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Iran J Parasitol
Iran J Parasitol
IJPA
Iranian Journal of Parasitology
1735-7020
2008-238X
Tehran University of Medical Sciences

10.18502/ijpa.v19i3.16398
IJPA-19-370
Case Report
Occurrence of Trypanosoma evansi Infection in Rottweiler Dog from Cauvery Delta Region of Tamil Nadu, India: A Case Report
Rangasamy Velusamy 1 *
Annamalai Latchumikanthan 1
Kanniappan Vijayasarathi Matheri 1
Mani Saravanan 2
Natarajan Babu Prasath 3
Chinnaswamy Prabu Ponnachipudur 3
Alagarsamy Alagesan 4
1. Department of Veterinary Parasitology, Veterinary College and Research Institute, Tamil Nadu Veterinary and Animal Sciences University, Orathanadu, Tamil Nadu, India
2. Department of Veterinary Medicine, Veterinary College and Research Institute, Tamil Nadu Veterinary and Animal Sciences University, Orathanadu, Tamil Nadu, India
3. Department of Veterinary Pathology, Veterinary College and Research Institute, Tamil Nadu Veterinary and Animal Sciences University, Orathanadu, Tamil Nadu, India
4. Poultry Disease Diagnosis and Surveillance Laboratory, Veterinary College and Research Institute, Tamil Nadu Veterinary and Animal Sciences University, Namakkal, Tamil Nadu, India
* Correspondence Email: velupara@gmail.com
Jul-Sep 2024
19 3 370375
10 4 2024
18 7 2024
Copyright © 2024 Rangasamy et al. Published by Tehran University of Medical Sciences.
2024
https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/by-nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited.
Trypanosomes are the extraerythrocytic haemoflagellate protozoan parasites of domestic and wild animals including dogs. A male Rottweiler dog was presented with the history of anorexia, cloudy eyes and vomiting for a week. The dog was examined both physically and clinically. Blood was collected and subjected to haematological and biochemical analysis. Echocardiography of the thoracic cavity and ultrasonography of the abdominal cavity were performed. Molecular confirmation of parasite was carried out by PCR and DNA was sequenced to identify strain variations. Upon clinical examination, the dog revealed emaciation with high fever, unilateral corneal opacity, anemia, enlarged lymph nodes and distended abdomen. Echocardiography of the thoracic cavity showed ventricular hypertrophy and cardiomyopathy. Ultrasonography of the abdominal cavity showed splenomegaly and renomegaly. Haematological findings revealed decreased haemoglobin and erythrocyte count and biochemical findings revealed increased BUN and creatinine, hypoglycemia and hypoalbuminemia. Wet film and blood smear examination revealed actively motile trypanosomes and massive infection of trypanosomes, respectively. Molecular confirmation of species was carried out by amplification of 227 bp partial VSG gene of Trypanososma evansi, subsequently sequenced and phylogenitically analysed. The infected dog was treated intramuscularly with diminazene aceturate @ 3.5 mg/kg b.wt along with the supportive therapy. The study described the occurrence and diagnosis of trypanosomosis in dog. Early diagnosis and timely treatment is the most reliable way to safeguard the companion animals. It is the first report on trypanosomosis in dog from Cauvery delta region of Tamil Nadu, India.

Dog
Trypanosoma evansi
Microscopy
PCR
Tamil Nadu
India
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pmcIntroduction

Trypanosomosis is an economically important haemoprotozoan diseases affecting wide range of domestic and wild animals including dogs (1). Trypanosoma evansi causes chronic disease (Surra) in camels and horses, but it is generally an acute and fatal disease in dogs and the infected dog may die within 2 - 4 weeks of infection if untreated (2). It is transmitted mainly by ingestion of infected herbivores meat and use of contaminated needles (3).

In India, the prevalence of T. evansi in dog was recorded in Madhya Pradesh (7.69%) (4), Andhra Pradesh (2.40%) (5), Kerala (2.7%) (6) and outbreak of canine trypanosomosis were also well documented in Calcutta, West Bengal (7). However, studies on trypanosomosis of dogs in Tamil Nadu are scanty.

We discuss about the diagnosis, clinical and therapeutic management of trypanosomosis in Rottweiler dog from Cauvery delta region of Tamil Nadu, India.

Case Report

An adult male Rottweiler dog was admitted to the Veterinary Clinical Complex (VCC) of Veterinary College and Research Institute (VCRI), Orathanadu, Thanjavur, Tamil Nadu, for the treatment with the history of anorexia, cloudy eyes and vomiting for a week. Upon physical examination, the dog was appeared dull and depressed, emaciated with high body temperature (40.1 o C), unilateral corneal opacity in the left eye, pale conjuctival mucous membrane, palpable prescapular lymph nodes, oedematous swelling of the hind limbs and distended abdomen. Echocardiography of the right parasternal short axis showed ventricular hypertrophy and cardiomyopathy (Fig. 1). Abdominal ultrasound examination of the kidney showed enlarged left kidney (Fig. 2) and cortical and medullary hyper echogenicity of the right kidney (Fig. 3), sagital view of the spleen showed splenomegaly with hypoechoic mass near the free border of the spleen (Fig. 4).

Fig. 1: M Mode Echocardiography - Right parasternal short axis- ventricular hypertrophy

Fig. 2: Left kidney - Sagittal view – Renomegaly (85.6 X 44.8mm)

Fig. 3: Right kidney - Sagittal view - Cortical and medullary hyper echogenicity (79.5 X 46.9 mm)

Fig. 4: Spleen - sagittal view - Splenomegaly with hypoechoic mass noticed on tail of the spleen, bladder and left kidney on same window

After clinical examination, blood was collected from cephalic vein and subjected to haematological and biochemical analysis. Hematological findings revealed severe anaemia as indicated by profound decrease in the haemoglobin value (4.2 g/dL) and total erythrocyte count of 2.7x106/mm3 whereas the total leucocyte count (14.6x103 /mm3) and differential leukocyte count (neutrophils - 74%, lymphocytes - 17% and monocytes - 6% were found normal (Table 1). The biochemical values of the serum samples revealed increased BUN (248 mg/dL) and creatinine (2.93 mg/dL) and decreased glucose (60.5 mg/dL) and albumin (2.16 g/dL) levels (Table 2).

Table 1: Haematological analysis of blood collected from dog infected with Trypanosoma evansi

Cell type	Subject’s values	Reference values	Findings	
Haemoglobin(g/dL)	4.2	12 – 18	Severe anaemia	
RBC (106/mm3)	2.7	5.5 – 8.5		
WBC (103/mm3)	14.6	4 – 15	Normal	
Neutrophils (%)	74	60 – 70		
Lymphocytes (%)	17	12 – 30		
Monocytes (%)	6	2 – 8		
Reference range: The Merck Veterinary Manual, 11th Edn.

Table 2: Biochemical analysis of serum from dog infected with Trypanosoma evansi

Parameters	Subject’s values	Reference values	Findings	
BUN (mg/dL)	248	6 – 30	Uremia	
Creatinine (mg/dL)	2.93	0.5 – 1.7	Azotemia	
Total Protein (g/dL)	8.14	5.4 – 7.5	Normal	
Glucose (mg/dL)	60.5	70 – 138	Hypoglycemia	
Albumin (g/dL)	2.16	2.3 – 3.1	Hypoalbuminemia	
Reference range: The Merck Veterinary Manual, 11th ed.

Additionally, wet film examination showed large numbers of motile trypanosomes, which was moving actively among the red blood cells (Fig. 5). Giemsa’s-stained thin blood smear revealed a numerous long slender trypanosome with central nucleus, small sub terminal kinetoplast, well developed undulating membrane and a long free flagellum. The length of the parasite was measured using micrometry and ranged between 19 - 31 μm. Severe parasitemia (+++) with the range of 9 -12 organisms per high power field was observed (Fig. 6).

Fig. 5: Wet film examination of blood showing numerous Trypanosoma organisms (x400)

Fig. 6: Photomicrograph of Giemsa’s-stained blood smear of dog showing numerous Trypanosoma organisms (x1000)

The clinical signs along with the microscopic examination of trypanosomes through blood smear examination suggested a case of trypanosomosis. However, it was difficult to distinguish the species of Trypanosoma by microscopical examination, hence, the species was confirmed by PCR amplification of 227 bp of the VSG gene of Trypanosoma evansi using species specific primers (Fig. 7), subsequently sequenced (Accession number - OP627198.1) and phylogenitically analyzed. Italics

Fig. 7: Agarose gel electrophoresis of parasite DNA, L 1: 100 bp DNA ladder, L 2 & 3 : Amplification of 227 bp fragment of VSG gene of Trypanosoma evansi L 4 & 5 : Negative control

The nucleotide sequences were aligned and edited using the BioEdit 7.0 software and compared with those available in the Gen-Bank database using Basic Local Alignment Search Tool (BLAST). To investigate the evolutionary relationship, phylogenetic analysis of twelve deduced nucleotide sequences in Gen-Bank was used to compare the relationship of T. evansi with other strains using the MEGA X software (8). The evolutionary relationship tree was inferred using the Maximum Parsimony method (9). The analysis revealed that T. evansi was grouped into two major clusters. It was observed that T. evansi sequences from the Philippines (JQ653273), and Orathanadu, TN, India (OP627198) were belonging to the cluster II, and the remaining ten sequences were clustered together exclusively in cluster I (Fig. 8). In the cluster I, various subspecies of T. evansi from Nigeria (MZ394795), Indonesia (MW489882), Chennai-India (MW489880), Kerala-India (MG600142), Malaysia (MW489885), Iraq (MH697863), Jabalpur-India (OK210088), Israel (HM209054), and Gujarat- India (MW221259) were belonging to the sub-cluster I. The overall mean distance was 0.370. T. evansi sequences of cattle in Philippines (JQ653273) and camel in Iran (MK752393) showed 100% and 98 % similarity with our sequences (OP627198), respectively. However, the other strain sequences were dissimilar with T. evansi sequences from various countries (37 - 100%).

Fig. 8: Maximum Likelihood phylogenetic analysis of VSG gene of T. evansi. Numbers at nodes represent bootstrap support (500 bootstrap replicates). Sequences obtained during this study are indicated with red dots

Discussion

Trypanosomosis is a highly pathogenic hemoprotozoan disease of vertebrate animals and is mechanically transmitted by biting insects such as Tabanus and Stomoxys species (3). The source of infection in dog is always plausible. In a survey conducted at Ludhiana, India, 4.68% (3/64) of dogs were sub-clinically infected through examination of blood smears during the rainy season and those dogs were kept mainly in an area near to the dairy cattle. The contamination could have been occurred either orally by ingestion of aborted placenta or foetuses eliminated by infected females (10). Therefore, livestock act as a reservoir and play an important role in the maintenance of trypanosomes in endemic areas. In our study, we also postulate that the infected dog might have contracted the infection from nearby livestock farms either through biting flies or by eating contaminated sheep and goat meat that was offered as regular diet. It is probable that the dog may be infected by Stomoxys spp. or Hippobosca spp. fly transmission from infected domestic animals such as cattle, buffaloes and horses, however, the epidemiological studies are needed to confirm this hypothesis.

The major clinical signs of canine trypanosomiasis were neurological, ocular and vascular. They occurred as acute, sub-acute and chronic infections, mostly seen in adult dogs and characterized by myocardial dilation and ventricular arrhythmias. This may be due to the release of proteolytic enzymes by the parasite such as proteases and phospholipases, which initiate a cascade of events leading to hemolytic anemia that eventually could lead to cardiovascular collapse (11). Anemia was the most characteristic feature of canine trypanosomosis (12). The mechanism of injuries on erythrocytes by contact movement of the flagellum was plausible and may be due to extra-vascular hemolysis because of adsorption of Trypanosoma antigen on RBC surface (3). Biochemical changes such as uremia and elevated creatinine observed in the study could be due to kidney dysfunction upon tissue damage caused by parasitemia. Hypoglycemia may be due to utilization of blood glucose by the parasites in the circulation. The decrease in serum albumin could be attributed to the decreased liver biosynthesis and progressive loss of albumin in urine.

Diminazene aceturate had been used as a drug of choice for the treatment of trypanosome infection in livestock, but its use was limited in canine and equine species (13). In the present study, the dog was treated with intramuscular injection of diminazene aceturate at the dose rate of 3.5 mg/kg b.wt along with the supportive therapy; however, the dog was collapsed two days later. This result was in contradictory with the other studies that the dogs were treated with a single dose of diminazene aceturate at 3.5 mg/kg b.wt and observed good clinical improvement (14). These effective results could be possible that the affected dogs may be treated at early stages of the infection. Mortality of the dog observed in the current study might be occurred due to multi-organ failure where the dog was brought for the treatment during an advanced stage of the Trypanosoma infection.

Conclusion

The present study described the occurrence and diagnosis of T. evansi in a Rottweiler dog. The etiological agent was confirmed by blood smear examination and further characterized by molecular technique. The standard treatment protocol was followed using diminazene aceturate and supportive therapy. Molecular techniques are warranted to differentiate species of Trypanosoma sp. in dogs. In spite of treatment, the dog found to have multiorgan failure and died two-days later. This study clarifies that prompt attention is needed to diagnose and treat trypanosomiasis in canines at earliest to overcome its systemic effects, multi-organ failure and death.

Acknowledgements

Authors are highly thankful to the Dean, Veterinary College and Research Institute, Orathanadu, Thanjavur, Tamil Nadu Veterinary and Animal Sciences University for providing necessary facilities to carry out the work.

Conflicts of interest

The authors declare that they have no conflict of interest.
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