Microscopic examination of stained blood smears revealed 11.8% (52/442) prevalence of
Babesia spp. infection in dogs in the study area, while 28.3% (125/442) of dogs were found to be positive for
Babesia spp. in the first
Babesia genus-specific PCR. The genus specific PCR amplified 619 bp partial 16S rRNA of
Babesia spp. (Plate 1). The microscopic examination of stained blood smear is the conventional method used for the specific differentiation between
B.
vogeli and
B.
gibsoni, which is based on the morphometric characteristics of each species. This is a rapid and low-cost method with high specificity, but lacks sensitivity to detect low-level parasitemia. Recently, molecular methods including the polymerase chain reaction (PCR) and sequence analysis have proven effective in some epidemiological studies of
Babesia infection in dogs. Out of these, the phylogenetic analysis of fragments of the SSU rRNA gene has been frequently applied for molecular studies of
B.
canis and
B.
gibsoni isolates
(Inokuma et al., 2004; Duarte et al., 2008).
Further analysis of babesiosis positive samples with species-specific PCR,
Babesia gibsoni and
B.
vogeli was conducted. Of 125 samples subjected to species-specific PCR, 87 (69.6%) were found to be positive for
B. gibsoni infection using primers Gib 599 and Gib 127. Thirty eight dogs (30.04%) were positive for
B. vogeli-specific PCR using primers BAB1 and BAB4. The parasite specific PCR amplified 546 bp partial and 671 bp partial 18S rRNA of
B.
vogeli and
B. gibsoni (Plate 2), respectively. Overall, the prevalence of
B. gibsoni and
B. vogeli in Andhra Pradesh was 19.9% (87/442) and 8.6% (38/442), respectively indicating higher percentage of
B.
gibsoni infection in dogs under study. Even though the virulence of this
Babesia spp. varies among species,
B.
gibsoni is known to cause highly severe disease (
Irwin and Hutchinson, 1991) and may represent a serious threat for dogs living in Andhra Pradesh.
Babesia vogeli is considered less virulent than its two sibling species (
B.
canis and
B.
rossi) and tends to produce subclinical disease, except in young or immunosuppressed dogs (
Penzhorn, 2011).
Muhammad et al., (2018) first reported optimization of loop-mediated isothermal amplification (LAMP) for the diagnosis of
B. felis in cats.
Babesiosis in dogs is endemic in tropical countries like India because of round the year presence of tick vectors and reservoir stray dog population (
Abd Rani et al., 2011). Some of the contemporary studies in India using microscopy and molecular techniques have shown that incidence of babesiosis due to
B.
gibsoni infection among dogs is increasing
(Vairamuthu et al., 2014; Singh et al., 2014; Mahalingaiah et al., 2017; Jain et al., 2018). Absence of accurate diagnosis, lack of knowledge among field veterinarians regarding the pathogen, poor prognosis and absence of effective treatment has made the disease more problematic
(Mittal et al., 2019). Contrary the prognosis remains good for
B.
vogeli infections, the large form of canine
Babesia infecting dogs in India
(Solano-Gallego et al., 2016). However,
Laha et al., (2014) and
Manoj et al., (2020) reported higher prevalence of
B.
canis/B.
vogeli infection (54.05% and 10.00%, respectively) in dogs in Assam and Tamil Nadu, respectively, compared to that of
B.
gibsoni infection (48.64% and 0.4%, respectively) using conventional PCR. Gautam
Patra et al., (2020) observed no significant difference in rate of prevalence of
B.
gibsoni (1.71%) and
B.
canis (1.52%) in different regions of Northeastern India.
Babesia canis reported from different parts of India is presumably
B.
vogeli.
B.gibsoni is the common cause of babesiosis in India. so, its diagnosis by conventional methods results in false negative results particularly in low parasitemic cases.
(Kushwaha et al., 2018). Presence of large pleomorphic single, paired and multiple pear shaped forms of
B.canis in the erythrocytes of peripheral heart and blood smears collected from a ten day old female Great Dane pup carcass, stained with LandG stain revealed by
Balachandran et al., (2010).
In the present study no dogs were found to be co-infected with either
B.
gibsoni and or
B.
vogeli. But concurrent infections of
B.
canis and
B.
gibsoni was noticed in dogs in Chennai
(Vairamuthu et al., 2014), Guwahati
(Laha et al., 2014) and Bengaluru
(Mahalingaiah et al., 2017). The present study supports and confirms that canine babesisois is endemic in different States of India including Andhra Pradesh, with a significantly higher proportion of animals harboring
B.
gibsoni, the small form of
Babesia piroplasm. The sequence analysis of Andhra Pradesh
B. gibsoni and
B. vogeli isolates formed a single major group with other Indian isolates and with other geographical isolates outside India. Studies conducted in different parts of India also indicated that Indian
B. gibsoni and
B. vogeli strains clustered into a single major group with other Indian isolates and with Asian countries isolates
(Mittal et al., 2019; Betgiri et al., 2019; Lavanya et al., 2019a; Lavanya et al., 2019b).
Effect of host factors and other variables on the prevalence of
B. gibsoni and
B. vogeli (Table 1) indicated that the prevalence of
B. gibsoni (P>0.05) was more in young dogs (≤ one year) than in adults (≥ one year). Infection with
B. gibsoni was more common (P<0.05) in male dogs than in female dogs. High prevalence of babesiosis in male is attributable to increased levels of testosterone associated with immunosuppression and increased susceptibility to parasites (
Zuk and McKean, 1996).
Kalaivanan et al., (2018) also noted higher prevalence of
B.
gibsoni in male dogs in Namakkal, Tamil Nadu. The prevalence of
B. gibsoni and
B. vogeli was significantly (P<0.05) high in mongrels than in pure breed. Similarly, pure breed dogs were less prone for infection in Lahore, Pakistan
(Bashir et al., 2009) agreeing the present findings. The prevalence of
B.
gibsoni and
B. vogeli was non-significantly (P>0.05) high in urban dogs compared to that of rural dogs. The prevalence of
B.
gibsoni and
B. vogeli was high in kennel dogs compared to that of pet and stray dogs. Due to close contact of dogs in kennels probably there may be the higher chance of transmission of babesiosis through ticks and bite wounds. The prevalence of
B. gibsoni and
B. vogeli was significantly high during summer (P<0.01) and rainy (P<0.05) seasons respectively, which might be due to high abundance of ticks in these seasons of the year (
Soulsby, 1982).
Risk factor analysis by binary logistic regression model revealed that the living condition of the dogs (kennel dogs), gender (male), medium (urban) and tick infestation as risk factors for
B.
gibsoni infection (Table 2). The variables such as living condition of the dogs (kennel dogs), age (young age) and tick infestation were identified as risk factors for
B. vogeli infection (Table 3). Kennel dogs were 5.2 (OR=5.17; P<0.01) and 5.4 (OR=5.429; P<0.01) times more prone for
B.
gibsoni and
B.
vogeli infection respectively, than stay dogs. Young dogs (≤ one year) were 39.5 (OR=39.453; P<0.001) times more likely to be infected with
B.
vogeli compared to that of adult dogs (≥ one year). In young animals immune system is immature and are less probably to have prior exposure to many infections, they are expected to be more susceptible.
Female dogs were 2.1 (OR=2.057; P<0.05) times less prone for
B.
gibsoni infection than male animals. The increased immune reactivity in females due to estrogens helps produce an effective resistance to infection and therefore females are less susceptible to infections (
Taneja, 2018). The prevalence of
B.
gibsoni was 2.1 (OR=2.057; P<0.05) times less in dogs living in rural areas than in dogs living in urban area that could be due to the low population size of dogs in rural area where infections cannot be continued for prolonged period
(Acosta-Jamett et al., 2010).
Dogs without tick infestation were 28.6 (OR=0.035; P<0.001) and 5.3 (OR=0.187 P<0.001) times less likely to be infected with
B.
gibsoni and
B.
vogeli respectively, than dogs with tick infestation. Dogs infected with
B.
canis were found to be infested with ticks in Nigeria
(Konto et al., 2014) indicating tick infestation as risk factor for babesiosis. The prevalence of
B.
gibsoni was two (OR=0.490; P<0.05) times more in dogs that were not treated for tick infestation previously than in dogs treated for tick infestation. Other variables such as gender, breed and season were not identified as risk factors for
B.
vogeli infection. In accordance, no variables were identified to be significant risk factors for testing positive
via PCR for
Babesia spp. in Nigeria, but dogs with ticks found on physical examination were 3.6 times more likely to test positive for hemoplasmas
(Happi et al., 2018).
