A total of 740 cattle were examined for tick infestation, (45.68%, n=338) from Kilombero district council and (54.32%, n=402) from Iringa district council. An overall tick prevalence of (41.08%, n=304) was recorded in the study areas. The tick prevalence in Kilombero district council was (41.42%, n=140) and (40.80%, n=164) in Iringa district council, both were nearly equal to the overall tick prevalence and no significant difference observed (p > 0.05) (Table 1). In total 1,889 ticks were collected from the infected cattle whereas, after counting ticks on one side of the animal’s body and doubled a total of 3,560 ticks were recorded. Season wise, tick infestation prevalence was (41.11%, n=148) during the wet season and (41.05%, n=156) during the dry season (Table 1). Tick infestation prevalence was highest in male (44.02%, n=92) than female cattle (39.92%, n=212). Based on cattle age groups, tick infestation prevalence was highest in calf (51.61%, n=16), followed by adult (40.91%, n=216) and juvenile (39.78%, n=72). With cattle health status, tick infestation was highest on cattle with average health condition (41.78%, n=216), and least in poor (39.82%, n=45) and cattle with good health condition (39.09%, n=43). Moreover, tick infestation prevalence was almost equal between the tick control frequency categories (Table 1). Therefore, there was no statistically significant difference in tick infestation prevalence between the two seasons, cattle sex, age groups, health categories and tick control frequencies observed in this study p > 0.05.
Of all tick species identified, Rhipicephalus microplus had the highest prevalence (48.1%, n=909), followed by Rhipicephalus evertsi (16.4%, n=310) and Amblyomma lepidum (16.4%, n=310) while, Hyalomma albiparmatum had the lowest prevalence (0.2%, n=3) (Table 2). Season wise, Rhipicephalus microplus was recorded with the highest proportions during the wet season 59.8% and 35.2% during dry season, followed by A. lepidum which had a higher proportion 19.3% during dry and 13.8% during wet season and R. evertsi had a higher proportion 20.8% during dry compared to 12.4% during wet season. The least tick proportion was observed in H. albiparmatum with a proportion of 0.3% during wet and 0% during dry season (Table 3). In general, there was statistically significant difference between the seasons on the proportion of R. microplus, R. evertsi, R. appendiculatus, H. rufipes, and A. lepidum (p < 0.05) (Table 3). On the other hand, Amblyomma gemma, A. variegatum and R. decoloratus had a higher proportion during wet than dry season however, the difference was not significant (p > 0.05) (Table 3).
For the case of mean tick burden, a higher overall mean tick burden of 12.07 ± 0.91 was observed in Kilombero compared to 11.40 ± 1.00 in Iringa district however, the difference was not statistically significant (CI = 95%, p = 0.640). Mean tick burden on cattle was higher during wet than dry season however the difference was not statistically significant (p = 0.436) (Table 4).
Table 1. Number of cattle examined, infested cattle and tick infestation prevalence with respect to district, season, cattle sex, age, animal health status and tick control frequency
Variables
|
Number of cattle examined
|
Number of cattle infested
|
Prevalence (%)
|
p- value
|
District
|
|
|
|
|
Iringa
|
402
|
164
|
40.80
|
0.864
|
Kilombero
|
338
|
140
|
41.42
|
|
Season
|
|
|
|
|
Dry
|
380
|
156
|
41.05
|
0.987
|
Wet
|
360
|
148
|
41.11
|
|
Cattle sex
|
|
|
|
|
Female
|
531
|
212
|
39.92
|
0.308
|
Male
|
209
|
92
|
44.02
|
|
Cattle age group
|
|
|
|
|
Adult
|
528
|
216
|
40.91
|
0.240
|
Calf
|
31
|
16
|
51.61
|
0.217
|
Juvenile
|
181
|
72
|
39.78
|
0.789
|
Cattle health status
|
|
|
|
|
Average
|
517
|
216
|
41.78
|
0.603
|
Good
|
110
|
43
|
39.09
|
0.911
|
Poor
|
113
|
45
|
39.82
|
0.702
|
Tick control frequency
|
|
|
|
|
Weekly
|
135
|
56
|
41.48
|
0.844
|
Biweekly
|
294
|
119
|
40.48
|
0.909
|
Monthly
|
154
|
65
|
42.21
|
0.901
|
Occasionally
|
133
|
54
|
40.60
|
0.884
|
Unknown
|
24
|
10
|
41.67
|
0.987
|
Table 2. Sex ratio and prevalence for the identified tick species collected on cattle from Kilombero and Iringa districts. Male (M); Female (F).
Tick species
|
Males
|
Females
|
M: F
|
Total ticks
|
Prevalence (%)
|
Amblyomma gemma
|
88
|
32
|
2.6:1
|
120
|
6.4
|
Amblyomma lepidum
|
247
|
63
|
3.9:1
|
310
|
16.4
|
Amblyomma variegatum
|
14
|
15
|
1:1.1
|
29
|
1.5
|
Hyalomma albiparmatum
|
3
|
0
|
3:0
|
3
|
0.2
|
Hyalomma rufipes
|
32
|
18
|
1.8:1
|
50
|
2.6
|
Rhipicephalus appendiculatus
|
45
|
95
|
1:2.1
|
140
|
7.4
|
Rhipicephalus decoloratus
|
0
|
18
|
0:18
|
18
|
1.0
|
Rhipicephalus evertsi
|
188
|
122
|
1.5:1
|
310
|
16.4
|
Rhipicephalus microplus
|
63
|
846
|
1:13.4
|
909
|
48.1
|
Table 3. The proportion and counts of hard ticks from Kilombero and Iringa District Councils during dry and wet season.
|
Season
|
|
District
|
|
Dry (%)
|
Wet (%)
|
p-value
|
|
Iringa (%)
|
Kilombero (%)
|
p-value
|
Tick genera
|
|
|
|
|
|
|
|
Amblyomma
|
235 (51.2)
|
224 (48.8)
|
0.021
|
|
447 (97.4)
|
12 (2.6)
|
0.000
|
Hyalomma
|
32 (60.4)
|
21 (39.6)
|
|
|
53 (100.0)
|
0 (0.0)
|
|
Rhipicephalus
|
630 (45.8)
|
747 (54.3)
|
|
|
501 (36.4)
|
876 (63.6)
|
|
Tick species
|
|
|
|
|
|
|
|
A. gemma
|
50 (5.6)
|
70 (7.1)
|
0.219
|
|
119 (99.2)
|
1 (0.8)
|
0.000
|
A. lepidum
|
173 (19.3)
|
137 (13.8)
|
0.001
|
|
306 (98.7)
|
4 (1.3)
|
0.000
|
A. variegatum
|
12 (1.3)
|
17 (1.7)
|
0.576
|
|
22 (75.9)
|
7 (24.1)
|
0.014
|
H. albiparmatum
|
0 (0.0)
|
3 (0.3)
|
0.252
|
|
3 (100.0)
|
0 (0.0)
|
0.252
|
H. rufipes
|
32 (3.6)
|
18 (1.8)
|
0.021
|
|
50 (100.0)
|
0 (0.0)
|
0.000
|
R. appendiculatus
|
121 (13.5)
|
19 (1.9)
|
0.000
|
|
137 (97.9)
|
3 (2.1)
|
0.000
|
R. decoloratus
|
6 (0.7)
|
12 (1.2)
|
0.246
|
|
14 (77.8)
|
4 (22.2)
|
0.055
|
R. evertsi
|
187 (20.8)
|
123 (12.4)
|
0.000
|
|
306 (98.7)
|
4 (1.3)
|
0.000
|
R. microplus
|
316 (35.2)
|
593 (59.8)
|
0.000
|
|
44 (4.8)
|
865 (95.2)
|
0.000
|
Table 4. Number of cattle infested, Total number of ticks, Mean tick burden per cattle ± standard error of mean (SE) with respect to district, season, cattle sex, age, animal health status and tick control frequency
Variables
|
No. of cattle
|
Tick counts
|
Mean tick burden ± SE
|
Std Dev
|
p-value
|
District
|
|
|
|
|
|
Iringa
|
164
|
1870
|
11.40 ± 1.00
|
12.76
|
0.625
|
Kilombero
|
140
|
1690
|
12.07 ± 0.91
|
10.80
|
|
Season
|
|
|
|
|
|
Dry
|
156
|
1746
|
11.19 ± 0.99
|
12.33
|
0.436
|
Wet
|
148
|
1814
|
12.26 ± 0.92
|
11.40
|
|
Cattle sex
|
|
|
|
|
|
Female
|
212
|
2286
|
10.78 ± 0.73
|
10.66
|
0.039
|
Male
|
92
|
1274
|
13.85 ± 1.47
|
14.14
|
|
Cattle age group
|
|
|
|
|
|
Adult
|
216
|
2654
|
12.29 ± 0.85
|
12.44
|
0.403
|
Calf
|
16
|
160
|
10.00 ± 2.98
|
11.91
|
|
Juvenile
|
72
|
746
|
10.36 ± 1.18
|
10.02
|
|
Cattle health status
|
|
|
|
|
|
Average
|
216
|
2394
|
11.08 ± 0.75
|
11.01
|
0.221
|
Good
|
43
|
624
|
14.51 ± 2.21
|
14.44
|
|
Poor
|
45
|
542
|
12.04 ± 1.95
|
13.09
|
|
Tick control frequency
|
|
|
|
|
Weekly
|
56
|
338
|
6.11 ± 0.77
|
5.75
|
0.000
|
Biweekly
|
119
|
1 516
|
12.77 ± 1.01
|
11.02
|
|
Monthly
|
65
|
970
|
14.83 ± 2.20
|
17.74
|
|
Occasionally
|
54
|
550
|
10.15 ± 0.95
|
6.99
|
|
Unknown
|
10
|
186
|
18.60 ± 2.91
|
9.19
|
|
Among the ticks collected from Kilombero district, Rhipicephalus microplus was the most abundant tick species (97.4%, n=865) while A. lepidum and R. evertsi were the most abundant species of all ticks collected on cattle from Iringa district each (30.6%, n=306). In addition, Hyalomma albiparmatum (100%, n=3) and H. rufipes (100%, n=50) were only recorded in Iringa district while none was recorded in Kilombero. For H. rufipes, the difference in proportion between the two districts was statistically significant (p < 0.05). The other six tick species including, A. gemma, A. lepidum, A. variegatum, R. appendiculatus, R. decoloratus and R. evertsi were all recorded with significantly higher proportions in Iringa district compared to Kilombero district (p < 0.05) (Table 3). Rhipicephalus microplus was the only species recorded with significantly higher proportion in Kilombero district than in Iringa district.
With regards to cattle sex, age group, health status and tick control frequency, a significantly high mean tick burden of 13.85 ± 1.47 was recorded in male as compared to female cattle 10.78 ± 0.73, (p < 0.05) (Table 4). The highest mean tick burden 12.29 ± 0.85 was recorded in adult cattle (>24 months) as compared to calf (<= 6 months) and juvenile (7 – 24 months) which had mean tick burden of 10.00 ± 2.98 and 10.36 ± 1.18 respectively. In general, there was no statistically significant difference in mean tick burden between the cattle age groups (p = 0.403) and between the cattle health status groups (p = 0.221). Based of tick control frequency categories, a significant low mean tick burden (6.11 ± 0.77) was recorded on cattle reported with weekly tick control frequency (p < 0.001) (Table 4).
With regard to tick distribution, there was high tick species diversity in Iringa than Kilombero district (Table 5). However, in Kilombero district, R. microplus was highly distributed in all sampled villages as compared to Iringa district. Among the five predilection sites on cattle’s body, ticks were distributed in all the five body zones. Tick distribution was highest on zone 4 (56.11%, n = 1,060) which includes (groin, flank, abdomen and around inner thigh of the hind legs), followed by zone 5 (23%, n = 451) and least on zone 2 (0.74%, n = 14) which includes back surface of the body. Amblyomma lepidum and Rhipicephalus microplus species were distributed in all the five body zones and recorded with the highest proportions on zone 4 (including, A. lepidum (67.42%, n=209) and R. microplus (78.55%, n=714) (Table 6).
Table 5. The distribution of the tick species collected on cattle from villages in Kilombero and Iringa Districts
|
Iringa
|
|
Kilombero
|
Tick species
|
Kisanga
|
Kitisi
|
Magombwe
|
Malizanga
|
|
Lufulu
|
Idunda
|
Merera
|
Sagama
|
A. gemma
|
45
|
23
|
5
|
46
|
|
0
|
0
|
1
|
0
|
A. lepidum
|
88
|
6
|
202
|
10
|
|
0
|
0
|
4
|
0
|
A. variegatum
|
2
|
9
|
9
|
2
|
|
0
|
0
|
2
|
5
|
H. albiparmatum
|
0
|
0
|
1
|
2
|
|
0
|
0
|
0
|
0
|
H. rufipes
|
27
|
7
|
14
|
2
|
|
0
|
0
|
0
|
0
|
R. appendiculatus
|
4
|
25
|
0
|
108
|
|
0
|
0
|
0
|
3
|
R. decoloratus
|
6
|
0
|
8
|
0
|
|
1
|
1
|
0
|
2
|
R. evertsi
|
104
|
32
|
154
|
16
|
|
0
|
0
|
4
|
0
|
R. microplus
|
2
|
3
|
39
|
0
|
|
119
|
299
|
293
|
154
|
*Name shortened: Sagama* (Sagamaganga).
Table 6. The distribution of hard tick species identified with respect to cattle body zones
|
Cattle body zones (Tick predilection site on cattle)
|
Tick species
|
Zone 1
|
Zone 2
|
Zone 3
|
Zone 4
|
Zone 5
|
A. gemma
|
6
|
0
|
36
|
59
|
19
|
A. lepidum
|
11
|
3
|
60
|
209
|
27
|
A. variegatum
|
0
|
4
|
12
|
10
|
3
|
H. albiparmatum
|
1
|
0
|
0
|
2
|
0
|
H. rufipes
|
0
|
0
|
2
|
3
|
45
|
R. appendiculatus
|
103
|
0
|
4
|
27
|
6
|
R. decoloratus
|
0
|
2
|
1
|
6
|
9
|
R. evertsi
|
4
|
0
|
2
|
30
|
274
|
R. microplus
|
12
|
5
|
110
|
714
|
68
|
Total ticks n (%)
|
137 (7.25)
|
14 (0.74)
|
227 (12.02)
|
1,060 (56.11)
|
451 (23.88)
|
For molecular identification of tick species, a total of 42 representative ticks, (1-5 ticks) from each species were randomly selected for molecular analysis. The nine tick species identified morphologically were also identified by molecular method however, during sequencing two species (Rhipicephalus appendiculatus and R. decoloratus) had poor quality sequences and were excluded from the analysis. The CO1 gene was successfully amplified from 92.86% (n=39) of the selected-on host ticks. The 16S rRNA gene was successfully amplified from 100% (n=8) of the samples with unreliable CO1 results. The amplification of approximately 455 bp sequence of 16S rRNA produced the expected amplification products. The nucleotide sequences of Tanzanian ticks obtained from this study were submitted at the GenBank and provided with accession numbers (OM974109 - OM974112 and OM978262 - OM978265).
Based on the CO1 gene sequences, Amblyomma gemma from this study (GenBank accession no. OM974111) was 100% identical to A. gemma isolate sequence from Kenya (BOLD: ARAK131-13). The A. lepidum sequence (GenBank: OM974112) from this study had the closest identical A. lepidum isolate sequence from Kenya (GenBank: KP987775). Hyalomma albiparmatum sequence from this study (GenBank: OM974110) had the closest identical H. albiparmatum isolate sequence from Israel (GenBank: KU130576), whereas, the H. rufipes sequence from this study (GenBank: OM974109) had the closest identical H. rufipes isolate sequence from France (GenBank: KX000643).
Based on the 16S rRNA gene sequences, Rhipicephalus evertsi sequence from this study (GenBank: OM978262) had the closest identical R. evertsi isolate sequence from Zambia (GenBank: LC634571). Amblyomma variegatum from this study (GenBank: OM978264) had the closest identical A. variegatum isolate sequence from Sao Tome and Principle and Ethiopia (GenBank: MF627697 and MN150175) respectively. Lastly, the 16S rRNA gene sequence of R. microplus from this study (GenBank: OM978265) had the closest identical R. microplus isolate sequence from Uganda (GenBank: KY688461).
Phylogenetic analysis based on mitochondrial CO1 and 16S rRNA nucleotide sequences of the identified tick species was performed to determine the genetic relationship between the nucleotide sequences obtained in this study and reference sequences obtained from GenBank. Alignment of CO1 gene nucleotide sequences obtained from each tick species in this study showed that the sequences were 100% identical. Similarly, the 16S gene nucleotide sequences obtained from each tick species were found to be 100% identical. Therefore, a single sequence from each tick species was selected for phylogenetic analysis. In both mitochondrial CO1 (Fig. 2) and 16S rRNA phylogenetic trees (Fig. 3), four major clusters were observed with all the nodes strongly supported by high bootstrap values.