Background

Tick-borne diseases have been extensively studied in companion animals; however, the epidemiology of protozoal diseases in the wild is understudied. Ticks transmit various infections in the tropical region. The hot and humid climate of the tropics is conducive to the propagation of ticks [1]. There are numerous reports of tick-borne diseases emerging from the Indian subcontinent every year. Hepatozoonosis is a tick-borne disease of companion and wild animals. Rhipicephalus and Amblyomma species are considered important transmitting agents for infection. Recently, the role of Dermacentor reticulatus has been investigated in the transmission of multiple infections, including Hepatozoonosis in red fox (Vulpes vulpes) [2]. The infection has been reported in dogs [3] and cats [4–7]; however, few reports from the wild have been documented. The reports of wild are primarily limited to canids [8–10], ursids [11], felids [12–17], rodents [18], reptiles [19], mustelids [20] and procyonids [21]. Dogs are considered reservoirs of H. canis; they can be infested with brown dog ticks (Rhipicephalus sp.), the definitive hosts. Many species of wild felids and canids are known to be infected with Hepatozoonosis. India has reported incidences of H. canis in wild dogs (Cuon alpinus) in the recent past; however, no reports in any other wild canid species have surfaced from the region [12]. The current case report highlights the clinical management and molecular investigation of Hepatozoonosis in an Indian jackal (Canis aureus indicus).

Case presentation

An adult male Indian jackal (Canis aureus indicus) was rescued from Melghat Tiger Reserve, Maharashtra, India with a history of paraplegia due to a possible automobile accident. The animal was rationally treated at the Transit Treatment Centre and was transferred to Wildlife Research & Training Centre, Gorewada, Nagpur, for further care and rehabilitation after a couple of months. On reception the vital parameters, blood and serum values were within the normal range. The radiological examination revealed an L6 spinal fracture with L6-L7 dislocation resulting in paraplegia. The animal was treated rationally with Injection Ceftiofur Sodium @ 22 mg/Kg, Injection Methyl Prednisolone 2.2 mg/ Kg, Injection Ranitidine HCl (Hydrochloride) @ 4.4 mg total dose once a day. This was supplemented with physiotherapy and infra-red therapy. Post 15 days into rehabilitation, the jackal was found to support his weight on the hind limbs and could walk. However, it was noticed that the jackal started knuckling during the walk and resulted in lacerations and wounds.

The jackal exhibited fever, anaemia, depression, weight loss, rough hair coat, lethargy and inappetence on day 55 of rehabilitation (Day 0). Blood investigation revealed decreased haemoglobin concentration (6.1 g/dL), thrombocytopenia, leucocytosis and neutrophilia; peripheral blood smear examination revealed anisocytosis and the presence of gamonts in neutrophils (Fig. 1). Serum biochemistry showed hypoglycemia and elevated AST (Aspartate Aminotransferase). On day 0, 12.5% of the circulating neutrophils were found to contain gamonts. Considering the low haemoglobin concentration and altered vitals, a mono antiprotozoal therapy was employed using Doxycycline @ 20 mg/Kg body weight once a day along with supportive therapy including hepatoprotectants, iron supplements, vitamins and probiotics. The mono antiprotozoal regime was continued for 45 days, during which vital parameters were monitored on days 0, 21, 45 (Table 1) and monthly thereafter. During the course of the therapy, blood indices and clinical presentation gradually improved, and the percentage of neutrophils containing gamonts gradually improved, and the percentage of neutrophils containing gamonts gradually tapered from 12.5%, 7.66% to 0% on days 0, 21 and 45, respectively.

Fig. 1
figure 1

Blood smear examination revealed presence of gamonts in the neutrophils (indicated by arrow). Normal neutrophils can be seen in the periphery

Full size image
Table 1 Haemato-biochemical values and peripheral blood smear studies evaluated on Day 0, 21 and 45 along with normal reference values
Full size table

To further investigate the infection, DNA (Deoxyribonucleic Acid) was isolated from 100 μl of whole blood using DNeasy\(\circledR \) Blood and Tissue Kit (Mfg. Qiagen Inc, MD, USA) as per the manufacturer’s instructions and Polymerase Chain Reaction (PCR) was performed targeting the 18S ribosomal RNA gene of Hepatozoon sp [22]. An amplification of 650 bp was obtained (Fig. 2), which was sequenced using HepF and HepR primer pairs on ABI 3130 automated DNA sequencer (Mfg. Applied Biosystems, CA, USA). H. felis amplicon reported from tiger was used as a positive control, while the negative control was devoid of any template. To monitor the progression of the infection, PCR studies were repeated on days 21 and 35 (Fig. 2, Lane 1, 2 and 3). To limit relapses, blood samples were examined every month and were found to be negative consistently. The obtained sequence was submitted to the NCBI nucleotide database using the nBLAST tool. It was found to be 99.68% identical to the previously reported H. canis in dogs from India (Accession No. MH922768, MN252045). A systematic phylogenetic analysis was undertaken by including sequences of Hepatozoon identified in wild canids, ursids and felids. The sequences were selected based on similarity with the query sequence (Details in Table2). Sequences reported in domestic and wild were included without preference. Neighbour joining phylogenetic trees using the bootstrap method were constructed using Mega X software to study the topologies of the phylogenetic trees (Fig. 3) [23]. The bootstrap values (1000 replications) were analysed to ensure tree consistency.

Fig. 2
figure 2

Gel electrophoresis on 1% agarose gel. Lane 1,2 and 3 PCR reaction performed on days 35, 21 and 0 respectively, L4 1 Kb ladder, L5 Day 45 and L6 Negative control. An amplification of 650 bp obtained using HEP-F and HEP-R primer (Inokuma et al., 2002)

Full size image
Fig. 3
figure 3

Phylogenetic Analysis of Sequence by Neighbour Joining Phylogenetic Tree using Bootstrap Method (1000 replications)

Full size image
Table 2 List of sequences along with their attributes used for the neighbour-joining phylogenetic studies
Full size table

Discussion and conclusion

Most of Hepatozoon spp. infection in wild and companion animals is subclinical, and it produces clinical outcomes only in very young and immunocompromised individuals [10]. Reports of death in hyenas [8] and coyotes [24] due to Hepatozoonosis highlight the need to investigate the disease among endangered wild canids. The impact of stress, habitat destruction and concurrent viral and bacterial infection on the spread of Hepatozoonosis under free-range has been studied [25]. Conventionally the diagnosis of Hepatozoonosis is primarily based on the blood smear examination; however, when the quantum of infection is scarce, the detection of infection becomes uncertain. Also, Rhipicephalus spp. transmit an array of protozoal infections, coinfection of Anaplasma platys, Ehrlichia canis, H. canis and Rickettsia monacensis has been reported in dogs [26] and wild canids [2, 27]. Molecular techniques can provide sensitive diagnostic tools for detecting protozoal diseases. When augmented with molecular tools like sequencing, qualitative inputs on the epidemiological aspects can be provided.

In the current case report, the jackal exhibited a steep fall in haemoglobin concentration, neutrophilia, leucocytosis, elevated AST levels indicative of hepatic involvement [28, 29]. During the treatment, blood parameters and corpuscular indices showed marginal improvement, with the haemoglobin concentration improving slightly from 6.1 gm/dl (Day 0) to 7.4 gm/dl (Day 45). Clinically, the animal regained its weight and began having a normal appetite and behaviour. The animal showed two instances of loose stools during treatment which was rationally treated with probiotics and diet management. The haemoglobin concentration returned to 10.5 after 90 days, probably due to concurrent blood loss from nursing injuries. Relapses of Hepatozoonosis have been reported in dogs [30, 31]. Hence, a monthly peripheral blood smear and PCR tests were conducted to rule out the relapse, which was consistently negative.

The selection of a therapeutic agent is critical in treating infection in patients with altered vital parameters. The therapeutic agent for treating Hepatozoonosis is selected based on the species of hepatozoon identified. The drugs employed vary from agent to agent; for H. americanum, a multi-drug combination of trimethoprim, pyrimethamine, clindamycin, decoquinate [32]. Also, anticoccidial agent toltrazuril has been found effective in the treatment of experimentally infected voles with H. erhardovae [33]. However, H. canis responds to a combination of imidocarb dipropionate and Doxycycline [29, 34]. Side-effects like hepatic necrosis, kidney failure and death have been reported in dogs treated with imidocarb [35], and the drug has failed to eliminate H. canis in naturally infected dogs [36]. Considering the safety and duration of treatment, mono drug therapy of Doxycycline for 45 days was opted for. The elimination of Hepatozoon infection on day 45 was confirmed by blood smear examination and PCR (Fig. 2 lane 5). It is worth pointing out that the jackal showed no deviations from normal during blood and serum examination on reception. The upsurge of infection was in coincidence with trauma and stress due to tissue injury, as reported in Lions [37]. The jackal recovered uneventfully; thus, Doxycycline has value in the clinical management of H. canis infection in wild canids with compromised vitals.

A Polymerase Chain Reaction (PCR) was carried out to confirm the finding further [22]. The amplicon of 650 bp was sequenced and submitted to National Center for Biotechnology Information (NCBI) database for reference. The sequence was allotted accession number MZ823614. In the current study, sequence analysis revealed 99.68% identity with previously reported sequences of H. canis in dogs from India (Accession No. MH922768, MN252045). There are only a couple of previously reported H. canis in wild dogs (Cuon alpinus) from India (Accession No. HQ829448, HQ829447). No reports of Hepatozoonosis from other wild species have been reported from the country. The phylogenetic analysis revealed the presence of the query sequence (MZ823614) in clade I A consisting of sequences of H. canis reported in dogs and other wildlife from India and abroad. The major clade II consisted mainly of the sequences of H. felis reported in wild felids from India, Korea, Bosnia & Herzegovina (Table2); H.silvestris and H. americanum isolated from a variety of host species including wild and domestic animals (Details provided in Table2). Plasmodium vivax formed a distinct outgroup.

The case report highlights the utility of oral Doxycycline in the clinical management of Hepatozoonosis in wild canids with altered vitals. Molecular techniques are sensitive and can be effectively utilised in the monitoring of the progression of the disease. The insights provided by high throughput technologies like sequencing can help understand the epidemiology and prevalence of Hepatozoonosis. It is essential to study the dynamics of the infection in the wild to ensure the survival of the endangered wildlife. Further investigation in sympatric canids species in the wild and captivity can provide critical insights on Hepatozoonosis.