites and attitudes of experimental and control SCID mice were normal
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until the end of the experiment. No diarrhea was observed in any mice. All immunosuppressed dogs inoculated with oocysts of C. canis showed signs of
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physical deterioration, exhibiting loss of sheen to the coat, loss of interest in the
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environment, and lethargy. These dogs began to have diarrhea around day 8 p.i. No
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diarrhea was observed in any control group dogs. No animals died during the experiment.
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3.2. Oocyst shedding Fecal examination of infected dogs revealed fully sporulated C. canis oocysts only
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in immunosuppressed dogs inoculated with C. canis oocysts. No Cryptosporidium
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oocysts were found in control or non-immunosuppressed dogs during the experiment.
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Likewise, no Cryptosporidium oocysts were detected in the feces of SCID mice in either
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group. C. canis oocysts found were ovoid, and 4.8 (4.2-5.2) µm x 4.4 (4.2-5.3) µm (n =
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50) (Fig. 1). Oocysts were first detected in feces on day 3 p.i., peaking twice on days 10
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and 17 p.i. The patterns of oocyst shedding in dogs in the test group are presented in Fig.
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2. Sequences of SSU rRNA gene, actin gene and HSP70 gene from experimentally
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infected dogs shared 100% identity with the isolate used in the inoculum.
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3.3. Histological observations of sites of C. canis infection Cryptosporidial infection in immunosuppressed dog (at 12 days p.i.) was found in
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the duodenum (Fig. 3A) and jejunum (Fig. 3B, C). However, no cryptosporidial
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developmental stages or pathologic changes were observed at any other anatomic sites,
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including the ileum (Fig. 3D), cecum, colon, or stomach. Likewise, no cryptosporidial
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developmental stages or pathologic changes were observed at any anatomic sites in dogs
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of control groups (data not shown). Histopathological changes in the intestinal tract of
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dogs with cryptosporidiosis were characterized by epithelial metaplasia and dilatation;
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the integrity of intestinal mucosal epithelial cells had been distinctly damaged, with
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whole sheets of cilia sloughed away. These changes were more pronounced in the middle
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and lower parts of duodenal mucosa epithelium, and the anterior and middle portions of
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jejunal mucosa epithelium.
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3.4. Ultrastructural observation
Scanning electron microscopy study showed that C. canis at various of
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developmental stages had adhered to the surface of the duodenum and jejunum in
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immunosuppressed dogs. Epithelial cell surfaces were swollen and disordered (Fig. 4A,
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B). The integrity of mucosal layer of the duodenum and jejunum had been damaged, and
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many cilia had fallen off or atrophied (Fig. 4C, D). Ultrastructural observation also
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showed that no parasites were observed in the ileum, cecum, colon, or stomach，
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consistent with the histological observations described above.
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4. Discussion
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The potential role of companion animals as reservoirs for zoonotic diseases has been
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recognized as a significant public health problem worldwide (Traub et al., 2005). Canids
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(wild and domestic) are recognized as the main reservoir of a vast range of parasites.
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Therefore, many studies with respect to Leishmania, Trypanosoma cruzi, Neospora
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caninum and Echinococcus multilocular have used the dog as an experimental model to
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investigate the reproductive potency, infectivity and pathogenicity of the parasite, as well
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as develop vaccines and assess drugs (Petitdidier et al., 2016; Padilla et al., 2009; Kapel
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et al., 2006; Cedillo et al., 2008;). Here, we established a canine model of infection with
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C. canis, with dexamethasone used to develop the experimental model of
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cryptosporidiosis as in previous reports (Surl and Kim, 2006; Certad et al., 2007). Experimentally inoculated immunocompetent dogs did not produce detectable C.
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canis oocysts by microscopy in faecal samples within 28 DPI. None of the SCID mice
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exhibited clinical signs or oocyst shedding at any point during the experiment. It is
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suspected that SCID mice were not infected with C. canis. Data from a previous study
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also indicate that oocysts of C. canis were not infectious for BALB/c neonatal mice or
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immunosuppressed C57 juvenile mice, but were infectious for calves (Fayer et al., 2001).
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In our study, infected dogs began shedding oocysts in low numbers on day 3 p.i.,
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with two peaks of infection intensity during the patent period. In immunosuppressed mice
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infected with C. parvum, oocyst shedding begins 6 days p.i., and these mice continue
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shedding until day 35 p.i. (Coco et al., 2012). The prepatent period was 4 days in the case
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of infection in immunosuppressed C57BL/6 mice with a single C. meleagridis oocysts
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(Huang et al., 2003). However, the prepatent period was 20-24 days, and the patent
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period varied between 46 and 59 days in neonatal and adult M. coucha infected with C.
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andersoni (Kvac et al., 2007). The differences in the prepatent and patent period probably
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depend on the experimental animals or species of Cryptosporidium.
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Variability of infection sites between different Cryptosporidium species and types
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has been reported (Plutzer and Karanis., 2009). Three of the four spieces of
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Cryptosporidium (C. canis, C. parvum and C. meleagridis) that infect dogs are detected
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in the small intestine, except C. muris which is detected in stomach; this difference could 11
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be associated with different patterns of colonization along the gastrointestinal tract. In
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this study, cryptosporidial developmental stages were observed in the middle and lower
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parts of duodenal mucosa epithelium, and anterior and middle parts of jejunal mucosa
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epithelium in histological sections and SEM study. This is similar to what has been
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described for naturally-occurring cryptosporidiosis in dogs (Greene et al., 1990; Wilson
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et al., 1983). However, one published case report indicates that C. canis was found both
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in the small intestine and gastric surface epithelium of an 8-week-old female Yorkshire
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terrier co-infected with Isospora spp. (Miller et al., 2003). Extraintestinal infection was
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not observed in our study, which could be related to the degree of immunosuppression,
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and/or the time post-infection that we evaluated.
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Cryptosporidium primarily infects the microvillous border of the intestinal
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epithelium, and to lesser extent extraintestinal epithelia, causing acute gastrointestinal
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disease in a wide range of mammalian hosts, including humans (Deng et al., 2004).
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Damage of host intestinal epithelial cells caused by cryptosporidiosis usually results in
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two outcomes: cell death and cell damage. Both produce architecture distortion and
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atrophy of the villus. In the present study, the surfaces of the epithelial cells were swollen
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and disordered. The integrity of mucosal layer had been damaged, and many cilia had
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fallen off atrophied in duodenum and jejunum of dogs infected with C. canis. Similarity,
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in chickens successfully infected with C. baileyi, the integrity of the epithelial cells of the
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BF (bursas of fabricius) was somewhat damaged (Yuan et al., 2014). In mice infected
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with C. parvum, cryptosporidium stages were detected on the brush borders and the
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intestinal epithelium showed loss of single epithelial cells, mild blunting, and shortening
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of villi (Sayed et al., 2016). In conclusion, we have established an experimental infection of C. canis in dogs
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immunosuppressed with dexamethasone. Parasitological and histopathological features of
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the infection were studied. These findings may foster further understanding of the biology
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of C. canis and lay the foundation to control cryptosporidiosis.
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Conflict of interests
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The authors declare that there are no conflicts of interest.
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Acknowledgments
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This study was partly supported by the National Key Research and Development
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Program of China (2017YFD0501305, 2016YFD0500707), the National Natural Science
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Foundation of China (31330079), and the Natural Science Foundation of Henan Province
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(162300410129).
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We thank Catherine Barnette, DVM, from Liwen Bianji, Edanz Group China
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(www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.
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Cryptosporidium parvum in immunosuppressed C57BL/6N mice. Journal of
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Wilson, R. B., Holscher, M. A., & Lyle, S. J. (1983). Cryptosporidiosis in a pup. J Am Vet Med Assoc, 183(9), 1005-1006, 1965.
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Yoshiuchi, R., Matsubayashi, M., Kimata, I., Furuya, M., Tani, H., & Sasai, K. (2010).
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Survey and molecular characterization of Cryptosporidium and Giardia spp. in
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owned companion animal, dogs and cats, in Japan. Vet Parasitol, 174(3-4), 313-316.
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Young, S. L., O’Donnell, M. A., & Buchan, G. S. (2002). IL 2 secreting recombinant
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bacillus Calmette Guerin can overcome a Type 2 immune response and
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corticosteroid induced immunosuppression to elicit a Type 1 immune response.
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Yuan, L., Yan, W., Wang, T., Qian, W., Ding, K., Zhang, L., . . . Shao, X. (2014). Effects
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of different inoculation routes on the parasitic sites of Cryptosporidium baileyi
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infection in chickens. Exp Parasitol, 145, 152-156.
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Figure legends
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Fig. 1. Microscopy of Cryptosporidium canis oocysts visualized by Modified acid-fast
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staining method (1000×).
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Fig. 2. Excretion of oocysts in one gram of feces in immunosuppressed dogs infected
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with C. canis during the experiment. Mean of all examined animals with standard errors.
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Fig. 3. Histological observation of C. canis infection in immunosuppressed dogs using H
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& E staining. High parasite burden in the duodenum (A, ×1000) and jejunum (B, C, 19
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×1000) (arrows). No cryptosporidial developmental stages and pathologic changes were
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observed in the ileum (D, ×400).
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Fig. 4. Scanning electronic microscopic observation of C. canis infection in
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immunosuppressed dogs. C. canis at a large number of developmental stage had adhered
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to the surface of duodenum and jejunum (A, B, ×3000) (arrows). The surfaces of the
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epithelial cells were swollen and disordered. The integrity of mucosal layer of the
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duodenum and jejunum had been damaged, and many cilia had fallen off or atrophied (C,
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D, ×5000).
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ACCEPTED MANUSCRIPT Highlights · This is the first canine model of experimental infection with C. canis. · C. canis was not infective for immunocompetent dogs and SCID mice. · Cryptosporidial infection was only found in duodenum and jejunum.
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· The histopathological changes were observed with HE and SEM.
S0014-4894(18)30008-0
DOI:
10.1016/j.exppara.2018.09.019
Reference:
YEXPR 7617
To appear in:
Experimental Parasitology
Received Date: 9 January 2018 Revised Date:
22 June 2018
Accepted Date: 23 September 2018
Please cite this article as: Cui, Z., Dong, H., Wang, R., Jian, F., Zhang, S., Ning, C., Zhang, L., A canine model of experimental infection with Cryptosporidium canis, Experimental Parasitology (2018), doi: https://doi.org/10.1016/j.exppara.2018.09.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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A canine model of experimental infection with Cryptosporidium canis
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Zhaohui Cuia,b#, Heping Donga,b#, Rongjun Wanga,b, Fuchun Jiana,b, Sumei Zhanga,b,
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Changshen Ninga,b, Longxian Zhanga,b*
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a
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Zhengzhou 450002, China
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b
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China
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International Joint Research Laboratory for Zoonotic Diseases of Henan, Zhengzhou,
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These authors contributed equally to this work.
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College of Animal Science and Veterinary Medicine, Henan Agricultural University,
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*Corresponding author. Longxian Zhang, College of Animal Science and Veterinary
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Medicine, Henan Agricultural University, No. 15 University Distract, Zhengdong Newly-
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developed Area, Zhengzhou 450046, China.
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Tel.: 86-371-56990163; Fax: 86-371-56990163;
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E-mail: [email protected]; [email protected].
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ABSTRACT
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Cryptosporidium is a genus of protozoal parasites that affects the gastrointestinal
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epithelium of a variety of hosts. Several models of experimental infection have been
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described to study the susceptibility, infectivity and pathogenicity among different
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Cryptosporidium species and isolates. This study aimed to establish an experimental
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infection of Cryptodporidium canis in canids. Infectivity and pathogenicity have been
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measured by evaluating the clinical status, pattern of oocyst excretion and histological
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examination. Results showed that C. canis was not infective for immunocompetent dogs
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or mice with severe combined immunodeficiency syndrome (SCID). Oocysts were first
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detected in the feces of immunosuppressed dogs on day 3 post-infection (p.i.), with levels
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peaking twice on days 10 and 17 p.i. during the patent period. cryptosporidial
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developmental stages were found in the duodenum and jejunum of dogs in histological
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sections stained with hematoxylin and eosin (H & E) and using scanning electron
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microscopy (SEM). Histopathological changes in the intestinal tract of infected dogs
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were characterized by epithelial metaplasia and dilatation; the integrity of intestinal
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mucosal epithelial cells was distinctly damaged with whole sheets of cilia sloughed away.
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Ultrastructural observation data were consistent with histological observations. Based on
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these findings, the canine model described in this work will be useful to evaluate clinical,
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parasitological and histological aspects of C. canis infection and will be useful for the
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further understanding of cryptosporidiosis, drug development, and vaccine development.
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Keywords: Cryptosporidium canis; Canine model, Experimental infection
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1. Introduction Cryptosporidium is a protozoal parasite that infects a variety of hosts, including humans, domestic and wild animals, worldwide (Ryan et al., 2016). Cryptosporidiosis
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characteristically results in watery diarrhea, but clinical symptoms can vary from
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asymptomatic shedding of oocysts to severe and life-threatening disease, depending on
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the immune status of the host (Bouzid et al., 2013). Immunocompetent individuals
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typically experience self-limiting illness (up to 2 to 3 weeks in duration) and recover
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without treatment. However, in immunocompromised patients, cryptosporidiosis can
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result in intractable diarrhea and may even be fatal in some cases (Chalmers and Davies,
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2010). An effective vaccine or drug treatment for cryptosporidiosis is not yet available.
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Dogs have been considered to be faithful friends and shared a close relationship with
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humans for over 10,000 years. Pet ownership has been recognizably proven to exert
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beneficial effects on human health, by improving physical condition as well as mental
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and emotional well-being (Hodgson et al., 2015). Consequently, pets are being
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increasingly used as a therapeutic option in healthcare facilities (Wells, 2007). However,
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dogs may not only act as intimate companions of humans but also as natural reservoirs of
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a large number of zoonotic pathogens, including Cryptosporidium spp. (Chomel, 2014;
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Esch and Petersen, 2013).
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Cryptosporidiosis has been reported in dogs worldwide (Jian et al., 2014). Currently,
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four species of Cryptosporidium, namely C. canis, C. parvum, C. muris, and C.
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meleagridis, have been detected in dogs (Lupo et al., 2008; Yoshiuchi et al., 2010; Scorza 3
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et al., 2011). Cryptosporidium canis causes the vast majority of infections in dogs (Ryan
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et al., 2014) and is considered to be potentially zoonotic due to its presence in human
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patients in a number of countries (Fayer, 2010; Gatei et al., 2008; Elwin K et al., 2012).
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Over the decades, different models of experimental infection have been described to study the susceptibility, infectivity and pathogenicity of Cryptosporidium (Ayinmode et
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al., 2017; Kvac et al., 2013; Modry et al., 2012; Masuno et al., 2014; Petermann et al.,
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2014). Nevertheless, information on cryptosporidiosis caused by C. canis infection in an
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experimental host has yet been reported.
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In this study, we developed a canine model of cryptosporidiosis caused by experimental infection with C. canis parasites. We analyzed the infectivity of C. canis
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using severe combined immunodeficiency (SCID) mice and immunosuppressed dogs,
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and described the clinical, parasitological, and histological aspects of experimental
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infection in immunosuppressed dogs, which provide a good model for the future testing
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of novel drugs or vaccines against cryptosporidisis.
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2. Materials and methods
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2.1. Ethics approval and consent to participate
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This study was conducted in accordance with the Chinese Laboratory Animal
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Administration Act (1988). The experimental protocol was approved by the Institutional
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Animal Care and Use Committee of Henan Agricultural University (authorization number
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IACUC-henau-20070503). 4
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2.2. Inoculum Cryptosporidium oocysts were obtained from a dog located in a pet hospital at the province of Henan, China. Oocysts were concentrated using the water ether technique
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(Bukhari and Smith, 1995), and purified by discontinuous sucrose density centrifugation
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(Heyman et al., 1986). Oocysts were counted using a Neubauer hemocytometer. A
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combination of streptomycin and penicillin was added and this oocyst suspension was
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kept at 4°C. C. canis oocysts for morphometry analyses were determined using digital
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analysis of images (Motic Images Plus 2.0 soft-ware). Length and width of oocysts (n =
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50) were measured under bright-field microscopy at 1000-fold magnification, and these
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were used to calculate the length-to-width ratio of each oocyst.
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Total DNA was extracted using an E.Z.N.A.® Stool DNA Kit (OMEGA Biotek Inc.,
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Norcross, GA, USA). To determine the species of Cryptosporidium for the isolate used in
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this study, nested PCR protocols were used to amplify the SSU rRNA gene, actin gene
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and HSP70 gene according to the previously-reported studies (Xiao et al.,1999; Sulaiman
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et al., 2002; Sulaiman et al., 2000). DNA sequencing indicated that the SSU rRNA and
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HSP70 nucleotide sequences had a 100% similarity with C. canis (GenBank accession
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number AB210854 and AY120920, respectively), while the actin nucleotide sequence had
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a 99% similarity with C. canis (GenBank accession number AY120927). The nucleotide
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sequences obtained in this study has been deposited in the GenBank database under
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accession numbers: EU754826, EU754835 and EU754843. 5
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2.23. Experimental animals Six 8-week old SCID mice were obtained from Shanghai SLAC Laboratory Animal
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Co., Ltd., Shanghai, China. All mice were housed individually in plastic cages with wire
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mesh tops, under pathogen-free conditions, and kept on daily 12 h cycles of light and
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dark. Mice received sterilized food and water, and were randomly divided into control and
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test groups with 3 mice per group. Each mouse in the test group was inoculated orally by
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a stomach tube with a dose of 1×106 oocysts.
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Nine beagle dogs, 7 weeks of age, were obtained from the Animal Lab Center of Henan Agricultural University. All dogs were confirmed to be free of C. canis infection
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by microscopic examination of feces. During the adaptation period, dogs were vaccinated
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against regional infectious diseases (Canine distemper, Parvovirus, Canine hepatitis,
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Leptospirosis, and Rabies). Animals received commercial dog food, according to their
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physiologic development, and water ad libitum.
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Experimental dogs (n = 3) were immunosuppressed according to the protocol as
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described previously (Young et al., 2002). Dogs were fed 6 mg of dexamethasone acetate
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per day for 5 days before infection. At day 8 of immunosuppression, each dog was
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inoculated orally by a stomach tube with a dose of 1×106 oocysts. The following two
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control groups were also included: immunocompetent dogs inoculated orally with 1×106
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oocysts of C. canis (n = 3), and immunocompetent, non-inoculated dogs (n = 3). At days
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12 and 28 post infection (p.i.), one dog per group was euthanized by an intravenous
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injection of a lethal dose of pentobarbital 100 mg/kg and their organs were removed.
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2.34 Clinical status
Dogs were examined daily for clinical signs including rectal temperature, respiratory
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rate, and heart rate. Additionally, the presence of diarrhea and any abnormal behaviours
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were observed.
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2.45. Oocyst excretion
To determine the prepatent period, daily fecal samples were collected from each mouse and dog from day 1 to 28 p.i. Briefly, 1g of the feces from each animal was
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examined using the Sheather’s sugar floatation method and counted using hemocytometer
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slide under bright-field microscopy at 400-fold magnification. Repeat the count three
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times and calculate the average. Infection intensity was determined by the number of
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oocysts per gram (OPG). The OPG was estimated on the basis of number of oocysts
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counted.
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2.56. Histological examination
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For histological analysis, tissue samples harvested from duodenum, jejunum, ileum,
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cecum, stomach, lungs, liver, spleen and kidney were fixed in 10% buffered formalin for
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24 h, dehydrated in absolute ethanol, cleared in xylene, and embedded in paraffin. Tissue 7
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sections were stained with hematoxylin and eosin (H & E), and observed microscopically
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at 400x and 1000x magnification. Tissue samples harvested from duodenum, jejunum, ileum and cecum were also
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prepared for scanning electron microscopy (SEM). Samples were fixed in 2.5%
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glutaraldehyde for one week at 4 , and then washed with 0.1 mol/L phosphoric acid
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buffer (pH = 7.4) three times for 10 min each. The dehydration procedure followed
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conventional methods in a graded ethanol series of 30%, 50%, 70%, 90%, 100%, and two
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more changes of 100%, each for 5 min followed by 50% isoamyl acetate solution (v/v,
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isoamyl acetate: ethanol = 1:1) and 100% isoamyl acetate solution for 10 min,
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respectively. After specimens were critically point-dried using CO2 and coated with gold,
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observations were made by an S-3400 SEM (HITACHI).
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3. Results
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3.1. Clinical status
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The appetites and attitudes of experimental and control SCID mice were normal
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until the end of the experiment. No diarrhea was observed in any mice. All immunosuppressed dogs inoculated with oocysts of C. canis showed signs of
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physical deterioration, exhibiting loss of sheen to the coat, loss of interest in the
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environment, and lethargy. These dogs began to have diarrhea around day 8 p.i. No
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diarrhea was observed in any control group dogs. No animals died during the experiment.
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3.2. Oocyst shedding Fecal examination of infected dogs revealed fully sporulated C. canis oocysts only
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in immunosuppressed dogs inoculated with C. canis oocysts. No Cryptosporidium
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oocysts were found in control or non-immunosuppressed dogs during the experiment.
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Likewise, no Cryptosporidium oocysts were detected in the feces of SCID mice in either
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group. C. canis oocysts found were ovoid, and 4.8 (4.2-5.2) µm x 4.4 (4.2-5.3) µm (n =
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50) (Fig. 1). Oocysts were first detected in feces on day 3 p.i., peaking twice on days 10
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and 17 p.i. The patterns of oocyst shedding in dogs in the test group are presented in Fig.
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2. Sequences of SSU rRNA gene, actin gene and HSP70 gene from experimentally
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infected dogs shared 100% identity with the isolate used in the inoculum.
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3.3. Histological observations of sites of C. canis infection Cryptosporidial infection in immunosuppressed dog (at 12 days p.i.) was found in
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the duodenum (Fig. 3A) and jejunum (Fig. 3B, C). However, no cryptosporidial
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developmental stages or pathologic changes were observed at any other anatomic sites,
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including the ileum (Fig. 3D), cecum, colon, or stomach. Likewise, no cryptosporidial
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developmental stages or pathologic changes were observed at any anatomic sites in dogs
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of control groups (data not shown). Histopathological changes in the intestinal tract of
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dogs with cryptosporidiosis were characterized by epithelial metaplasia and dilatation;
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the integrity of intestinal mucosal epithelial cells had been distinctly damaged, with
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whole sheets of cilia sloughed away. These changes were more pronounced in the middle
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and lower parts of duodenal mucosa epithelium, and the anterior and middle portions of
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jejunal mucosa epithelium.
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3.4. Ultrastructural observation
Scanning electron microscopy study showed that C. canis at various of
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developmental stages had adhered to the surface of the duodenum and jejunum in
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immunosuppressed dogs. Epithelial cell surfaces were swollen and disordered (Fig. 4A,
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B). The integrity of mucosal layer of the duodenum and jejunum had been damaged, and
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many cilia had fallen off or atrophied (Fig. 4C, D). Ultrastructural observation also
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showed that no parasites were observed in the ileum, cecum, colon, or stomach，
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consistent with the histological observations described above.
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4. Discussion
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The potential role of companion animals as reservoirs for zoonotic diseases has been
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recognized as a significant public health problem worldwide (Traub et al., 2005). Canids
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(wild and domestic) are recognized as the main reservoir of a vast range of parasites.
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Therefore, many studies with respect to Leishmania, Trypanosoma cruzi, Neospora
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caninum and Echinococcus multilocular have used the dog as an experimental model to
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investigate the reproductive potency, infectivity and pathogenicity of the parasite, as well
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as develop vaccines and assess drugs (Petitdidier et al., 2016; Padilla et al., 2009; Kapel
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et al., 2006; Cedillo et al., 2008;). Here, we established a canine model of infection with
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C. canis, with dexamethasone used to develop the experimental model of
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cryptosporidiosis as in previous reports (Surl and Kim, 2006; Certad et al., 2007). Experimentally inoculated immunocompetent dogs did not produce detectable C.
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canis oocysts by microscopy in faecal samples within 28 DPI. None of the SCID mice
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exhibited clinical signs or oocyst shedding at any point during the experiment. It is
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suspected that SCID mice were not infected with C. canis. Data from a previous study
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also indicate that oocysts of C. canis were not infectious for BALB/c neonatal mice or
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immunosuppressed C57 juvenile mice, but were infectious for calves (Fayer et al., 2001).
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In our study, infected dogs began shedding oocysts in low numbers on day 3 p.i.,
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with two peaks of infection intensity during the patent period. In immunosuppressed mice
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infected with C. parvum, oocyst shedding begins 6 days p.i., and these mice continue
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shedding until day 35 p.i. (Coco et al., 2012). The prepatent period was 4 days in the case
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of infection in immunosuppressed C57BL/6 mice with a single C. meleagridis oocysts
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(Huang et al., 2003). However, the prepatent period was 20-24 days, and the patent
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period varied between 46 and 59 days in neonatal and adult M. coucha infected with C.
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andersoni (Kvac et al., 2007). The differences in the prepatent and patent period probably
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depend on the experimental animals or species of Cryptosporidium.
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Variability of infection sites between different Cryptosporidium species and types
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has been reported (Plutzer and Karanis., 2009). Three of the four spieces of
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Cryptosporidium (C. canis, C. parvum and C. meleagridis) that infect dogs are detected
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in the small intestine, except C. muris which is detected in stomach; this difference could 11
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be associated with different patterns of colonization along the gastrointestinal tract. In
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this study, cryptosporidial developmental stages were observed in the middle and lower
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parts of duodenal mucosa epithelium, and anterior and middle parts of jejunal mucosa
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epithelium in histological sections and SEM study. This is similar to what has been
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described for naturally-occurring cryptosporidiosis in dogs (Greene et al., 1990; Wilson
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et al., 1983). However, one published case report indicates that C. canis was found both
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in the small intestine and gastric surface epithelium of an 8-week-old female Yorkshire
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terrier co-infected with Isospora spp. (Miller et al., 2003). Extraintestinal infection was
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not observed in our study, which could be related to the degree of immunosuppression,
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and/or the time post-infection that we evaluated.
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Cryptosporidium primarily infects the microvillous border of the intestinal
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epithelium, and to lesser extent extraintestinal epithelia, causing acute gastrointestinal
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disease in a wide range of mammalian hosts, including humans (Deng et al., 2004).
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Damage of host intestinal epithelial cells caused by cryptosporidiosis usually results in
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two outcomes: cell death and cell damage. Both produce architecture distortion and
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atrophy of the villus. In the present study, the surfaces of the epithelial cells were swollen
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and disordered. The integrity of mucosal layer had been damaged, and many cilia had
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fallen off atrophied in duodenum and jejunum of dogs infected with C. canis. Similarity,
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in chickens successfully infected with C. baileyi, the integrity of the epithelial cells of the
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BF (bursas of fabricius) was somewhat damaged (Yuan et al., 2014). In mice infected
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with C. parvum, cryptosporidium stages were detected on the brush borders and the
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intestinal epithelium showed loss of single epithelial cells, mild blunting, and shortening
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of villi (Sayed et al., 2016). In conclusion, we have established an experimental infection of C. canis in dogs
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immunosuppressed with dexamethasone. Parasitological and histopathological features of
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the infection were studied. These findings may foster further understanding of the biology
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of C. canis and lay the foundation to control cryptosporidiosis.
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Conflict of interests
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The authors declare that there are no conflicts of interest.
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Acknowledgments
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This study was partly supported by the National Key Research and Development
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Program of China (2017YFD0501305, 2016YFD0500707), the National Natural Science
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Foundation of China (31330079), and the Natural Science Foundation of Henan Province
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(162300410129).
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We thank Catherine Barnette, DVM, from Liwen Bianji, Edanz Group China
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(www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.
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Figure legends
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Fig. 1. Microscopy of Cryptosporidium canis oocysts visualized by Modified acid-fast
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staining method (1000×).
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Fig. 2. Excretion of oocysts in one gram of feces in immunosuppressed dogs infected
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with C. canis during the experiment. Mean of all examined animals with standard errors.
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Fig. 3. Histological observation of C. canis infection in immunosuppressed dogs using H
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& E staining. High parasite burden in the duodenum (A, ×1000) and jejunum (B, C, 19
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×1000) (arrows). No cryptosporidial developmental stages and pathologic changes were
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Fig. 4. Scanning electronic microscopic observation of C. canis infection in
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immunosuppressed dogs. C. canis at a large number of developmental stage had adhered
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to the surface of duodenum and jejunum (A, B, ×3000) (arrows). The surfaces of the
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epithelial cells were swollen and disordered. The integrity of mucosal layer of the
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duodenum and jejunum had been damaged, and many cilia had fallen off or atrophied (C,
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D, ×5000).
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ACCEPTED MANUSCRIPT Highlights · This is the first canine model of experimental infection with C. canis. · C. canis was not infective for immunocompetent dogs and SCID mice. · Cryptosporidial infection was only found in duodenum and jejunum.
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· The histopathological changes were observed with HE and SEM.