Incidence of thrombocytopenia
Out of 1320 dogs screened during the study period, thrombocytopenia was identified in 450 dogs. This accounted to 34.09 per cent incidence of thrombocytopenia in critically ill dog population in our study period. The incidence recorded in our study was higher than the earlier reports on canine thrombocytopenia wherein
Grindem et al. (1991) have documented 5.2% incidence of thrombocytopenia and
Botsch et al. (2009) have reported 6.7 per cent incidence of thrombocytopenia in their study on thrombocytopenia in dogs.
Thrombocytopenia was recorded in twenty-four different breeds in our study. The highest incidence was recorded in Labrador 135(30%) followed by Non-descript109 (24.22%), Spitz 50 (11.11%), German Shepherd 36 (8%), Pug 22 (4.89%), Golden Retriever 19 (4.22%), Rottweiler 18 (4%), Doberman 16 (3.56%), Cocker Spaniel 8 (1.78%), Siberian Husky 7 (1.56%), Beagle 6 (1.33%) and 3 (0.67%) each in Pomeranian, Chippiparai, Dachshund, Pitbull and Lhasa Apso breeds. Two cases (0.44%) were recorded in Dalmatian and one each (0.22%) was recorded in Great Dane, Kanni, Bull Mastiff, Miniature Pinscher, BulliKutta, Boxer and Shih Tzu. Similar to our findings,
Khan et al. (2021) recorded the highest prevalence of thrombocytopenia in Labrador retriever (41.6%). An increase in the occurrence of thrombocytopenia in Labrador breed in our study could be attributed to the higher proportion of Labrador dogs reared in and around Chennai due to preference of Labrador breed by the clients.
In sexwise analysis, thrombocytopenia was recorded predominantly in males 248 (55.11%) than in females 202 (44.89%). In contrast to our findings,
Schneider and Mischke (2016) reported a higher occurrence of thrombocytopenia in females (320) than in males (272) in their study. The gender bias in our findings favouring thrombocytopenia in males could be attributed to the pet owners’ favouritism for having male dogs over female dogs in Chennai and its outskirts.
In age wise incidence, thrombocytopenia was recorded in critically ailing dogs of all age groups from the lowest age of 37 days in a Beagle to the highest age of 15 years in a German Shepherd dog. The incidence was highest in dogs belonging to the age group 1-5 years (219, 48.67%) and adult dogs of age group 5-10 years (129, 28.67%). The incidence was relatively less in young dogs of <1 year (61, 13.55%) and in older dogs of age group >10 years (41, 9.11%). However, published reports on agewise incidence of canine thrombocytopenia are not available for the comparison of agewise incidence recorded in our study.
Grades of thrombocytopenia
Of the 450 dogs diagnosed with thrombocytopenia, severe thrombocytopenia(less than 50000 platelets/cmm) was observed in 190 (42.22%) dogs, moderate thrombocytopenia (50000 to 100000 platelets/cmm) was observed in 166(36.89%) dogs and mild thrombocytopenia (100,000 to 150000 platelets/cmm) was observed in 94 (20.89%) dogs. Comparison of mean±SE values of platelet count in thrombocytopenic groups with the control group revealed a highly significant decrease (p<0.01) in mean± SE values of platelet count in thrombocytopenic dogs compared to the control group and a highly significant difference was also observed between the three groups of thrombocytopenia. Haemoparasitic diseases (35.26%, 67/190) followed by miscellaneous etiology(16.84%, 32/190), renal failure (12.10%, 23/190) and sepsis (8.95%, 17/190) were identified as the major causes that caused severe thrombocytopenia in our study.
Clinical signs in thrombocytopenic dogs
The predominant clinical signs observed in thrombocytopenic dogs were anorexia (401/450, 89.11%), pyrexia(208/450, 46.22%), epistaxis (198/450, 44%) and petechiation and ecchymoses on the skin of ventral abdomen (151/450, 33.55%) (Table 1 and Fig 1).
Similar to our findings,
Kohn et al. (2000) also reported the classic signs of thrombocytopenia in dogs
i.e. petechiation, ecchymosis, epistaxis and gastrointestinal blood loss. Among the three grades of thrombocytopenic dogs, clinical presentations such as epistaxis, petechiation and ecchymoses, haematemesis, reddish discolouration of urine and vaginal bleeding were manifested more in dogs with severe thrombocytopenia, compared to the dogs with moderate and mild thrombocytopenia.
Aetiology of thrombocytopenia
The causes of canine thrombocytopenia recorded in our study were haemoparasitic diseases 25.11% (113/450), renal disorders 17.55% (79/450), hyperthermia 10.44% (47/450), sepsis 8.44% (38/450), neoplasia 8.44% (38/450), hepatic disorders 6.89% (31/450), viral diseases 3.55% (16/450), immune mediated thrombocytopenia 3.11% (14/450), leptospirosis 2.44% (11/450), pneumonia 1.55% (7/450), dilated cardiomyopathy 0.55% (1/450) and ivermectin toxicity 0.55% (1/450). Fifty-four (12%) cases recorded were grouped under miscellaneous category as the cause of thrombocytopenia could not be ascertained.
Our research identified haemoparasites as the major cause of thrombocytopenia (25.11%) in critically ailing dogs. This could be attributed to the tropical climate of our geographical region which is conducive to the propagation of tick population which serve as vectors for the transmission of haemoparasitic diseases. In close concordance with our findings,
Khan et al. (2021) also reported haemoprotozoans, followed by renal failure and neoplasia as the major etiologic groups of canine thrombocytopenia.
Haematology
Haematological parameters
Comparison of mean ± SE values of hemoglobin, PCV and RBC in thrombocytopenic groups revealed a highly significant decrease (p<0.01) in the mean ± SE values of hemoglobin, PCV and RBC in critically ill dogs with thrombocytopenia compared to the control group. No significant difference was observed in WBC count between thrombocytopenic groups and reference range in our study. Changes in WBC count as reported in perusal of literature such as elevation of WBC count was recorded in individual cases of thrombocytopenia with various etiologies such as sepsis, haemoparasitic infection etc and a decrease in WBC count was recorded in individual cases of ehrlichiosis, canine parvo viral enteritis
etc. Monocytosis and neutrophila were recorded in dogs with
Ehrlichia canis and
Hepatozoon canis infection respectively which was in agreement with
Thongsahuan et al. (2020).
Platelet indices
Regarding the platelet indices, no significant difference was recorded on comparison of mean ± SE values of MPV in thrombocytopenic dogs with control group as well as between the three grades of thrombocytopenic dogs. However, a highly significant increase (p<0.01) in the mean ± SE values of PDW was recorded in thrombocytopenic dogs which was in agreement with
Souza et al. (2016) who also observed significant increase in the mean values of PDW in dogs with thrombocytopenia when compared to dogs with normal platelet counts. An increase in PDW can be considered as a positive indicator in thrombocytopenic dogs as increase in PDW is attributed to the release of different sized platelets resultant to increased thrombopoiesis and platelet activation to combat the platelet demand in the body. Highly significant reduction (p<0.01) in the mean ± SE values of PCT was recorded in thrombocytopenic dogs and between the three thrombocytopenic groups which agreed with the findings of
Schwartz et al. (2014).
Blood picture findings
Microscopic examination of LG stained blood smears revealed, hypochromasia, neutrophilia, monocytosis and severe to moderate thrombocytopenia in
Ehrlichia canis positive blood smears.
Babesia gibsoni and
Babesia vogeli positive blood smears revealed severe thrombocytopenia, presence of leptocytes, nucleated RBCs, leukopenia, polychromasia and hypochromasia. In case of
Trypanosoma evansi positive blood smear, hypochromasia and poikilocytosis were noticed and neutrophilia was present in
Hepatozoon canis positive smears.
Microscopic examination of blood smears revealed changes in erythrocytes such as hypochromasia, leptocytes, poikilocytosis, echinocytes and codocytes. Signs of regenerative anaemia such as nucleated RBCs, Howell Jolly bodies and polychromasia were more evident in blood smears positive for babesiosis.
The abnormalities in WBCs recorded in our study were leukocytosis in 125 cases, of which, neutrophilia was recorded in 65 cases, neutrophilia with shift to left in 57 cases and monocytosis in 60 cases. Leukopenia was observed in 54 thrombocytopenic dogs.
In LG stained blood smears, platelets appeared as pink round, ovoid, rod, discoid structures and as pleomorphic cellular fragments. The size of the platelets varied from 2µ to 4µ approximating to one-fourth to one fifth of the RBC in most of the cases. Occasionally, platelets appeared bigger, approximating the size of RBCs and were even found as giant or macroplatelets (more than 5µ) appearing bigger than the RBCs.
Souza et al. (2016) reported on macroplatelets in thrombocytopenic animals as seen in our study and stated that large platelets can be observed in the peripheral blood when bone marrow is intact and when it is overstimulated to produce platelets. In contrary to this, platelets also appeared very small as microplatelets in smears screened for detection of thrombocytopenia.
Regarding the distribution, platelets were found as randomly scattered individual element in different fields and as few to many platelets without adherence in linear or random fashion in different fields. The platelets were also seen as small aggregates in a field as well as small to big clumps or clusters. The blood picture findings are presented in Fig 2.
The platelet clumps were either seen as an exclusive platelet cluster or as neutrophils aggregated with platelet clusters. These findings were in close agreement with
Jones (2009) and Villiers and
Ristic (2016).
Blood smear examination for the detection of blood parasites
Ehrlichia canis 54.87% (62/113),
Babesia gibsoni 17.70% (20/113),
Babesia vogeli 11.5% (13/113),
Hepatozoon canis 6.19% (7/113),
Trypanosoma evansi 4.42% (5/113) and Microfilaria 0.88% (1/113) were the haemoparasites (113/450) identified in the present study based on blood smear examination (Fig 3).
Severe thrombocytopenia was recorded in 51 out of 62 dogs diagnosed with ehrlichiosis, 14 out of 20 dogs diagnosed with
Babesia gibsoni, 12 out of 13 dogs diagnosed with
Babesia vogeli and two out of seven dogs diagnosed with
Hepatozoon canis. The thrombocytopenia was of mild grade in a dog diagnosed with microfilaria in blood smear. In agreement with our finding of a single case of microfilaria in thrombocytopenic dogs,
Souza et al. (2016) have recorded microfilaria in blood smears of two thrombocytopenic dogs.
Biochemical findings
Comparison of mean±SE values of serum biochemical parameters of thrombocytopenic groups with the control group revealed a highly significant increase in the mean±SE values of blood urea nitrogen (BUN), creatinine, ALP, total bilirubin, direct bilirubin and phosphorus. Lactate was significantly higher (p<0.01) in non-survivor thrombocytopenic dogs. A highly significant decrease (p<0.01) in ionized calcium was observed in critically ill thrombocytopenic dogs and among the thrombocytopenic groups, dogs with sepsis etiology had higher incidence of ionized hypocalcemia. of the 48 thrombocytopenic dogs that died, 14 dogs had ionized calcium concentration <1 mmol/L prior to death in our study. Hence, ionized calcium concentration of <1 mmol/L can be used in future as an indicator of grave prognosis in critically ailing thrombocytopenic dogs. Similar to our observation,
Sharp et al. (2009) and
Holowaychuk and Monteith (2011) also favoured the evaluation of iCa concentrations rather than tCa in critically ill dogs.
Goggs et al. (2017) also reported that the case fatality rate was higher in low concentration of ionized calcium and attributed that alteration in the electrolyte concentrations was associated with mortality in critically ill dogs.
Polymerase chain reaction for the detection of blood parasites and canine parvo viral enteritis
Polymerase chain reaction performed to identify haemoparasitic infections in 60 dogs with severe thrombocytopenia revealed positive infections in 47 cases (78.33%).
Happi et al. (2018) also detected 89/116 (76.7%) positive samples of blood borne pathogens by PCR analysis.
Ehrlichia canis (33/60, 55%),
Babesia gibsoni (12/60, 20%) and
Babesia vogeli (2/60, 3.33%) were the haemoparasites identified. Earlier,
Macieira et al. (2005) in their study on the prevalence of
Ehrlichia canis infection in thrombocytopenic dogs from Rio de Janeiro, Brazil reported that 26.8% of thrombocytopenic dogs were positive for
E.canis in polymerase chain reaction.
Anaplasma platys is the exclusive parasite of platelets which cause thrombocytopenia.
Atif et al. (2021) stated that
Anaplasma platys chiefly infects the canine platelets and leads to severe thrombocytopenia by causing direct damage to platelets and by immune-mediated mechanisms. But,
A.platys organisms were not detected in blood smear examination as well as by molecular study in the thrombocytopenic dogs in our research.
Canine parvo viral enteritis was diagnosed in 16 thrombocytopenic cases by Polymerase chain reaction.
Earlier, Castro et al. (2013) also observed that thrombocytopenia were significantly more frequent among dogs infected with canine parvo viral enteritis than the control group in their study on clinical, hematological and biochemical findings in canine parvovirus enteritis.
Mortality in thrombocytopenic dogs
Of the 450 thrombocytopenic dogs recorded in our study, 134 dogs recovered completely with restitution of adequate platelet counts when evaluated concurrently by automated blood analyser and blood smear examination. 268 dogs were not reported for follow up. Unfortunately, 48 thrombocytopenic dogs (10.67%) succumbed to death. Majority of the non-survivors belonged to the severe thrombocytopenia group (37/48).
Post mortem examination was conducted on six out of the 48 dogs to study the pathological changes of non-survivor thrombocytopenic dogs. Post mortem examination could not be conducted on rest of the dogs (n=42) due to unwillingness of the pet owners. Post mortem examination revealed varying degrees of congestion and haemorrhage of the visceral organs in all the cases and based on the gross and histopathological changes, ehrlichiosis (n=2), babesiosis (n=2), chronic kidney disease (n=1) and sepsis (n=1) were confirmed (Fig 4).
