Infection by haemopathogens and tick infestation of sheep during summer season in Constantine region, Northeast Algeria

Abstract A study on tick infestation of 43 sheep with clinical symptoms of piroplasmosis and anaplasmosis was carried out during summer seasons of 2016 and 2017 in 34 sheep flocks from Beni Hamidene locality, district of Constantine, Northeast Algeria. Only animals with clinical symptoms of piroplasmosis and/or anaplasmosis were checked for tick infestation. Among the 43 examined sheep, 58 ± 15% were infested by ticks. A total of 185 adult ticks (100 males and 85 females) were collected from the 25 sheep. Two tick genera, Rhipicephalus and Hyalomma consisting of four species were collected, Rhipicephalus bursa was the most frequent tick (164/185; 88.6 ± 4.6%), followed by Rhipicephalus sanguineus sensu lato (16/185; 8.6 ± 4%), Rhipicephalus (Boophilus) annulatus (4/185; 2.2 ± 2.1%) and Hyalomma scupense (1/185; 0.5 ± 1%). Mean overall tick infestation intensity was 7.4. Mixed infestations with two tick species were found in 10 sheep (23.3 ± 12.6%). All farmers (34/34) used ivermectin, and only 11% of them used acaricide to control ticks. The majority of R. bursa ticks (87/185; 45 ± 7%) were located on the ears 37/185; 20 ± 6%) and the testicles (50/185; 27 ± 6%). Giemsa‐stained blood smears examination showed the presence of three haemopathogens: Anaplasma spp. (19/43; 44 ± 15%); Babesia spp. (6/43; 14 ± 10%) and Theileria spp. (1/43; 2 ± 4%). These results showed the presence of several low infestation burdens by ticks transmitting three haemopathogens.

Ovine anaplasmosis represents a major health problem in sheep flocks in several tropical and subtropical regions (Shi et al., 2020). This rickettsia is transmitted by ticks and mechanically by serval haematophagous insects. Symptoms are not specific: fever, apathy, anorexia, anaemia, icterus, nasal discharge, weight loss, hypogalactia and abortion Tibbitts et al., 1992;Yasini et al., 2012).
In North Africa, several studies reported the presence of Theileria spp. in sheep specially Theileria lestoquardi and Theileria ovis (Al-hosary et al., 2021;Hussein et al., 2017;. Theileria lestoquardi causes malignant ovine theileriosis (MOT) which is by far more pathogenic than T. ovis (Hassan et al., 2019;Yaghfoori et al., 2016). The clinical symptoms of MOT are similar to tropical theileriosis in cattle (Theileria annulata infection) and can cause death in severe cases (Ge et al., 2012).
We aimed herein to study the tick infestation during the tick season in sheep with clinical episodes or heamopathogen infections in Beni Hamidene locality, district of Constantine, Northeast Algeria.

Farms and sheep
The present survey was performed in 34 sheep flocks containing a total number of 3200 sheep with a mean sheep population of 74 (range: 14-150). The majority of sampled animals were maintained under semi-intensive (91 ± 9%; 31/34;), followed by extensive (6 ± 8%; 2/34) and intensive systems (3 ± 6%; 1/34 The farmers were questioned about the symptoms expressed by their animals and the use of acaricides. Animal owners were questioned about the application of acaricides on their animals, and a verbal consent was obtained from each farmer. Sheep were clinically examined; their ages were estimated by examining their teeth.

Blood samples collection
Blood samples were collected from the ear capillaries of each sheep using a disposable scalpel; blood smears were immediately made in the farms, fixed in the methanol for 3 min and Giemsa-stained.
Blood smears were examined under a microscope with immersion oil at x1000 magnification.

Ticks collection and identification
All ticks fixed on the examined sheep were collected and stored in identified tubes containing 70% ethanol. Ticks were identified under a stereomicroscope according to the key of Walker et al. (2014).

Parasitological indictors and statistical analyses
Three parasitological indicators were estimated (Bush et al., 1997) as The infection intensity by haemopathogens was estimated by calculating the number of infected-red blood cells in 50 microscope fields (on average, each microscopic field contains 350 red blood cells).

Treatments
All sheep with clinical symptoms of anaplasmosis and/or piroplasmosis were treated. Oxytetracycline (Longicine ® , Vetoquinol, France) was injected at the dose of 20 mg/kg to animals suffering from anaplasmo-

Parasitological indicators
More than half of sheep were infested by at least one tick (
Among the 43 sheep with clinical symptoms, almost half of them (20/43; 46 ± 15%) were infected by at least one haemopathogen.

Lethality
Among the 43 sheep suffering from piroplasmosis and/or anaplasmosis 4 (9 ± 9%) died just after treatment. Two dead sheep were co-infected by Anaplasma spp. and Babesia spp. The two others died but the blood smears microscopic examination did not show any haemopathogen.

DISCUSSION
In the present study, the overall infestation prevalence by ticks was estimated to 58%, it was higher to the prevalence reported in Adrar In Northeast Algeria, Leulmi et al. (2016)  Only adult ticks were collected, the absence of immature instars could be explained by the fact that the work was carried out in summer, generally only adults are present (Boulkaboul, 2003;Laamari et al., 2012). Babesia spp.

Other tick species
Overall

Other tick species
Overall to ticks. However, Papadopoulos et al. (1996) reported that anus, vulva, udder and scrotum are the attachment sites of R. bursa.
Giemsa-stained blood smear was used to confirm the clinical suspicion. This technique is used in sick animals since they have relatively high parasitaemia (Altay et al., 2012). Out of the 43 sampled sheep, 47% were infected by at least one haemopathogen.
Anaplasma spp. was the most frequent haemopathogen (12/25) in the 25 tick-infested sheep. Anaplasma is transmitted mechanically by several tick species. In Algeria, Anaplasma ovis was detected in sheep by the quantitative real-time PCR (qPCR) in the northeast of Algeria (Aouadi et al., 2017;Sadeddine et al., 2020). Anaplasma ovis was identified in sheep in Senegal (55.9%) (Dahmani et al., 2019). A study on sheep anaplasmosis was carried out in Kairouan (central Tunisia, Kairouan); of the 8049 sheep examined by Giemsa-stained blood smears, 282 were infected by A. ovis . Using PCR, Belkahia et al. (2014)  we worked only on sheep with clinical symptoms of anaplasmosis and/or piroplasmosis and the sampled animals was low. The prevalence was lower than the estimation reported by Naderi et al. (2017) in Iran (38/306; 9.9%) who worked on babesiosis-suspected sheep.
Only one sheep (2%) was infected by Theileria spp. This rate is similar to that reported by  in Tunisia (2/166) by blood smears examination. However, G. R. Razmi et al. (2006) reported a rate of 11.9% (100/840) in South Khorasan province (Iran) using microscopic examination and concluded that Theileria infection was spreading in this region. In Turkey, the reverse line blotting (RLB) method detected that 28.9% (37/128) of apparently healthy sheep were infected by Theileria spp. (Altay et al., 2012). In Kenya, polymerase chain reaction revealed that 51.3% (39/76) of sheep were infected by Theileria spp. (Ringo et al., 2019). This could be explained by the diagnostic methods; PCR, qPCR and RLB are more sensitive and specific than Giemsa-stained blood smears Shayan & Rahbari, 2005;Theodoropoulos et al., 2006;Wang et al., 2015). The majority of sheep with clinical symptoms of piroplasmosis and/or anaplasmosis (32/43; 74 ± 13) showed prescapular lymph node enlargement.
Further studies are needed to identify the tick fauna infesting sheep in order to implement the correct tick-control strategy and also to identify the species of haemopathogens infecting sheep.