Babesiosis and theileriosis were detected in significant amount in all three zones that advocate the endemicity of these infections (Rehman et al., 2019). However, subtle differences in intensity of the infection have been detected i.e., highest in SSZ, following in order by NAZ and CPZ (Fig. 2). A range of versatile determinants was observed for the persistent incidence of babesiosis and theileriosis includes more infection rate observed in cattle than buffalo, indigenous breed was more resistant than cross-bred and exotic, female were more prone to infection than male, and adults were at less risk of infection than calves (Li et al., 2014; Tumwebaze et al., 2020; Siddique et al., 2020).
Previous prevalence reports recorded in Pakistan include 18.42%, 29%, 6.57%, and 25.26% in Lahore, Okara, Rawalpindi, and, Chakwal respectively for TBP (Nasir et al., 2000; Chaudhry et al., 2010; Hassan et al., 2018).
Through Giemsa staining, it was observed 19.35%, 23.08%, and 21.93% for babesiosis and 15.89%, 15.25%, and 15.16% for theileriosis, whereas, PCR-based surveillance showed 35.43%, 36.40%, and 36.95% for babesiosis and 42.21%, 41.01%, and 41.52% for theileriosis in SSZ, CPZ and NAZ, respectively (Table 1). 278 bp and 393 bp length of PCR product were observed for B. bigemina and T. annulata, respectively. Overall, PCR-based prevalence found much higher than Giemsa staining. This trend is the same as observed in many previous reports showing PCR is a more sensitive diagnostic tool as compared to Giemsa staining (Jaimes-Duenez et al., 2017; Alade et al., 2019).
Table 1
Comparison of prevalence of Babesia bigemina and Theileria annulata detected through polymerase chain reaction and Giemsa staining procedure in Southern Sandy Zone, Central Plain Zone and Northern Arid Zone of Pakistan
Location
|
Total Animals Screened
|
Analysis
|
Babesia bigemina Positive
|
Prevalence %
|
Confidence Interval (95%)
|
Odds Ratio
|
P-Value
|
Z-Value
|
Lower Limit
|
Upper Limit
|
Southern Sandy Zone
(Multan)
|
2170
|
PCR
|
767
|
35.43
|
1.17
|
2.27
|
> 1
|
< 0.05
|
3.5
|
Giemsa staining
|
420
|
19.35
|
Central Plain Zone (Toba Tek Singh)
|
2170
|
PCR
|
790
|
36.40
|
1.07
|
1.95
|
> 1
|
< 0.05
|
2.41
|
Giemsa staining
|
501
|
23.08
|
Northern Arid Zone (Rawalpindi)
|
2170
|
PCR
|
802
|
36.95
|
1.00
|
1.86
|
> 1
|
< 0.05
|
1.99
|
Giemsa staining
|
476
|
21.93
|
|
|
Theileria annulata Positive
|
|
Southern Sandy Zone
(Multan)
|
2170
|
PCR
|
916
|
42.21
|
1.12
|
2.01
|
> 1
|
< 0.05
|
3.5
|
Giemsa staining
|
345
|
15.89
|
Central Plain Zone (Toba Tek Singh)
|
2170
|
PCR
|
890
|
41.01
|
1.07
|
1.90
|
> 1
|
< 0.05
|
2.41
|
Giemsa staining
|
331
|
15.25
|
Northern Arid Zone (Rawalpindi)
|
2170
|
PCR
|
901
|
41.52
|
1.30
|
2.07
|
> 1
|
< 0.05
|
1.99
|
Giemsa staining
|
329
|
15.16
|
(Values in the row of Giemsa staining are indicating prevalence of babesiosis and theileriosis since Giemsa staining only discriminate genera) |
A significantly higher prevalence (P < 0.05) of TBP was observed in cattle than buffalo in the three zones (Table 2 and Table 3). This trend is consistent with previous findings (Elsify et al., 2015; Farooqi et al., 2017). It is attributed to the fact that cattle have a delicate skin texture than buffalo that makes them more vulnerable to tick infestation (Iqbal et al., 2013; Jaimes-Dueñez et al., 2018). Moreover, less parasitemia in buffalo is due to skin color preferences for the attachment of ticks marked more fabric in cattle than buffalo (Anna Szczepańska et al., 2017; Benitez et al., 2018). Babesiosis and theileriosis were observed more in exotic, cross-bred than Sahiwal among cattle breeds (Table 2). Multiple exposures of infection to indigenous breeds, climatic conditions for tick growth whereas hygienic living conditions for the exotic and cross-bred animals lead to less transfer of placental and maternal antibodies results in weak immunity development in young ones are the possible reasons for the present findings (Rehman et al., 2019; Zeb et al.,2019).
Table 2
Prevalence of Babesia bigemina and Theileria annulata based on PCR with reference of risk factors in cattle
Risk Factors
|
Sub-division of Risk Factors
|
Cattle
|
P-Value
|
Odds
Ratio
|
Babesia
bigemina %
|
Theileria annulata %
|
Prevalence
|
|
20.43
|
20.0
|
< 0.05
|
> 1.00
|
Breeds
|
Exotic breed
|
31.57
|
37.47
|
< 0.05
|
> 1.00
|
Cross breed
|
22.16
|
21.21
|
< 0.05
|
> 1.00
|
Sahiwal
|
11.79
|
13.60
|
< 0.05
|
> 1.00
|
Sex
|
Female
|
22.91
|
24.10
|
> 0.05
|
< 1.00
|
Male
|
10.95
|
14.38
|
< 0.05
|
< 1.00
|
Age
|
Young
|
23.67
|
26.98
|
< 0.05
|
> 1.00
|
Adult
|
16.40
|
19.90
|
< 0.05
|
> 1.00
|
Housing system
|
Tethered
|
24.95
|
25.29
|
< 0.05
|
> 1.00
|
Open
|
13.78
|
19.90
|
< 0.05
|
> 1.00
|
Season
|
Summer
|
23.78
|
25.92
|
< 0.05
|
> 1.00
|
Autumn
|
21.20
|
15.84
|
< 0.05
|
> 1.00
|
Spring
|
14.38
|
29.93
|
< 0.05
|
> 1.00
|
Winter
|
18.43
|
21.20
|
< 0.05
|
> 1.00
|
Feed
|
Supplemented
|
10.26
|
12.90
|
< 0.05
|
< 1.00
|
Non-supplemented
|
22.41
|
13.21
|
< 0.05
|
< 1.00
|
Table 3
Prevalence of Babesia bigemina and Theileria annulata based on PCR with reference of risk factors in Water Buffalo
Risk Factors
|
Sub-division of Risk Factors
|
Water Buffalo
|
P-Value
|
Odds
Ratio
|
Babesia bigemina %
|
Theileria annulata %
|
Prevalence
|
|
14.03
|
10.66
|
< 0.05
|
> 1.00
|
Breeds
|
Kundi
|
25.45
|
26.23
|
< 0.05
|
> 1.00
|
Nili Ravi
|
15.13
|
14.58
|
< 0.05
|
> 1.00
|
Sex
|
Female
|
22.91
|
24.10
|
> 0.05
|
< 1.00
|
Male
|
10.95
|
14.38
|
< 0.05
|
< 1.00
|
Age
|
Young
|
23.67
|
26.98
|
< 0.05
|
> 1.00
|
Adult
|
16.40
|
19.90
|
< 0.05
|
> 1.00
|
Housing system
|
Tethered
|
24.95
|
25.29
|
< 0.05
|
> 1.00
|
Open
|
13.78
|
19.90
|
< 0.05
|
> 1.00
|
Season
|
Summer
|
23.78
|
25.92
|
< 0.05
|
> 1.00
|
Autumn
|
21.20
|
15.84
|
< 0.05
|
> 1.00
|
Spring
|
14.38
|
29.93
|
< 0.05
|
> 1.00
|
Winter
|
18.43
|
21.20
|
< 0.05
|
> 1.00
|
Feed
|
Supplemented
|
10.26
|
12.90
|
< 0.05
|
< 1.00
|
Non-supplemented
|
22.41
|
13.21
|
< 0.05
|
< 1.00
|
Kundi was observed more prone to babesiosis and theileriosis than Nilli Ravi among the wild-buffalo breeds showing a significant (P < 0.05) relationship between all breeds, which is consistent with Atif et al. (2012b) and Siddique et al. (2020).
The sex of cattle and WB has been found significantly associated (P < 0.05) for babesiosis and theileriosis being recorded higher in female than in the male population in the study zones, which is consistent with some earlier reports (Alim et al., 2012; Atif et al., 2012a), and inconsistent with some previous studies (Hussein et al., 2017; Idris et al., 2018). This inconsistency might be due to tradition of use of female animal for transportation and other field ploughing purposes. Breeding stress, milk production, pregnancy, parturition, poor feeding, older age, hormonal changes, higher medication stress, and use for draught purpose in older age are possible reasons for the higher infection rates in female than male (Maharana et al., 2016; Bary et al., 2018). Calves were seen more prone to babesiosis and theileriosis (P < 0.05) than adult animals, which is consistent with the previous reports of Muhanguzi et al. (2010) and Parmar et al. (2019) and inconsistent with Kamini et al. (2010) and Kaur et al. (2016). Softer skin of a young animal, lesser immunity, underfeeding, poor housing management, and endemicity of disease in certain zones are the possible reasons for the higher infection rate in calves than adults (Iqbal et al., 2013; Lawrence et al., 2019).
The housing of cattle and WB, either tethered or open, also have a significant relationship (P < 0.05) with the prevalence of babesiosis and theileriosis. Cattle and WB kept tethered were seen more infected than kept open which is due to possible reasons includes tethering stress leads to more infection (lesser immunity), more prone to tick infestation if tethered near to infested animals, reuse of syringe from infected animals to nearly tethered animals (Iqbal et al., 2013; Siddique et al., 2020).
Highest distributions of babesiosis and theileriosis were seen in summer (P < 0.05) followed by autumn (P < 0.05), spring (P < 0.05), and winter, which might be due to favorable environmental condition for growth and development of the vectors and somewhere slightly different trends were observed which might be due to fomites (Qayyum et al., 2010; Naz et al., 2012; Patel et al., 2017). The month-wise distribution of babesiosis and theileriosis in SSZ, CPZ and NAZ have been given in Figs. 3, 4 and 5, respectively.
Cattle and WB fed with nutritional supplements along with the fodder were less likely to have the infection (P < 0.05) as compared to animals without any supplementation. Supplements were usually given in injectable forms like vitamins b12, Vit-E Selenium, Vit-C, and Vit-D or mixed in the feed or spread in powder form on the fodder. Oxidative stress and lipid peroxidation lead to the fragility of the red blood cells and lysis of the membrane (hemolytic anemia) resulting from parasitemia caused by Babesia and Theileria sp. The administration of vitamins and minerals lesser this antioxidant effect, thus heal the blood cells leads to improve the immunity level to combat the infection (Nayak et al., 2018; Dhanasree et al., 2019). More researches are still needed to evaluate all possible reasons contributing to this fact as very less literature is available regarding this aspect. Association of all the determinants for babesiosis and theileriosis in cattle and WB have been shown in Tables 2 and 3, respectively.
Only two genera of Ixodidae ticks i.e., Hyalomma and Rhipicephalus were found. As ticks were not focused in this study, so other details were not noticed.
Concluding, B. bigemina and T. annulata have a strong association with animal species, breed, sex, age, housing, season, and supplementation. Moreover, Giemsa staining technique should be followed by LAMP and/or PCR to avoid false negative results, in case of suspected animals. WB, being naturally resilient, should be preferred on HF cattle as far as Babesiosis and Theileriosis is concerned, at the study areas. A complete genomic study is encouraged to devise comprehensive control of Babesiosis and Theileriosis.