Introduction
Domesticated dogs (Canis
lupus familaris) have become an essential part of people around the world.
They influence people’s daily lives as they impart delight, diminish friendlessness
and psychological issues and give people emotional support (Deng et al. 2018). Pakistan has a huge
population of livestock and other domestic animals such as dogs. Dogs possess
peculiar attributes like guarding, sniffing, hunting and retrieving. These
specialties of dogs are utilized by military, rangers, police, anti-narcotic
forces, and other agencies. There are three million dogs in Pakistan (Towakal et al. 2010). Canine coronavirus (CCoV) was regarded as a pathogen of
dogs in 1971 (Binn et al. 1974).
Viruses of the coronaviridae are single stranded RNA viruses with a genomic
length of 30 kbp. CCoV infection is very common in younger dogs, especially
those kept in large groups, breeding facilities, shelters and kennels (Stavisky
et al. 2012). Canine Corona
Virus (CCoV) infects epithelial cells of
intestinal villi causing mild to severe diarrhea (Saif and Heckert 1990). Pups
are highly prone to develop severe and fatal disease. The infected dog is dull,
lethargic, may or may not be febrile, anorectic, shows vomiting, bloody
diarrhea and dehydration, Coronavirus infection is not diagnosed accurately as
it mimics another viral infection caused by Canine Parvovirus (CPV). Thus,
clinicians fail to educate the dog owners confidently about the prognosis of
the disease and it causes great economic loss to dog breeders (Sulehria et al. 2020). CCoV can be diagnosed by
cell-line culture method, PCR and immune-chromatography based test kits (Yoon et al. 2018). Chloroquine is
known since 1934. Besides from its reputed antimalarial action, it has good
antiviral effects especially against viruses like coronaviruses, retroviruses
& flaviviruses and HIV (Savarino et
al. 2003).
Materials and
Methods
Experimental
details and treatments
Experimental
material: A total of thirty (n = 30) dogs (C. lupus familaris) positive for CCoV reported from different
private and public veterinary clinics in Lahore were included in the study from
January 2019 to December 2019. CCoV was identified from diarrheic dogs that
were presented at different private and public veterinary clinics. For this
purpose fecal samples were collected from the morbid dogs and were rendered for
rapid detection (Fig. 1) using immuno-chromatography based rapid detection test
kits (Sulehria et al. 2020)
manufactured by Quicking Biotech China (Pvt. Ltd.). (Fig. 1).
%20426-430_files/image002.png)
Fig. 1: A CCoV
positive sample (Left Column with double bands) using a Rapid Detection Test
Kit
%20426-430_files/image004.png)
Fig. 2: PCR results
for M-gene amplification of CCoV showing DNA ladder, positive and negative
controls and positive and negative samples
%20426-430_files/image006.png)
Fig. 3: No. of dogs
alive at the start and completion of the trial
Confirmation
of CCoV by PCR
PCR assay was performed for the confirmation of CCoV. The CCoV RNA was extracted through RNA
Fast Extraction stool Kit (Cat # RP8001, Bioteke Corporation China). To confirm
the correct extraction and quality of the RNA, all samples were quantified by
using a Nano drop 2000 spectrophotometer. To confirm the presence of CCoV, a
321 (bp) fragment of the M-gene of CCoV was targeted (Figs. 2, 3) by using
protocols & PCR conditions as described by (Agnihotri et al. 2018; Sulehria et al.
2020).
Treatments
A total of 30 dogs (CCoV positive) were randomly
selected in the study (Table 2). These 30 dogs were further divided into two
groups; A & B containing 15 dogs each. Group-A was taken as control
group whereas Group-B was taken as treatment group. The dogs in both the groups
A and B were given fluid therapy, anti-diarrheal medicine (Metronidazole @ 15
mg/kg q12h IV), anti-emetic (Metoclopramide @ 0.4 mg/kg q8h IV) medicine along with
antibiotic (Ceftriaxone Sodium @ 50 mg/kg q12h). The treatment group (Group B)
was given the same medicines and, additionally, Chloroquine @ 10 mg/Kg SQ q24h
for consecutive 3 days.
Blood
collection for hemato-biochemical analysis
Blood samples of the morbid dogs were collected
aseptically from the cephalic or saphenous veins into EDTA coated (purple cap)
and non-EDTA (yellow cap) coated vacutainers. 4 mL of blood was collected from
each dog, 1 mL for Complete Blood Count (CBC) and 3 mL for Liver Function Tests
(LFTs) & Renal Function Tests (RFTs). The samples were transported to the
laboratory of Dairy Health Research Lab (DHRL), Department of Veterinary
Medicine and Surgery (CMS), UVAS, Lahore, Pakistan by maintaining the cold
chain 4şC. The VET hematology analyzer (Model No. DW-3680/DW-36) was used for
performing the CBC. While the serum samples were analyzed for estimation of
biochemical parameters using a Semi-automated clinical chemistry analyzer
machine (Model URIT-810).
Post
treatment examination of dogs
The treated animals after the drug administration were
re-examined after 14th day for rapid detection test using the kit.
Simultaneously, the blood samples were also collected for hematological and
serum biochemical analysis to check the hemato-biochemical changes.
Drug’s efficacy formula
drug efficacy formula was taken from Asmaa et al. (2014);
%20426-430_files/image008.png){kind=small}
.
Table 1: Clinical
Scoring of the Patients
|
S. No. |
Clinical sign |
No. of dogs |
Percentage (%) |
|
|
1 |
Anorexia |
Yes |
30 |
100 |
|
No |
0 |
0 |
||
|
2 |
Temperature |
Below 101şF |
10 |
33.3 |
|
101şF to 102.5şF |
4 |
13.3 |
||
|
Above 102.5şF |
16 |
53.3 |
||
|
3 |
Vomiting |
Yes |
21 |
70 |
|
No |
9 |
30 |
||
|
4 |
Diarrhea |
Yes |
30 |
100 |
|
No |
0 |
0 |
||
|
5 |
Mucous Membrane |
Reddish Pink |
4 |
13.3 |
|
Pale |
26 |
86.7 |
||
|
6 |
Dehydration |
4-5% |
24 |
80 |
|
6-7% |
2 |
6.7 |
||
|
More than 7% |
4 |
13.3 |
||
Statistical analysis
For statistical analysis, Chi-square (χ2) Test and
Sampled paired t-test was applied to various hemato-biochemical
parameters to determine the significant (P
< 0.05) difference of mean and standard deviation before and after the
treatment. All the statistical analyses were carried out using IBM® SPSS
(statistical product and service solutions) Statistics® version 21.0.
Results
Clinical condition and scoring
The current study showed that
corona virus infected dogs showed different clinical signs (Table 1). Prior to
treatment, clinical examination revealed that all the dogs (100%, 30/30) were
anorectic. Among the dogs (33.3%, 10/30) showed low body temperature i.e., less than 101şF, fever
(temperature more than 102.5şF) was observed in 16 out 30 dogs (53.3%) whereas
4 out of 30 dogs (13.3%) had normal body temperature, but harbored the canine
corona virus infection. Among the dogs, 21 out of 30 (70%) had been confirmed
to have been vomiting. All the canine corona virus infected dogs (100%) showed
diarrhea. The dogs showed pale mucous membrane were 26 out of 30 (86.7%), while
4 out of 30 dogs (13.3%) showed pink mucous membrane. Dogs appeared dehydrated
with varying degrees. Out of 30 infected dogs, 24 (80%) were 4–5% dehydrated, 2
dogs (6.7%) were 6–7% dehydrated while 4 out of 30 (13.3%) were more than 7%
dehydrated. It was observed that all the 30 dogs (100%) dogs had a poor body
condition.
Survival
rate
The clinical trial of chloroquine showed significant
results. The Table 2 shows the comparison of the results between Group A
(control group) and Group B (treatment group). In group A, 5 out of 15 dogs
recovered and remained alive while 10 died, the mortality rate 66.7% in this
group. On the other hand, in the group B, 10 out 15 dogs recovered and 5 out of
15 died during the course of this study. The mortality rate was observed to be
33.7% in this group suggesting that the mortality rate was higher in the
control group where there was no administration of chloroquine.
Dynamics
of hemato-biochemical parameters
There was significant improvement (P < 0.05) in RBCs, neutrophils, monocytes, eosinophil,
lymphocytes, MCH and platelets count on 14th day post-treatment in
Group B compared to the dogs in Group A, indicating a significant increase
(Table 3). Table 4 clearly shows that in treatment group (B), the values of Aspartate Aminotransferase (AST), Alanine Transaminase (ALT), bilirubin, alkaline
phosphatase, total protein, globulin and urea were significantly lower as compared
to the control group (A).
Discussion
This study was a novel attempt
to diagnose canine coronavirus at molecular level and to treat this disease
with chloroquine, an antimalarial drug. It was observed that CCoV infected dogs
showed different clinical signs. The clinical examination revealed that all the
dogs were anorectic, ten out of thirty dogs showed a decreased body
temperature, sixteen out of thirty dogs showed fever, while the remaining four
out of thirty dogs showed normal body temperature upon presentation but they
harbored the canine corona virus infection. These same non-specific clinical
signs have been observed by (El-Neshwy et
al. 2019). Among the thirty dogs twenty-one dogs had been confirmed to have
been vomiting. All the CCoV infected dogs showed bloody mucoid diarrhea and all
of them had poor body condition. Dogs appeared dehydrated with varying degrees.
Out of 30 dogs, 24 were 4–5% dehydrated, two were 6–7% dehydrated while four
dogs were more than 7% dehydrated. Twenty-six out of 30 dogs showed pale mucous
membrane whereas four dogs showed pink mucous membrane. The same has been
observed by (Thomson and Gagnon 1980; Naylor et al. 2001; Godsall et al.
2010; Schultz et al. 2010; Kalli et al.
2010; Stavisky et al. 2012).
This study was a clinical trial, conducted to evaluate
chloroquine as an antiviral drug against CCoV. The results suggested that in
group A, 5 out of 15 dogs recovered and remained alive while 10 died, the
mortality rate 66.7% in this group. On the other hand, in the group B, 10 out
15 dogs recovered and 5 out of 15 died during the course of this study. The
mortality rate was observed to be 33.7% in this group suggesting that the
mortality rate was higher in the control group where there was no
administration of chloroquine. These results are in accordance with (Pardridge et al. 1998; Keyaerts et al. 2009;
Kaptein and Neyts 2016) who proved the antiviral effects of chloroquine against
human coronavirus, dengue virus and HIV respectively. As seen in Table 3, there
was a significant improvement (P < Table 2: Treatment Plan of Control and
Treatment Groups
|
S. No. |
Fluid
Therapy + Antidiarrheal + Anti-emetic + Antibiotic |
Fluid
Therapy + Antidiarrheal + Anti-emetic + Antibiotic+ Chloroquine |
||
|
|
Group (A) CCoV |
Group (B) CCoV |
||
|
1 |
Died |
Recovered |
||
|
2 |
Died |
Died |
||
|
3 |
Recovered |
Recovered |
||
|
4 |
Died |
Recovered |
||
|
5 |
Recovered |
Died |
||
|
6 |
Died |
Recovered |
||
|
7 |
Died |
Recovered |
||
|
8 |
Recovered |
Recovered |
||
|
9 |
Recovered |
Recovered |
||
|
10 |
Recovered |
Recovered |
||
|
11 |
Died |
Died |
||
|
12 |
Died |
Recovered |
||
|
13 |
Died |
Recovered |
||
|
14 |
Died |
Died |
||
|
15 |
Died |
Died |
||
|
Mortality
Rate |
10/15 (66.7
%) |
5/15 (33.3%) |
5/15 (33.3%) |
10/15
(66.7%) |
|
Drug
Efficacy |
33.3% |
66.7% |
||
Table 3: Comparison of Hematological
Parameters Before and After Trial
|
Parameters |
Control Group (A) |
Treatment
Group (B) |
P-Value |
|
|
Before (Day 0) |
After (Day 14) |
|||
|
Hb (G/dL) |
9.19 ± 0.70 |
8.47 ± 0.92 |
9.87 ± 0.54 |
0.243 |
|
RBCs x10^6/µL |
4.67 ± 0.31 |
4.27 ± 0.34 |
4.95 ± 0.14 |
0.000 |
|
PCV (%) |
30.85 ± 2.00 |
28.44 ± 1.82 |
33.03 ± 2.82 |
0.921 |
|
MCV fl |
74.25 ± 2.24 |
79.36 ± 1.65 |
71.48 ± 1.47 |
0.109 |
|
MCHC (G/dL) |
29.06 ± 1.37 |
28.15 ± 1.27 |
31.18 ± 0.96 |
0.515 |
|
TLC (x103 /µL) |
12.25 ± 0.41 |
11.53 ± 0.61 |
12.09 ± 0.52 |
0.548 |
|
Neutrophils % |
70.51 ± 2.10 |
72.91 ± 2.68 |
68.53 ± 2.09 |
0.015 |
|
Monocytes % |
4.32 ± 0.24 |
4.03 ± 0.18 |
5.11 ± 0.25 |
0.000 |
|
Eosinophils % |
0.86 ± 0.14 |
0.49 ± 0.018 |
1.01 ± 0.18 |
0.017 |
|
Lymphocytes % |
14.88 ± 0.44 |
13.99 ± 0.70 |
16.12 ± 0.76 |
0.000 |
|
MCH Pgs |
21.65 ± 0.93 |
20.75 ± 1.50 |
22.69 ± 0.50 |
0.001 |
|
Platelets (x 105/µL) |
386.24 ± 41.81 |
230.19 ± 14.93 |
419.23 ± 24.74 |
0.014 |
Table 4: Comparison
of Biochemical Parameters Before and After Trial
|
Parameters |
Groups |
Day 0 |
Day 14 |
P-Value |
|
AST U/L |
Group A |
70.53 ± 4.11 |
57.40 ± 5.71 |
0.269 |
|
Group B |
71.86 ± 1.95 |
48.82 ± 5.06 |
0.000* |
|
|
ALT U/L |
Group A |
141.51 ± 1.97 |
115.70 ± 4.69 |
0.182 |
|
Group B |
143.38 ± 4.95 |
106.62 ± 3.57 |
0.000* |
|
|
Bilirubin Total mg/dL |
Group A |
0.61 ± 0.25 |
0.51 ± 0.034 |
0.533 |
|
Group B |
0.60 ± 0.26 |
0.42 ± 0.014 |
0.000* |
|
|
Alkaline Phosphate U/L |
Group A |
279.08 ± 7.88 |
231.42 ± 18.25 |
0.559 |
|
Group B |
277.35 ± 8.09 |
193.15 ± 10.77 |
0.000* |
|
|
Total Protein G/dL |
Group A |
8.57 ± 0.32 |
7.83 ± 0.37 |
0.050* |
|
Group B |
8.42 ± 0.12 |
7.07 ± 0.16 |
0.002* |
|
|
Albumin G/dL |
Group A |
2.97 ± 0.11 |
3.10 ± 0.07 |
0.667 |
|
Group B |
3.01 ± 0.13 |
3.08 ± 0.06 |
0.908 |
|
|
Globulin G/dL |
Group A |
6.16 ± 0.08 |
5.73 ± 0.24 |
0.005* |
|
Group B |
6.18 ± 0.04 |
5.16 ± 0.11 |
0.017* |
|
|
Urea mg/dL |
Group A |
55.36 ± 0.99 |
47.08 ± 1.11 |
0.731 |
|
Group B |
55.12 ± 1.01 |
34.42 ± 1.71 |
0.030* |
|
|
Creatinine mg/dL |
Group A |
1.58 ± 0.57 |
1.48 ± 0.49 |
0.560 |
|
Group B |
1.54 ± 0.47 |
1.48 ± 0.47 |
0.714 |
0.05) in RBCs, neutrophils, monocytes, eosinophil,
lymphocytes, MCH and Platelets count on 14th day post-treatment in
Group B compared to the dogs in Group A indicating a significant increase. The
same has been reported by (Sharma et al.
2008; Dongre et al. 2015; Agnihotri et al. 2017; Sulehria et al. 2020). It was uncovered in
the current study that in the treatment group B, the values of AST, ALT,
bilirubin, alkaline phosphatase, total protein, globulin and urea were
significantly lower (better) as compared to the control group (A), (Shaker and
Carey 1990; Berghoff and Steiner 2011; Bhat et al. 2013) were also of the same view.
Conclusion
The findings of our study conclude that canine coronavirus is circulating in the dog
population of Pakistan. Chloroquine
is a good and cost effective drug to treat canine coronavirus infection
in dogs. The study has set a more authentic and reliable way to diagnose canine
coronavirus infection in dogs. The advantage of using this line of
diagnosis is that it will give more confidence to the practicing vets and will
improve their clinical skills. Chloroquine may be referred to as a
potential and novel drug for the treatment of canine coronavirus infection in
dogs.
Acknowledgments
The first author acknowledges the financial aid from the
Higher Education Commission (HEC) of Pakistan for funding us through the
National Research Programme for Universities (NRPU) project # (9719/NRPU/R&
D/HEC).
Author Contributions
Conception, US, SS; methodological approach, US, SS, MI;
critical examination, US, SS, MI and HM; writing initial draft preparation, US,
SS; writing, assessment, and proofreading, US, SS and MI.
Conflict
of Interest
All the authors have declared no conflicts of interest.
Data
Availability
Data presented in this study will be available upon a
fair request to the corresponding author.
Ethics
Approval
The study design was approved by the Ethics Committee of the University of
Veterinary and Animal Sciences Lahore, Pakistan under diary No.
502/dated 02.03.2018.
Funding
Source
National Research Programme for Universities
(NRPU) project # (9719/NRPU/R& D/HEC).
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