J Arthropod-Borne Dis, June 2022, 16(2): 173–179 H Abbas et al.: Concurrent Trypanosomosis, … 173 http://jad.tums.ac.ir Published Online: June 30, 2022 Case Report Concurrent Trypanosomosis, Babesiosis and Toxocarosis in a Dog: a Case Report *Haider Abbas1, Hafiz Muhammad Rizwan1, Muhammad Younus2, Muhammad Sohail Sajid3, Shahid Hussain Farooqi4, Zeeshan Iqbal5, Muhammad Imran6, Usman Amanat7, Nida Wazir7, Muhammad Ahsan Naeem8 1Department of Pathobiology (Parasitology Section), KBCMA College of Veterinary and Animal Sciences Narowal, Sub-campus of University of Veterinary and Animal Sciences (UVAS) Lahore, Pakistan 2Department of Pathobiology (Pathology Section), KBCMA College of Veterinary and Animal Sciences Narowal, Sub-campus of UVAS Lahore, Pakistan 3Department of Parasitology, Univesity of Agriculture Faisalabad, Pakistan 4Department of Clinical Sciences (Medicine Section), KBCMA College of Veterinary and Animal Sciences Narowal, Sub-campus of UVAS Lahore, Pakistan 5Department Animal Sciences (Livestock Management Section), KBCMA College of Veterinary and Animal Sciences Narowal, Sub-campus of UVAS Lahore, Pakistan 6Department of Animal Sciences (Animal Nutrition Section), KBCMA College of Veterinary and Animal Sciences Narowal, Sub-campus of UVAS Lahore, Pakistan 7KBCMA College of Veterinary and Animal Sciences Narowal, Sub-campus of UVAS Lahore, Pakistan 8Department of Basic Sciences, KBCMA College of Veterinary and Animal Sciences Narowal, Sub-campus of UVAS Lahore, Pakistan *Corresponding author: Dr Haider Abbas, E-mail: haider.abbas@uvas.edu.pk (Received 05 Mar 2022; accepted 25 Jun 2022) Abstract Background: Among the blood-borne parasitic infections of dogs, trypanosomosis and babesiosis are highly prevalent in the Indian subcontinent and highly pathogenic. Methods: A six-month-old dog was presented with complaint of lethargy, anorexia and loss of body weight over the previous 15 days on October 11, 2021. A full clinical and targeted haematologic and parasitological examination was undertaken. Results: Wet smears were positive for the motile trypanosomes while stained thin blood smears showed large number of extracellular trypanosomes and intra-erythrocytic ring-like inclusion bodies of Babesia spp. A high burden of Toxo- cara canis was identified on coprological examination. Conclusion: This report describes the clinical manifestation of concurrent infection of haemoparasites and gastrointes- tinal nematodes in Pakistani dogs, and highlights the importance of prompt veterinary intervention. Keywords: Trypanosoma; Babesia; Toxocara canis; Dog; Pakistan Introduction The haemoprotozoan parasites like Babesia (B.) canis, B. gibsoni and Trypanosoma (T.) ev- ansi are common causes of life-threatening dis- eases in wide range of animals including dogs throughout the Indian subcontinent and in other parts of world (1-5). Salivarian trypanosomes are flagellate haemoprotozoa which belong to family Trypanosomatidae and the genus Tryp- anosoma (6), and are biologically transmitted by biting flies (7). Trypanosoma evansi is wide- ly recognized as a cause of neuropathy, immune suppression and anemia in camels and horses, referred to as surra (8, 9); but adult dogs are also widely infected in Pakistan (10, 11). The Copyright © 2022 The Authors. Published by Tehran University of Medical Sciences. 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. http://jad.tums.ac.ir/ mailto:haider.abbas@uvas.edu.pk https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, June 2022, 16(2): 173–179 H Abbas et al.: Concurrent Trypanosomosis, … 174 http://jad.tums.ac.ir Published Online: June 30, 2022 clinical signs of trypanosomosis in dogs in- clude fever, anemia, superficial lymph node en- largement, loss of appetite, lethargy and pro- gressive loss of bodily condition, leading to fa- tal outcomes if untreated (12). Babesia spp. are intraerythrocytic parasites, biologically trans- mitted by Haemaphysalis or Rhipicephalus spp. ticks and infecting a wide variety of hosts in- cluding humans as well as domestic and wild animals (13). Many species of Babesia can in- fect dogs, causing anemia and hemoglobinu- ria due to haemolysis as the parasite replicates asexually in the red blood cells (14). The par- asites complete their life history through sex- ual reproduction in the tick host mid gut and the formation of sporozoites in the salivary gland (15). Canine gastrointestinal nematode (GIN) in- fections, especially Toxocara (T.) canis and An- cylostoma spp., are important causes of retard- ed growth and general malaise (16). Dogs and humans have a close association (17), and their GINs are of great concern due to their serious zoonotic potential (16). Toxocara canis is dis- tributed worldwide, causing diarrhea, poor growth and death if present in large number (18). Environmental contamination with faeces containing vast number of eggs which shed during patent infections presents a serious pub- lic health risk (19), resulting in human disease referred to as visceral larval migrans. This case report presents the co-infection of Trypano- soma spp., and Babesia spp. haemoparasites along with the GIN, Toxocara canis, and de- scribes the therapeutic response to treatment in a dog. Case presentation A six-month-old male pet dog (Pointer cross- bred) was brought to the outdoor clinic at the Khan Bahadhar Chaudhry Mushtaq Ahmad (KBCMA) College of Veterinary and Animal Sciences, Narowal, sub-campus of University of Veterinary and Animal Sciences Lahore, Pakistan on October 11, 2021. The owner re- ported that the dog had been anorexic, dull and depressed for the past 15 days, and that the onset of clinical signs coincided with the dog having consumed a putrified dead lamb carcass. On examination, the dog was thin, weak and did not respond well to its surroundings (Fig. 1). The rectal temperature was normal (38.8 oC), oral mucous membranes were pale and the con- junctivae were pale and jaundiced (Fig. 2). There were no swollen lymph nodes, but signs of tachypnoea (38 breaths per minute), coughing and a frothy nasal discharge were observed. With the consent of the owner, a 5ml blood sample was collected from the cephalic vein into an EDTA coated vacutainer to investigate hematologic parameters and detect any haemo- parasites. Sample was collected for coprologi- cal examination and processed qualitatively by the faecal floatation technique and quantitative- ly by a McMaster egg counting method using three-chambered slide having threshold of 8 eggs per gram (EPG) (Chalex, USA). Wet mounts were prepared for motile trypanosomes and the thin blood smears were stained with Field’s stain and examined at 1,000x under oil immersion to detect haemoparasites. The haematologic parameters (Table 1) showed significantly low packed cell volume (PCV) and haemoglobulin (Hb) values indicat- ing severe anemia, and marked eosinophilia, lymphocytosis and neutropenia. The wet mount of blood sample showed the presence of large number of motile extracellular trypanosomes. The Field-stained thin smear confirmed their identity (Fig. 3) and showed the presence of multiple intracellular Babesia spp. with ring- like forms in the red blood cells (Fig. 4). Toxocara canis eggs were identified on cop- rological examination, being about 75μm x 90 μm in size and having a dark brown colour, a thick pitted shell and sub-globular shape (Fig. 5). An adult worm of T. canis was also found in the faeces measuring approximately 18cm in length. The T. canis faecal egg count was 2900 EPG, indicating a moderate to high level of infection. The disease history and clinical signs were consistent with chronic haemoprotozoal infec- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 173–179 H Abbas et al.: Concurrent Trypanosomosis, … 175 http://jad.tums.ac.ir Published Online: June 30, 2022 tion and GIN infection (12); hence the dog was treated with intramuscular injection of dimi- nazene aceturate (Pronil® Injection, Selmore) at a dose rate of 3.5mg/kg body weight on the first day and same dose was repeated after 72 hours. Doxycycline (Vibramycin®, Pfizer) was given orally at a dose rate of 5mg/kg of body weight, along with a liver tonic (1teaspoon per day, Hepa-Merz® Syrup, Brookes) and multivitamins (1 teaspoon per day, ViDayLin-L® Syrup, Abbott) for 5 days. A single treatment of an- thelmintic 1 tablet per 10kg body weight (Drontal® Plus, Bayer) was given. The dog did not respond to supportive therapy and drug treat- ments, and died 20 days after clinical presen- tation. Fig. 1. Dog showing emaciation and persistent recum- bency Fig. 2. Dog showing anaemic conjunctiva and an ab- sence of corneal opacity Fig. 3. Trypanosoma spp. (arrows) on a Field-stained thin blood smear (1000 x magnification) of an infected dog. The genus identity was confirmed by the mor- phology of extracellular protozoa with a flagellum, kinetoplast and undulating membrane Fig. 4. Erythrocytes showing ring-like forms of Babe- sia spp. (arrows) on a Field-stained thin blood smear (1000 x magnification) of an infected dog Fig. 5. Toxocara canis eggs at 40 x identified on cop- rological examination of an infected dog http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 173–179 H Abbas et al.: Concurrent Trypanosomosis, … 176 http://jad.tums.ac.ir Published Online: June 30, 2022 Table 1. Summary of haematologic parameters of the infected study dog Hematologic parameters Observed values Reference values Erythrocytes (1012/L) 1.69 5.5-8.5 Hemoglobin (g/dL) 3.70 12-18 Haematocrit (%) 14.0 37-55 Leucocytes (109/L) 17.0 6.0-17 Neutrophils (109 /L) 1.50 2.9-12 Eosinophils (109 /L) 2.0 0.0-1.3 Granulocytes (%) 41.1 60–70 Lymphocytes (%) 49.7 8.0–21 Platelets (109 /L) 33.0 200–500 Discussion Disruption of the erythrocytes by the re- lease of Babesia spp. piroplasms and mechan- ical injury to erythrocytes by circulating tryp- anosomes would have accounted for the clini- cal signs that were observed (20, 21). Eosino- philia, and lymphocytosis are typical in case of trypanosomosis (22, 23). Trypanosomal infections in dogs have been reported in different countries in Asia (10, 21, 24), southern Europe (25), Africa, north Amer- ica (26) and South America (27). Salivarian tryp- anosomiasis in various animal hosts caused by T. evansi, T. brucei, T. vivax, T. congolense or T. theileri, is transmitted by biological vectors such as tsetse fly, and tabanids or Stomoxys spp. biting flies, depending upon the Trypanosoma spp. and geographical location (28). In Pakistan, the predominant species is T. evansi, which is considered to be the result of the bites of the flies of family Tabanidae and Stomoxidae, and the molecular studies should be performed to confirm the presence of species of this parasite in animals (29, 30). Babesia canis and B. gib- soni are important causes of disease in dogs with a similar global distribution to Trypanosoma spp. but dependent on the distribution of Der- macentor spp., Haemaphysalis spp. or Rhip- icephalus tick vectors. The ring-like morphol- ogy of the piroplasms identified in this case are consistent with B. gibsoni, which is particularly pathogenic and prevalent in the Indian subcon- tinent, causing mainly chronic disease. The treat- ment response of these cases to antiprotozoal drugs is generally poor (31). Male dogs are con- sidered more susceptible to Babesia infection due to more exposure to tick infestation because of their roaming behaviour in search of mates (32), but the dog in the present case was a pet dog, and the development of infection of Tryp- anosoma and Babesia might be due to its being less trypanotolerant and having had less expo- sure to ticks at a young age (due to provision of more care and hygienic environment as com- pared to roaming dogs), resulting in less protec- tive immunity. Babesiosis may also be predis- posed to immunosuppression caused by Trypa- nosoma spp. (33, 34). Toxocara canis infection is common in dogs worldwide, and particularly common in the In- dian subcontinent (35, 36). Patent infections are usually seen in young puppies, following pre- natal placental infection, lactogenic infection, ingestion of eggs or ingestion of a paratenic host. From about 3 months-old, second stage larvae ingested with eggs usually migrate to various tissues, where they become dormant. The high level of infection in a six month-old dog was unusual, and may have been predis- posed to by immunosuppression due to trypa- nosomosis. Conclusion The poor response to diminazene aceturate and doxycycline was not unexpected, due to the advanced stage of disease in the dog, and http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 173–179 H Abbas et al.: Concurrent Trypanosomosis, … 177 http://jad.tums.ac.ir Published Online: June 30, 2022 highlights the importance of seeking veterinary advice and treatment immediately when signs of ill health are observed. 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