Journal of Threatened Taxa |
www.threatenedtaxa.org | 26 January 2022 | 14(1): 20494–20499
ISSN 0974-7907
(Online) | ISSN 0974-7893 (Print)
https://doi.org/10.11609/jott.7578.14.1.20494-20499
#7578 | Received 18
July 2021 | Final received 03 September 2021 | Finally accepted 04 January 2022
Trypanosoma evansi infection in
a captive Indian Wolf Canis lupus pallipes – molecular diagnosis and therapy
Manojita Dash 1, Sarat Kumar Sahu 2,
Santosh Kumar Gupta 3, Niranjana Sahoo 4 & Debarat
Mohapatra 5
1,3 Centre for Wildlife Health,
College of Veterinary Science & Animal Husbandry, Odisha University of
Agriculture and Technology, Bhubaneswar, Odisha 751003, India.
2,5 Nandankanan Zoological Park, Po-Barang, Khordha District, Odisha
754005, India.
4 Department of Preventive
Veterinary Medicine and Epidemiology, College of Veterinary Science &
Animal Husbandry, Odisha University of Agriculture & Technology,
Bhubaneswar, Odisha 751003, India.
1 manojita.dash@gmail.com, 2 sahu.sarat77@gmail.com
(corresponding author), 3 santoshkumargupta67@ymail.com, 4 niranjanasahoo@hotmail.com,
5 debabrat73@gmail.com
Editor: Rajeshkumar G. Jani, Anand Agricultural University, Anand,
India. Date of publication:
26 January 2022 (online & print)
Citation: Dash, M., S.K. Sahu, S.K. Gupta, N. Sahoo & D. Mohapatra (2022). Trypanosoma evansi
infection in
a captive Indian Wolf Canis lupus pallipes – molecular diagnosis and therapy. Journal of Threatened Taxa 14(1): 20494–20499. https://doi.org/10.11609/jott.7578.14.1.20494-20499
Copyright: © Dash et al. 2022. Creative Commons Attribution
4.0 International License. JoTT allows unrestricted use, reproduction, and
distribution of this article in any medium by providing adequate credit to the
author(s) and the source of publication.
Funding: This study is
a part of normal healthcare
activity of the zoo (Nandankanan Zoological Park) which is under the control of ‘Forest and Environment Department, Government of Odisha’. No separate fund has been received
from any agency for this study.
Competing interests: The authors
declare no competing interests.
Author details: Manojita Dash, MSc Biotechnology—She is
working as Senior Research Fellow at Centre for Wildlife Health (CWH), CVSc & AH, OUAT, Bhubaneswar. She is actively involved
in the processing of bio-samples collected from Nandankanan
Zoological Park as well as various other zoos and sanctuaries of Odisha. She is
well versed with handling of sophisticated instruments used in analysis of the
bio-samples, data interpretation, statistical analysis. Sarat Kumar Sahu, MVSc, Veterinary
Microbiology—He is working as Veterinary Assistant Surgeon at Nandankanan Zoological Park since more than 11 years. He is
actively involved in treatment, preventive health care, conservation and
scientific research related to captive and free ranging wild animals. Santosh Kumar Gupta—He has completed M.V.Sc. in Veterinary Clinical Medicine, Ethics and
Jurisprudence from College of Veterinary Science, Guwahati, Assam and has been
engaged as Research Associate at Centre for Wildlife Health, OUAT. He has keen
interest in Wildlife and conservation science and has been carrying out
scientific research work at Nandankanan Zoological
Park. Niranjana Sahoo, MVSc, PhD—He is the Professor and Head, Department of
Veterinary Epidemiology and Preventive Medicine, and Director, Teaching
Veterinary Clinical Complex, CVSc & AH, OUAT. He
is also the Coordinator, Centre for Wildlife Health (CWH), OUAT. CWH is serving
as the microbiological, molecular and forensic diagnostic hub for captive and
free ranging wild animal diseases of the entire Odisha state. Debabrat
Mohapatra—He has done MVSc in Veterinary Clinical
Medicine. He is currently working as Medicine Specialist at Nandankanan
Zoological Park since last six years. He has keen interest in treatment and
preventive healthcare of captive wild animals of the zoo.
Author contributions: SKS, SKG and DM have executed the
treatment, collected samples and documented the clinical findings. MD and NS
have carried out molecular screening, interpreted the results and guided the
treatment. All authors formulated and revised the manuscript, and approved the
final version.
Acknowledgements: Authors are thankful to the
Director, Nandankanan Zoological Park, Odisha for
extending necessary facilities to carry out this in situ study and to publish
this manuscript.
Abstract: A five-year old, apparently
healthy male Indian Wolf Canis lupus pallipes of Nandankanan
Zoological Park, Odisha became ill with acute signs of anorexia, lethargy,
staggering gait, and was non-responsive to external stimuli. Microscopic
examination of Giemsa stained blood smear revealed presence of extracellular
flagellates having morphological similarity to Trypanosoma spp. Haematological parameters showed anaemia
(Hb 6.0 g%), mild leucopenia (total leukocyte count 5
× 103 / mm3) and thrombocytopenia (180 x 103 /
µl). Serum biochemistry revealed high aspartate aminotransferase (AST) (830
IU/L), blood urea nitrogen (BUN) (178.2 mg/dl), creatinine (4.44 mg/dl), and
low glucose (25.7 mg/dl) levels. Polymerase chain reaction (PCR) analysis
targeting internal transcribed spacer (ITS1) region followed by National Centre
for Biotechnology Information blast confirmed Trypanosoma evansi infection in the captive Indian Wolf. The animal
showed clinical recovery with the administration of single dose of quinapyramine sulphate and quinapyramine chloride @ 4.0 mg/kg b wt
subcutaneously. The wolf started taking meat from the very next day with
improved activity. No trypanosomes could be detected in the stained blood
smears as well as through PCR carried 25 days post treatment. The occurrence
became an eye opener for the zoo and henceforth, all canids were included under
chemoprophylaxis protocol against trypanosomosis.
Keywords: Anemia, Canids, captivity
stress, Chemoprophylaxis, PCR, Quinapyramine salts.
INTRODUCTION
Trypanosomosis, caused by an unicellular,
eukaryotic haemoprotozoan of different Trypanosoma
spp., is an important disease of domestic and wild animals (Aulakh
et al. 2005; Gupta et al. 2009). A number of trypanosomes exist worldwide;
however, Trypanosoma evansi is the only
pathogenic species prevalent in India (Desquesnes et
al. 2001; Kumar et al. 2021). Sengupta (1974), Ziauddin et al. (1992),
and Shukla (2002) reported trypanosomosis in Indian
Wolves in Indian zoos at Kolkata, Mysore, and Lucknow, respectively. This
extra-cellular haemoparasite is transmitted by biting
flies of genera Tabanus, Stomoxys,
and Haematobia (Parashar et al. 2006,
2018). The disease is characterized by anaemia,
anorexia, intermittent fever, generalised weakness,
conjunctivitis, corneal opacity, oedema of head and
throat, difficulty in swallowing, hoarse voice, and staggering gait (Chaudhuri
et al. 2009). The disease can be diagnosed by direct demonstration of trypomastigote forms of the parasite in the stained blood smears,
but the polymerase chain reaction (PCR) has an increased diagnostic potential
with high sensitivity and specificity to detect parasite DNA (Eloy & Lucheis 2009). Trypanosomosis has
been successfully treated with a single dose of diminazine
aceturate @ 3.5 mg/kg body weight intramuscular (Rani & Suresh 2007) or sulphate and chloride salts of quinapyramine
@ 4.0 mg/ kg bw subcutaneous (Singh et al. 1993).The
present case study documents molecular diagnosis through PCR and successful
therapy of Trypanosoma evansi infection in a
captive Indian Wolf at Nandankanan Zoological Park
(NKZP), India.
CASE HISTORY AND OBSERVATION
The NKZP received a pair of
wolves during September 2018 from Sri Chamarajendra
Zoological Gardens, Mysuru under an animal exchange program. Both were housed
in an open air enclosure of 28 sq meters attached to
a feeding cell of 15 sq meters. Regular prophylactic
measures included annual vaccination against rabies, parvo, distemper,
parainfluenza, adenovirus type I and II, hepatitis and Leptospira spp.,
fecal sample examination followed by deworming with albendazole/ fenbendazole
at three month intervals and ground spray of enclosure with ectoparasiticides
deltamethrine/cypermethrine
in alternate months. The female partner died on 07 March 2019 due to cardiac
dysfunction leaving the male wolf alone.
On 24 September 2019, the 5-year
old apparently healthy male partner (approximate body weight 20.0 kg) was
noticed anorectic, debilitated, non-responsive to external stimuli, reduced
activity levels with staggering gait. Close examination inside a squeeze cage
revealed shallow breathing and pale conjunctiva. Body temperature was 103.2°F.
Peripheral blood samples were collected on the same day from the left saphenous
vein in ethylene diamine tetraacetic acid @ 1.5 mg/ml
(EDTA) and clot activator vials for haemato-biochemical and parasitological
examination. Faecal samples were collected for
detection of gastrointestinal infection.
DIAGNOSIS AND TREATMENT
Coprological examination did not
reveal the presence of any endoparasite ova or cyst. Blood smear stained with Giemsa stain and
examined under oil immersion showed the presence of extracellular flagellated
Trypanosomes (Image 1). Molecular test was performed for confirmation of the
species. DNA was extracted from the EDTA
blood sample using Qiamp DNA blood Mini kit (M/S
Qiagen, Germany) according to the manufacturer’s instructions. PCR was carried
out in 50 µl reaction volumes containing 10X reaction buffer with KCl, 25 mM MgCl2, 2 mM dNTPs, 3 units of Taq DNA
polymerase, 2 µM of each primer (Njiru et al. 2005),
nuclease free water and 2 µl of template DNA. PCR was programmed to perform a
denaturation step at 950 C for 10 mins followed by 35 cycles
consisting of 30 secs at 940C, 30 secs at 550 C, and 30
secs at 720 C. The last extension step was 10 mins at 720
C. The PCR product was run in 2% agarose gel with ethidium bromide-stain using
an electrophoresis system (M/S BIO-RAD, USA) along with one positive (1 µg of
DNA) and one negative control (Image 2). After getting the desired band at 480 bp, the PCR product was sequenced and the data was compared
in National Centre for Biotechnology Information (NCBI) database. The sequenced
data matched with T. evansi with 93.6%
identity and 97.0% query cover. The consensus sequence (generated in BIOEDIT
software) was submitted in genbank (NCBI) and the
assigned accession number was MZ321577.
Analysis results depicted in
Table 1 revealed decrease in certain haemato−biochemical values like haemoglobin (6.0 g%), total leukocyte count (5.0 103/mm3)
neutrophil (56%), platelets count (180 × 103/µl) and glucose (25.7
mg/dl). Increased values in both haematological and
biochemical parameters included lymphocyte (41%), AST (830.4 IU/l), total
protein (7.63 g/dl), urea (178.2 mg/dl), creatinine (4.44 mg/dl), cholesterol
272.7 mg/dl), triglyceride (418.8 mg/dl), calcium (11.1 mg/dl), phosphorous
(11.4 mg/dl), magnesium (2.7 mg/dl), and total billirubin
(0.80 mg/dl)
Quinapyramine sulphate
and chloride @ 4.0mg/ kg b wt (Injection Triquin of M/S Vetoquinol India
Animal Health Pvt Ltd., Thane) was administered
subcutaneously. As supportive therapy, the Indian Wolf was administered with
paracetamol inj (Injection Fevastin
of M/S Tablets India Limited, Chennai) @ 2.0 ml intramuscular and electrolytes
with 20% dextrose infusion @ 300 ml (Rintose of M/S Vetoquinol India Animal Health Pvt
Ltd.). The Indian Wolf started responding to treatment from the very next day
itself. Body temperature dropped to 101.4°F with signs of improvement in the
activity and appetite.
DISCUSSION
NZKP had the earlier records of trypanosomosis among white Tigers Panthera
tigris, Bengal Tigers Panthera
tigris tigris, and
Jungle Cat Felis chaus
(Parija & Bhattacharya 2001; Sahoo et al. 2009).
Hence, the NKZP is following a chemoprohylaxis
protocol against trypanosomosis for all large felids
(N= 46) and calculated doses of quinapyramine salts
(Injection Triquin of M/S Vetoquinol
India Animal Health Pvt Ltd, Maharashtra) are being
administered subcutaneously at every four month intervals. But the canids were
not included in this chemoprophylaxis protocol, as there was no incidence of
the said disease amongst canids at NKZP.
It is quite challenging to
ascertain the species of Trypanosoma spp. from the blood smear. PCR is
the ultimate diagnostic protocol to reveal the fact. PCR targeting internal
transcribed spacer (ITS1) region is highly sensitive and reliable for the
diagnosis of pathog-enic Trypanosoma spp. such
as T. evansi, T. brucei brucei,
T. b. rhodesiense, T. b. gambiense, T. congolense, T. savannah, T. congolense
kilifi, T. congolense
forest, T. simiae, T. simiae
tsavo, T. godfreyi, and
T. vivax (Njiru et al. 2005). Successful
detection of Trypanosoma spp. has been reported using ITS1 CF and BR PCR
primers in cattle, tsetse fly, sand fly, dogs, equids, monkeys, and camels (Thumbi et al. 2008; Alanazi
et al. 2018; Gaithuma et al. 2019; Medkour et al. 2020). The current study unveiled incidence
of T. evansi in a captive Indian Wolf at NKZP.
Wild animals often exhibit
moderate levels of trypano-tolerance with their
innate ability to co-exist with trypanosomes without showing overt disease
(Sudan et al. 2017). The disease flares up when the animal gets exposed
to physiological and somatic stress following concurrent infection, capture,
translocation and captivity that often compromises their innate resistance
(Fowler 1986; Singh et al. 2003).
The clinical signs in the present
case were high rise of temperature (103.20F), pale mucous membrane,
bilateral lacrimation, and generalised debility.
These observations were in agreement with the findings of Rani & Suresh
(2007). The fever might be due to the effects of toxic metabolites produced by
dying trypanosomes (Tizard et al. 1978).
Anemia was a consistent finding
as reported earlier in different hosts including dogs infected with Trypanosomosis (Moreira et al. 1985; Monzon et al. 1991; Silva et al. 1995; Gurtler
et al. 2007). The anaemia is attributable to
extravascular destruction of RBC which may be through the process of
erythrophagocytosis or metabolic product and toxins liberated from the
parasites. Blood cellular changes revealed leucopenia along with reduced
neutrophil count. Similar findings were recorded by Barr et al. (1991).
Increase in AST, ALT, ALP, urea,
creatinine level as compared to reference level corroborated with findings of
Barr et al. (1991) who reported a similar pattern of changes in a dog
during the acute phase. Marked elevation in the level of total protein values
were recorded as compared to reference level. Hyperproteinemia found in this
study could be associated with hypergammaglobulinemia due to antigenic
stimulation provoked by the parasite, as seen in canines (Aquino et al. 2002).
There was a decrease in the albumin and globulin ratio. The fall in albumin
levels was secondary to hyperglobulinemia as a compensatory mechanism for
maintenance of normal blood viscosity increased by globulin levels (Aquino et
al. 2002). Hyperbilirubinemia has been reported in naturally infected dogs as a
consequence of an increase in unconjugated bilirubin (Sandoval et al. 1994) and
conjugated bilirubin. There was decrease in serum glucose (25.7 mg/dl) level.
Hypoglycemia has been shown to be an important clinical laboratory finding in naturally
infected animals, and it is inversely proportional to blood trypanosome count.
Diminazine aceturate is a commonly used
drug in the treatment of trypanosomosis (Rani &
Suresh 2007). However, a combination of quinapyramine
sulphate and quinapyramine chloride
(3:2 w/w) at dose rate 4.0 mg/kg b wt is also
effective in achieving complete recovery (Singh et al 1993). Shukla (2002) did
not get a complete cure with diminazine@ 0.8g/ 100 kg
b. wt in case of an Indian Wolf, rather, quinapyramine sulphate @ 5.0mg/
kg b wt resulted in complete recovery. In a similar
line, combination of quinapyramine sulphate and quinapyramine
chloride @ 4.0mg/kg b wt administered subcutaneously
as a single dose showed uneventful recovery in the present case.
The incidence of trypanosomosis in an Indian Wolf became an eye opener for
the zoo to extend the chemoprophylaxis to other hosts. As per the
recommendation, the susceptible species, viz., Indian Wolf, Jackal, Dhole, and hyenids of NKZP are being included in the preventive protocol
against trypanosomosis now.
CONCLUSION
Molecular diagnosis of Trypanosoma
evansi infection in an Indian Wolf followed by
successful treatment with a single injection of quinapyramine
sulphate and quinapyramine
chloride @ 4.0 mg/kg b wt subcutaneously was recorded
at Nandankanan Zoological Park.
Table 1. Pre- and post-treatment
haemato-biochemical values of an Indian Wolf with Trypanosoma evansi infection.
Parameter |
Days of blood collection |
|
|
24.ix.2019 (Pre-treatment) |
18.x.2019 (Post-treatment) |
Reference range |
|
Hematology |
|||
Haemoglobin (g %) |
6.0 |
13.0 |
10.5-15a |
Total leucocyte count (103/mm3) |
5.0 |
5.6 |
5-14.1 b |
Neutrophil (%) |
56.0 |
70.0 |
58-71a |
Eosinophil (%) |
3.0 |
3.0 |
0-4a |
Lymphocyte (%) |
41.0 |
26.0 |
28-39a |
Monocyte (%) |
- |
1.0 |
0-2a |
Basophil (%) |
- |
0 |
0a |
Platelet (×103/µl) |
180.0 |
226.0 |
211-621b |
Biochemistry |
|||
ALT(IU/L) |
10.3 |
331.1 |
24-64a |
AST(IU/L) |
830.4 |
159.8 |
23-66b |
ALP(IU/L) |
96.1 |
26.3 |
20-156b |
BUN (mg/dl) |
178.2 |
63.8 |
16-41a |
Creatinine (mg/dl) |
4.4 |
2.18 |
0.5-1.5b |
Glucose (mg/dl) |
25.7 |
117.2 |
58.2 - 91a |
Total protein(g/dl) |
7.63 |
6.4 |
5.07- 6.49a |
Albumin (g/dl) |
1.5 |
2.7 |
2.92-3.53a |
Globulin (g/dl) |
5.0 |
3.6 |
2.03- 3.16a |
Cholesterol (mg/dl) |
272.7 |
178.5 |
138-198a |
Triglyceride(mg/dl) |
418.7 |
39.7 |
20-112b |
Calcium (mg/dl) |
11.1 |
10.9 |
5.58-7.94a |
Phosphorous (mg/dl) |
11.4 |
2.6 |
4 – 5.32a |
Magnesium (mg/dl) |
2.7 |
2.4 |
1.8-2.4 b |
Total Billirubin
(mg/dl) |
0.8 |
0.8 |
0.10-0.50 b |
a Sabapara
& Vadalia(1999) | b Kaneko et
al.(2008)
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