Molecular evidence of hepatozoonosis in tigers of Vidarbha region of Maharashtra State of India

Abstract

Background

Hepatozoonosis has been reported in many species around the world. Few incidences have been reported in various species of wild felids. Tigers are endangered large cats and are protected under the Wildlife Protection Act, 1972 under Schedule I. The study was carried out to estimate the positivity rate of hepatozoonosis in tigers of the Vidarbha region of Maharashtra, India.

Methods

Blood (n = 21) or tissue samples (n = 5) were collected from 26 wild captured / zoo-born or dead tigers during the quarantine period/post-mortem examination. Blood smear examination along with Polymerase Chain Reaction (PCR) studies were conducted for the detection of hepatozoonosis. All the amplicons from the positive samples were purified and sequenced, and the sequences were subjected to nBLAST analysis to detect the species of Hepatozoon. The sequences were deposited into public domain database of National Center for Biotechnology Information (NCBI) and accession numbers were allotted. A phylogenetic study was undertaken to understand the evolutionary lineage of the pathogen. Tissue distribution studies were carried out on tissue samples received during post mortem. A clinical case in a tiger cub was managed and sub-clinical cases were monitored for relapse. Age-wise, sex-wise, region-wise and captive time-wise positivity rate was estimated. The data was analyzed using statistical tools.

Results

A total of 12 tigers were found positive for H. felis during the screening. A clinical case was diagnosed and successfully treated. The age group of 0–3 years reported a positivity rate of 66.66%, and all the cases found positive were reported between the age group of 0–7 years. Males reported a positivity rate of 58.33 per cent, while females reported 35.71%. Taboba and Andhari Tiger Reserve of the state had a positivity rate of 52.94 per cent. However, the statistical analysis for blood parameters and positivity rate by ‘t’ test and Chi-squared test were found to be non-significant.

Conclusions

An overall positivity rate of 46.15% indicates the wide distribution of hepatozoonosis among wild tigers of the Vidarbha region of Maharashtra, India, which is strategically important considering the gene flow and migration of tigers. Hepatozoonosis can progress to clinical outcomes in young animals and require veterinary intervention. Molecular tools and phylogenetic studies can supplement important data on circulating species of Hepatozoon in the field. Further studies on the clinical management and epidemiology of the infection in wild felids will comprehend the cause of wildlife conservation.

Background

India is a megadiversity hotspot that is home to three-quarters of the world’s tiger population. According to the 2018 census, the tiger population is estimated to be 2967 [1]. The tropical climate of the Indian subcontinent is not only conducive to the proliferation of flora and fauna but also to many disease vectors such as ticks. Rhipicephalus and Amblyomma spp. are commonly detected in large cats in this region. A wide array of diseases transmitted by ticks has been identified in companion species, such as dogs and cats [2]; however, India has reported only a few cases of tick-borne illnesses in the wild population [3,4,5]. Hepatozoonosis is a tick-borne infection transmitted by Rhipicephalus spp. and Amblyomma spp. ticks. Recently, Rhipicephalus turanicus was identified as a vector for transmitting H. canis [6]. The dog is the definitive host, and reports of co-infection with other tick-borne diseases have been reported in the species [7, 8], and wildlife [9,10,11]. Various protocols have been designed to treat hepatozoonosis in companions and wild animals. Monodrug therapies with oxytetracycline and doxycycline [12] have been found to be effective in eliminating infection. It has been opined that mono-drug therapies fail to eliminate the tissue forms of Hepatozoon spp., leading to relapses in many cases in companion animals [13]. Stress, in any form, is known to trigger relapse of infection. Hence, it is recommended to follow up on cases of relapse for at least a couple of years [14]. In the current veterinary management, patients are regularly followed up for relapses by undertaking blood and molecular tests. Wild animals exhibit a decent tolerance to many protozoan diseases, including hepatozoonosis, and the infection does not progress to clinical stages. Such carrier animals lead a normal life but may act as a source of infection to other animals in the captive and free range [15]. Animals with hepatozoon infections are prone to opportunistic diseases [16]. Reports from Serengeti highlight the impact of protozoal diseases in co-infection with common viruses such as the Canine Distemper Virus (CDV) [17, 18]. Critical care and needful health management of captive wild animals limit the flaring up of the disease. However, no such provisions are available in the wild, and wild animals in the free range face critical challenges in securing food, mate, and territory [19]. In the presence of such challenges, thoughts on whether the disease is subclinical, even in the wild, need to be investigated in light of evidence-based studies [20]. A few reports of hepatozoonosis in large felids have been reported in India [3,4,5, 21, 22]; however, very little is known about the impact of this disease in the free range. Very few reports have focused on wild or wild-captured animals from this perspective. The subclinical nature of the disease, limited active sampling and limited post-mortem investigation of dead large wild felids are the primary causes of the limited information available on the disease. There are definite research gaps in the assessment of the impact and prevalence of hepatozoonosis in the free range, as most available data have emerged from captive wildlife. Based on the data, the disease has often been described as self-limiting and effectively treated with drugs such as doxycycline [4, 23]. Typically, clinical disease in felids is characterised by fever, anaemia, lethargy, wasting, muscle pain and bone deformities [4]. Death and clinical disease have been reported in coyotes and hyenas [18]. However, no report of clinical progression in large wild felids has been encountered [24]. However, it is worth pointing out that many studies in companion animals have indicated relapse in cases of hepatozoonosis due to the failure to eliminate the protozoan’s tissue forms [25]. Regressions in wild felids can be critical, as stress is a consistent feature in the free range. Baseline data on the prevalence of the infection in wild felids in the region is lacking. In order to build baseline data, a non-probablility convenience sampling method was utilised to screen wild/orphan or wild captured tigers presented to the Wildlife Research & Training Centre, Gorewada, Nagpur, for hepatozoonosis, trypanosomosis, babesiosis, theileriosis, ehrlichiosis and Anaplasma platys by blood smear and molecular methods.

Methods

Blood/ tissue sample collection, smear and PCR examination

Blood samples (n = 21) and tissue samples (n = 5) were collected from 26 wild capture/zoo-born or dead tigers during the quarantine period/postmortem examination. Permission for the same was obtained from the Principal Chief Conservator of Forests & Chief Wildlife Warden, Maharashtra State, as per the provisions of the Wildlife Protection Act, 1972. Blood was aseptically collected from the lateral coccygeal vein following physical restraint in a squeeze cage. Blood smear examination using Leishman and Giemsa staining along with Polymerase Chain Reaction (PCR), was conducted for the detection of hepatozoonosis. Alongside the samples were also screened for trypanosomosis, babesiosis, theileriosis, ehrlichiosis and Anaplasma platys. Blood and serum samples collected between January 2020 and January 2023 were examined using an automated hematological and biochemical analyzer (Abaxis Ltd, California, USA) for routine blood and serum parameters. DNA was isolated from the blood sample using the DNeasy^® Blood and Tissue Kit (Qiagen Inc., MD, USA) according to the manufacturer’s instructions. Polymerase Chain Reaction (PCR) was performed using the primer pair Hep-F 5’-ATACATGAGCAAAATCTCAAC-3’ and Hep-R 5’-CTTATTATTCCATGCTGCAG-3’ targeting the 16 S ribosomal RNA [26]. Details of the primers employed for the detection of protozoan diseases under the study are provided in Table 1. Tissue samples were collected during post-mortem examination of five carcasses (Table 2 Column No. 9). Organ-wise distribution of the protozoan was studied by DNA isolation and PCR studies using the primers mentioned above. The amplicon was purified using the QIAquick PCR Purification Kit (Qiagen Inc, MD, USA).

Table 1 The details of the primers used in the study for the detection of various protozoan diseases

Table 2 The details of the tigers selected for the study along with the results of the peripheral blood smear examination and polymerase chain reaction (PCR). The amplicon sequenced were submitted to NCBI and were allotted accession number as indicated in column 14

Sequencing, phylogenetic and statistical analysis

The purified amplicon was sequenced by an outsourced service provider (Eurofins Pvt. Ltd., Bangalore, India), and the sequences were compared with other reported sequences using the nBLAST tool from the National Center for Biotechnology Information (NCBI) public domain DNA database. The sequences were deposited in the NCBI database using BankIt, and accession numbers were assigned to the sequences. Phylogenetic analysis was performed using Mega X software using neighbour joining method with a bootstrap value of 1000 replications to ensure tree consistency [27]. The data for blood parameters was analyzed by using independent ‘t’ test and a Chi-squared test was employed to estimate the significance of the data for positivity rates.

Clinical management

The clinical management of the tiger cub (TTC1) showing clinical signs was undertaken with a regime consisting of oxytetracycline (10 mg/ kg) and supportive therapy in the form of hepatoprotectants, hematinics, vitamins and probiotics. The tigers (n = 7) found positive were monitored closely for the changes in feeding, behaviour, activity and alertness. In case of any deviation, samples were collected and screened for routine blood and serum examination and molecular assay for protozoans. The samples were otherwise screened quarterly. The tigers under observation received routine vaccination (feline calicivirus, feline rhinotracheitis, feline panleukopenia and rabies) deworming, vitamin and mineral supplementation as per the Central Zoo Authority (CZA) norms.

Results

Blood smear examination with Leishman staining revealed the presence of gamonts in the neutrophils in the blood smear of two tigers (2/26) (Fig. 1), the smear was found to be negative for presence of any intermediate forms of Hepatozoon spp. in rest of the tigers. The PCR analysis produced expected amplification of approximately 650 bp in twelve samples indicating a positive result. The tissue distribution studies indicated uniform distribution of the protozoan in the tissue of the tiger including the liver, spleen, heart, intestine, kidney, lungs and bone marrow (Fig. 2). As shown in Table 2, the accession numbers were assigned to twelve H. felis sequences that were the subject of the investigation.

Fig. 1

Peripheral blood smear stained with Giemsa’s stain indicating the presence of gamont (Arrow) in the neutrophils of the infected tiger. Normal neutrophil (Arrow Head)

Fig. 2

Tissue-wise distribution of the protozoan was studied with Polymerase Chain Reaction using primers Hep-F and Hep-R [11] (Inokuma et al. [26]) yielding an amplicon of 650 bp. (Lane L: 1 Kb Ladder, Lane 1: Liver, Lane 2: Spleen, Lane 3: Heart, Lane 4: Intestine, Lane 5: Kidney, Lane 6: Lungs and Lane 7: Bone marrow.)

Phylogenetic analysis was carried out using sequences of Hepatozoon spp. reported from companion and wild animals. The sequences reported from tigers were preferentially included in the study. A total of 46 sequences were included in the study (Table 3). The phylogenetic study identified six major clades with each sub-species maintaining its intraclade identity. Plasmodium vivax formed a distinct outgroup (Fig. 3).

Table 3 List of sequences along with the attributes used for the phylogenetic study by Neighbour Joining Method using boot strap value of 1000 to ensure tree consistency (* = current study)

Fig. 3

Phylogenetic analysis using Neighbour Joining Method with bootstrap value of 1000 replications to ensure tree consistency. Red arrows indicating query sequences generated under the current study

The positivity rate was estimated based on age, sex, geographical origin and time spent in captivity. The age group of 0–3 years reported the highest positivity rate of 66.66%, while 3–7 years reported a positivity rate 42.85%. All the cases found positive were reported from the age group of 0–7 years; Males exhibited a positivity rate of 58.33% while it was 35.71% in females (Table 4). The statistical analysis revealed no significant difference between various blood parameters (Table 5). Also, there was no significant difference in positivity rates between sex, age, geographical origin and time in captivity by Chi square test (Table 6).

Table 4 The estimation of positivity rate and P value based on the sex, age, geographical origin and time in captivity

Table 5 Selected haemato-biochemical values of blood samples of tigers found positive for hepatozoonosis. Tiger cub TC1 has been included for the comparison with TC2 that was positive for hepatozoonosis and both the tigers are from the same litter. References [46]

Table 6 Statistical analysis of the blood and biochemical parameters (minimum 5 readings) using‘t’ test (2 tailed method). All values except blood Urea Nitrogen (BUN) are non-significant. (NS = non-significant, S = significant)

The tiger cub (TTC1) treated with oxytetracycline and supportive therapy recovered from the infection after 40 days. The cub tested negative by PCR test and was continuously monitored for two years. Seven tigers that tested positive only by PCR test continued to be positive at each screening without any clinical signs. All the tigers tested negative for trypanosomosis, babesiosis, theileriosis, ehrlichiosis and Anaplasma platys.

Discussion

In majority of the countries in Europe, America and Africa, randomised samples can be collected during the hunting season, road kills, post mortem examination and other such avenues. Under the Indian context, hunting of wildlife is totally prohibited and there is a limited access to road kills and post mortem of wildlife due to remote location and stringent law enforcement. As a result the current study was designed to provide the basic positivity rate for the Vidarbha region of Maharashtra. Previous studies have clearly pointed out that hepatozoonosis is majorly subclinical unless age, stress, co-infection and immunosuppression dictate otherwise [28, 29]. Recent reports on African Wild Dogs from Kruger National Park revealed a high prevalence of 88.88% (n = 54) [30]. Deaths due to the infection have been reported in young hyena cubs [15]. Prevalence studies reported from South Africa have pointed out the high prevalence of 40.99% (n = 23) among wild felids in the region [31]; also, similar studies have pointed out a high prevalence (38–61%) of hepatozoonosis in wild carnivores from Zambia [32]. Reports from Serengeti highlight the role of stressors in the clinical outcome of protozoal diseases in case of co-infection with other pathogens, including viruses [33]. Factors like co-infection, climatic extremes, the season of tick abundance and immunosuppression play a vital role in the outcome of the infection. The outcome of the disease also seems to be governed by the cub’s age and its immune status. Maternal immunity appears to play an essential role in the upsurge of infection in young cubs, and cubs that are separated from their mothers or orphans were found to be more prone to the infection, indicating the vital role of passive innate immunity [34]. Tissue forms have been considered important in the relapse of infection. Relapses have been reported in many companion animals, like dogs and cats. The tissues collected from five dead tigers were processed to study the tissue distribution of Hepatozoon spp. The PCR studies indicated uniform tissue distribution throughout the visceral organs. Tissue forms are crucial in the clinical management of hepatozoonosis as they trigger relapses. Relapses can be critical in endangered wildlife, and such animals can act as a source of infection to others in the wild or captivity. Various molecules like toltrazuril have also been used as therapeutic agents with limited success [35]. Ponazuril has been used successfully in treating H. americanum [36].

One tiger (TTC1) showed clinical signs characterised by anaemia, hypoproteinemia, and elevated AST and ALT values (Table 4). The animal was treated with oxytetracycline 10 mg/ kg IV owing to the low haemoglobin concentration and inability to administer doxycycline orally. The blood smear and PCR test was negative on day 40, and there was a significant improvement in the blood parameters in the affected tiger. The tiger was found active with the restoration of appetite and normal behaviour. The tiger was regularly screened for relapses considering the nature of the disease [25]. Also, a nine-month-old tiger cub (TCP) found dead in one of the protected areas of India was found to be positive for hepatozoonosis, though the tiger’s death could not be attributed to hepatozoonosis. The consideration of hepatozoonosis as a subclinical diseases based on the data available for companion or captive wild animals needs serious reconsideration. In the current study a clinical case in a tiger cub was diagnosed and successfully treated providing evidence that hepatozoonosis can lead to clinical manifestation in young large cats.

The phylogenetic study revealed species specific clade distribution with H. felis occupying the clade I and clade V. It is worth mentioning that sequences OK036954, OK036951, OK036961, OM462674, OM462703 and OM462842 were placed in clade I along with sequences of H. felis reported from India including the Trivandrum Zoo, (Accession No. OL852087 [Panthera tigris]) and Laboratory for Conservation of Endangered Species, Hyderabad (LaCONES) (Accession No. HQ829445 [Panthera tigris]; Accession No. JN584475 [Felis domesticus]) and Etawah Accession No. KX017290 [Panthera leo] including sequences of H. felis from a diverse range of taxa. However the clade V was dominated by sequences reported in tigers under this study. Previous research has suggested that domestic and wild cats can be infected by different genotypes of H. felis. The results align with the findings of the reference study, which suggested that cats on Maio Island, Republic of Cape Verde, have two distinct circulating genotypes of H. felis [37]. The statistical analysis using‘t’ test revealed no significant difference between various blood parameters among the male and female tigers. The Chi- squared test for positivity rates between sex, age, geographical origin or time in captivity was non-significant probably due to small data set.

Out of the nine subclinical tiger cases, seven were monitored for clinical progression or otherwise. The blood and serum parameters were normal except for slight elevation of liver function parameters AST (Aspartate Transaminase) and ALT (Alanine Transaminase). The spike in liver enzymes can be attributed to the inflammatory response to the hepatic invasion by the intermediate forms of Hepatozoon sp. The AST elevation in wild cats can be in response to muscle injury or kidney injury. Haemolysis due to improper blood collection technique could also result in the elevation of AST values. In the current context, the elevation was considered in context with the kidney function test and visible clinical presentation of the patient. The tigers were clinically normal and did not exhibit any illness or distress. During the follow-up screening all the tigers tested positive by PCR with no deviation in the complete blood count and serum biochemistry. The subclinical tigers were observed in captivity since January 2020; blood samples of the animals were screened every quarterly to monitor the progression of the disease. The tigers continued to harbour the infection without any clinical manifestations. The tigers received all management care like vaccination, health screening and deworming, and a balanced diet during captivity. The infection in adult tigers is majorly subclinical, and good managemental practices in captivity can prolong life and ensure a quality life. However, in the free range, uncertain factors like circulating viral diseases, tick population upsurge, vector-borne diseases and environmental stressors etc., can influence the outcome of the disease. Thus, whether the disease is subclinical even in the free-range needs affirmation through a detailed investigation of tigers at all possible avenues.

Conclusion

Hepatozoonosis has always been considered a subclinical infection, and the current study provides evidence of clinical disease in a wild-captured tiger cub. Though the disease in rest of the observed tigers was subclinical, the study clearly opines that the outcome depends on many concurrent factors like age. The status of the infection in wild animals in the free range needs to be reviewed as the disease is widely prevalent in the free range. The current study determined the positivity rate and provided the basic data on the impact of hepatozoonosis on tigers from Central India. The data on the clinical outcome of the infection in wild tigers is definitely lacking. Studies that can further clarify the impact of the infection in wild endangered felids have considerable implications for conservation efforts.