Hematological characteristics of autochthonous hypermicrofilaremic dirofilariosis in dogs in Kannur District of Kerala

This study aimed to determine whether autochthonous high-intensity D. repens microfilaremia leads to haematological changes in companion dogs. Dogs with hypermicrofilaremia presented to the District Veterinary Centre, Kannur were selected for the study. A total of 100 dogs were positive for hypermicrofilariaemia with D. repens based on peripheral blood smear examination and Knott’s test. Of these, 3 dogs were concurrently positive for sheathed microfilaria (MF) of Brugia spp. Interestingly, an adult live worm of D. repens approximately eight cm in length was detected on a subcutaneous mass on the hind limb of a hypermicrofilaremic dog in the study. We compared the hematological parameters of 100 autochthonous hypermicrofilaremic dogs and 15 uninfected dogs. The hematological findings in the study are mild to moderate anemia, moderate to severe thrombocytopenia, leucocytosis with granulocytosis, lymphocytosis and monocytosis as compared to the uninfected group of dogs. The pathogenicity of naturally occurring hypermicrofilaremia is poorly studied, and knowledge of its epidemiology, host-parasite relationship and impact on various organs is warranted for better prevention and control, especially in hot-spot areas.


Introduction
Canine dirofilariosis is a non-contagious parasitic disease caused by filarial nematodes of the genus Dirofilaria that naturally affects various domestic and wild animals.Among the nearly 40 species of Dirofilaria identified, only a few have been reported in India causing infection in canines.Two forms of the disease are reported in dogs: pulmonary dirofilariasis caused by Dirofilaria immitis (the dog heartworm) and subcutaneous dirofilariasis caused primarily by D. repens (Ravindran et al. 2014;Kini et al. 2015;Capelli et al. 2018).In India, D. repens is the most common species affecting both dogs and humans (Joseph et al. 2011).Mosquitoes of the genera Aedes, Culex, and Anopheles are the vectors that transmit these parasites from host to host (Mirahmadi et al. 2017).The increased geographical spread of dirofilariasis can be related to climatic changes favoring seasonal transmission and the resultant establishment of mosquito vectors (Genchi et al. 2009;Morchon et al. 2012).The vectors take up the microfilaria (L1 stage) while feeding on an infected host.Infective third-stage larvae (L3) develop in the Malpighian tubules and then migrate to the proboscis through the body cavity.Transmission to dogs or other hosts, including humans, takes place through a potential vector bite during a subsequent blood meal (Sabu et al. 2005).The species of MF are differentiated by morphology based on peripheral blood smear examination, modified Knott's test and molecular methods.
The pathogenicity of high microfilaremic infections of D. repens is poorly studied.D. repens has a pejorative influence on the health of dogs, and its pathogenicity is dependent upon the number of MFs rather than the presence of adult parasites (Niwetpathomwat et al. 2007;Wysmołek et al. 2020a).It has been postulated that hypermicrofilaremia might be associated with multiple organ dysfunction or could be associated with a state of immunosuppression complicating the course of other ailments (Wysmołek et al. 2020b).Hence, there is an increased need to identify the pathogenesis caused by hypermicrofilaremia in the canine host for effective treatment, prevention, and control of canine filariasis.This study aimed to understand the species of filarial canine parasite in northern Kerala and to elucidate the haematological profile of dogs with hypermicrofilaremia.

Materials and methods
Cases of canine dirofilariosis were conveniently sampled for the study, and the inclusion criteria for the infected group was asymptomatic dogs that tested positive for high microfilariaemia with more than four actively moving MFs per 10× microscopic fields on routine wet blood film evaluation (Monobe et al. 2017).Dogs of different ages, sexes, breeds, and body weights that presented to the District Veterinary Hospital, Kannur, from January 2020 to January 2021 and met the inclusion criteria were enrolled in this study.Dogs that had other diagnosed concurrent diseases were excluded from the study.Dogs brought to the centre for vaccination or regular health check-up or spaying and castration with negative wet film results were included as the non-infected group.
Blood smears were prepared from peripheral blood taken from the ear tip of the infected dogs.The smears stained using Giemsa stain (Himedia, India) were evaluated for identification of MF under 100× magnification by light microscopy.Around 5 mL of blood was collected from the cephalic vein/saphenous vein/recurrent tarsal vein in EDTA vials for modified Knott's technique and haematological analysis.Modified Knott's technique was performed for the morphological identification of MF according to Magnis et al. (2013).Haematology was performed by a haematology analyser (Exigo EOS, Sweden), and the hypermicrofilaremic dogs were further classified based on haemoglobin and platelet values according to Preena et al. (2021).The data obtained were represented as the mean ± standard deviation.Haematological parameters were evaluated statistically by the Mann-Whitney U test using the Statistical Package for Social Sciences (SPSS Version 20.0.0).Variables with p < 0.01 were considered highly significant.

Results and discussion
Kerala is an Indian state considered endemic for D. repens, and its prevalence in domestic dogs may range from 7 to 24% (Sabu et al. 2005).Though it is presumed that these canine infections are non-pathogenic and asymptomatic, several reports suggest that the condition goes either undetected or neglected.Hence, hematological biomarkers may help to diagnose the condition and to estimate the severity of infection for managing the disease condition (Wysmołek et al. 2020b).A total of one hundred dogs that were positive for high microfilariaemia based on wet film examination (Supplementary video 1) were studied.All dogs were confirmed to be positive for hypermicrofilaremia of D. repens infection based on peripheral blood smear examination (Fig. 1) and Knott's tests.MFs of D. repens were found to be unsheathed, having a short cephalic space terminated by a distinct pair of nuclei separating the anterior end of the nuclear column.The nerve ring, excretory pore and anal pore region were well appreciated in stained smears as previously described by Liotta et al. (2013).It was also stated by them that the other canine Dirofilaria spp., D. immitis, had a longer cephalic space and did not have distinct paired nuclei near the anterior end.
On Knott's test, the MFs of D. repens were 6.0-8.2 μm wide and 330-380 μm long with a rounded anterior extremity, well-developed cephalic space and curved caudal extremity (Fig. 2).The measures found in our study were in agreement with the results of comparable studies by Magnis et al. (2013), Liotta et al. (2013) and Deak et al. (2021).It should be noted that the 24-hour microfilarial periodicity is an influential factor that makes the time of sampling an important factor in Knott's test result (Hawking 1967).Co-infection with sheathed microfilaria of Brugia spp. was detected microscopically in three dogs (Fig. 3).In an endemic area of Kerala state, a very high prevalence of MF (42.68%) was observed, with D. repens as the most commonly detected species, followed by B. pahangi and Acanthocheilonema reconditum.However, D. immitis was not detected in any of the samples examined similar to the present study (Ravindran et al. 2014).A similar study in the northeastern state of India revealed a prevalence of 9.02% D. immitis infection in pet dogs and 25.90% in stray dogs.In addition, D. repens was detected in four stray dogs from Guwahati, Assam, in the same study.They also reported high occult infections (amicrofilaremic infections), which could arise as a result of factors such as low parasite burdens, prepatent infection by young adults, infection by only male worm, geriatric female worm, host immune response and microfilaricidal therapy (Borthakur et al. 2015).
Interestingly, an adult live filarial worm approximately eight cm in length was detected on a subcutaneous mass on the hind limb of a hypermicrofilaremic dog in the study (Supplementary image 1a).On microscopic examination, a striated cuticle with longitudinal ridges resembling tree bark could be detected on the surface of the nematode characteristic of D. repens (Supplementary image 1b).Leishangthem et al. (2016) reported the presence of nematodes enclosed within the fibrous capsule or freely present in the testicular tunica vaginalis or body cavities or subcutaneously in the tissue of the canine forelimb.Well-developed multilayered thick longitudinal ridges in the outermost cuticular layer continued by transverse smooth muscle striations were detected in adult worms identical to the present study (Krishna et al. 2015).The length of females may vary from 8 to 13 cm, and males may vary from 4 to 4.8 cm, in accordance with the present study (Nadgir et al. 2001).
The hematological parameters showed significant differences between the infected and noninfected groups of dogs based on the Mann-Whitney U test (Table 1).More than 65% of hypermicrofilaremic dogs were found to be anaemic (low haemoglobin & HCT) in the study, as shown in Fig. 4. Severe intravascular haemolysis caused by the destructive potential of MF could have attributed to the anemia in accordance with the reports of Dipetalonema reconditum infected dogs (Hashem and Badawy 2007).Similarly, Madril et al. (2020) reported that anaemia in dirofilariosis could be due to the mechanical trauma of the red blood cells and vascular walls caused by the movement of the parasites.Anaemia of inflammation is characterized by mild to moderate non-regenerative anemia and neutrophilic leukocytosis that occurs due to multiple downstream effects of released cytokines, which shift granulocytic cell production in bone marrow, decreasing erythroid production (Grimes and Fry 2015).A normocytic and normochromic (normal MCV & MCHC) type of anemia was found in the study, in contrary to the reports from Rath et al. (2014) and Bendas et al. (2022).A severe hyperchromic, macrocytic regenerative anaemia was described in a clinical case of autochthonous heartworm infection in a cat in Austria due to haemolysation (Kulmer et al. 2021).
45% of hypermicrofilaremic dogs were found to have less than 2 lakhs platelets/µl in the study, as shown in Fig. 4. The mild to severe thrombocytopenia noticed in affected dogs in our study may be attributed to the immune-mediated destruction of platelets, as previously reported in dogs infected with D. immitis (Niwetpathomwat et al. 2007).This may be due to platelet consumption or the presence of antiplatelet antibodies, as observed in dogs with other vectorborne diseases (Shaw et al. 2001).Contrary to our findings, thrombocytosis was noticed in dogs with filariasis (Hashem and Badawy 2007).This difference in findings could be Leucocytosis with granulocytosis, lymphocytosis and monocytosis was observed in 65% of hypermicrofilaremic dogs in the study, in contrast to Wysmołek et al. (2020b), who reported lymphopenia in infected dogs.It could be due to the production of endogenous glucocorticoids during an infectious stimuli or stress, which can result in apoptosis or sequestration of lymphocyte leading to lymphopenia (Harvey 2012).Leucocytosis, monocytosis and eosinophilia are all commonly associated with chronic stress in helminth infections, and neutrophilia can inhibit a Th1-type response, all these abnormal changes could be expected as a squeal to increased MF count (Wysmołek et al. 2020a;Monobe et al. (2016).Also, neutrophilia and monocytosis are attributed to the phagocytic removal of tissue breakdown products or MF (Hashem and Badawy 2007).In our study, three dogs out of 100 infected dogs were concurrently positive for sheathed MF of Brugia spp.Since the sample size for dogs infected with sheathed is very low, the comparisons between the haematological parameters of the two groups were not considered here.A study conducted in Thrissur, Kerala, revealed that out of 100 dogs with symptoms of filariaisis, such as fever, anorexia, conjunctivitis, edema of the limb, and scrotum, 16 cases with sheathed MF were identified as B. malayi (Ambily et al. 2011).
In conclusion, the study gives an insight about the haematological characters of hypermicrofilaremic cases of dogs, which showed anaemia, mild to moderate thrombocytopenia and leucocytosis with granulocytosis, lymphocytosis and monocytosis.The majority of the dogs involved in the study were infected with MF of D. repens and only three dogs were found infected with Brugia spp.Future research focusing on the epidemiology and impact on different organ systems of canines as a result of high intensity microfilaremia is recommended.The impact of concurrent infection of sheathed and unsheathed microfilaria or its nematodes in canines may be studied.The increased geographical spread makes it a global concern, and further studies are necessary for a better understanding of the disease.

Fig. 1
Fig. 1 MFs (6 in number) observed on a single field (red arrows) on peripheral blood smear examination of a hypermicrofilaremic. (10X magnification)

Fig. 2
Fig. 2 MF of D. repens with a rounded anterior extremity, welldeveloped cephalic space and curved caudal extremity on Knott's test

Fig. 4
Fig. 4 Severity of anaemia and thrombocytopenia in hypermicrofilaremic dogs

Table 1
Haematological findings in blood samples from hypermicrofilaremic dogsDifferent superscripts indicate that the mean value differs significantly at P < 0.01.The superscript 'NS' indicates that the mean value does not differ significantly at P < 0.01