Cross‐sectional survey of cattle haemopathogens in Constantine, Northeast Algeria

Abstract This aim of the present study was to estimate the prevalence of haemopathogens in cattle in Beni Hamidene locality, district of Constantine (Νortheastern Algeria). Between June and October 2014, 169 bovines from 25 farms were included in this survey, 32 (18.9%) among them were suspected of piroplasmosis and/or anaplasmosis. Infection prevalences were estimated by microscopic examination of Giemsa‐stained blood smears and blood samples from all included cattle (n = 169). Animals were infected by Theileria annulata (65/169; 38.46%), Anaplasma marginale (22/169; 13%) and Babesia bovis (5/169; 3%). Two co‐infection patterns were found: Theileria annulata/Anaplasma marginale (7.69%) and Theileria annulata/Babesia bovis (1.18%). Only one farm had no cattle infected by any of the haemopathogens. There was a signification difference of T. annulata infection prevalence according to age category (p =.04). These results emphasised mainly the presence of bovine tropical theileriosis in northeastern, Beni Hamidene locality, province of Constantine, Algeria.

Four techniques are used for diagnosis of haemopathogens: (1) Giemsa stained blood smears is the quickest and the cheapest technique. It allows the estimation of the parasitaemia and detects the presence of any co-infection by haemopathogens. It allows also the detection of leukocytes infected by T. annulata schizonts in lymph-node biopsies (Bilgic et al., 2016). However, it has low sensitivity in detecting carrier animals (Alvarez et al., 2019;Ashuma et al., 2013;M'ghirbi et al., 2008;Uilenberg, 2004). (2) Enzyme Linked Immunosorbent Assay (ELISA) is used to detect specific antibodies (Al-Hosary et al., 2015; it has a high sensitivity and specificity (Santamaria et al., 2020) and many samples can be easily tested (Salih et al., 2007). As ELISA becomes positive approximately 3 weeks after infection, this technique is only used for epidemiological studies to detected carrier animals with low parasitaemia.
(3) The indirect immunofluorescent antibody test (IFAT) is also used to detect carrier animals (Nayel et al., 2012). This technique is very time-consuming and, in some cases, the interpretation of the fluorescence is difficult.
(4) Several PCR techniques are used for the detection of haemopathogens (conventional PCR, nested PCR, Real-time PCR, PCR multiplex, PCR-RFLP, reverse line blot); they have high sensitivity and specificity (Wang et al., 2019) but they are relatively expensive (Liu et al., 2014). Moreover, these techniques don't differentiate between carrier and clinically infected animals.
In Algeria, tick-borne diseases represent a real constraint for cattle owners. For example, the mean milk yield decreases to 319 L/cow that suffers tropical theileriosis during 2 months following the infection (Benchikh Elfegoun et al., 2017). Ayadi et al. (2016) estimated that the mean daily milk yield decreases during 2 months to 2.76/ day/cow presenting clinical theileriosis.
The aim of the present study was to estimate the prevalence of piroplasmosis and anaplasmosis in cattle, in Beni Hamidene locality, province of Constantine, Northeast Algeria.

| Study area
The present survey was carried out in Beni Hamidene locality (36°30' S; 6°31'W) located in northwest of Constantine district (Northeast Algeria) and has 131 km 2 area ( Figure 1). Beni Hamidene has an altitude that varies between 300 and 1,364 m, a sub-humid and semi-arid climate and a mean annual pluviometry of 761 mm.
There are 171 cattle breeders in Beni Hamidene, owning approximately 1,640 animals, among them were 783 dairy cows (Direction des Services Agricoles de la Wilaya de Constantine, 2015).

| Characteristics of studied farms and animals
A cross-sectional study was carried out between June and October 2014 in 25 cattle farms containing between 2 and 40 cattle (average population per farm: 10 cattle). Among the 248 cattle in these farms, 169 were included in this study. Animals that received piroplasmicides during the 2 months before our visit were excluded from the study. Almost half of the farms were dairy (12/25) or mixed (12/25), and there was one beef cattle breeder. All farms except one were managed under a semi-intensive system (24/25). The majority of farms (24/25) had cracked walls and were poorly maintained (16/25).

| Sample collection
Among the 169 studied cattle, 32 (18.9%) presented clinical signs of piroplasmosis and/or anaplasmosis (anaemia, icterus, ocular /vaginal petechiae, hyperthermia, swelling of lymph nodes, apathy, anorexia, hypogalactia and weight loss) The animals were clinically examined and blood samples were collected from the ears capillaries with sterile disposable scalpels. Blood smears were done in the farm and immediately fixed in 100% methanol for 3 min, air-dried and then F I G U R E 1 Geographical location of Beni Hamidene locality, Wilaya of Constantine, Algeria transported to the laboratory. Blood smears were Giemsa-stained for 10 min, air-dried and then rinsed with tap water and air-dried. For each slide, 50 microscope fields were examined at 1000X magnification under microscope with immersion oil. The infection intensity by haemopathogens was semi-quanlitatively estimated, and animals were ranked into four categories: low (0.2-1 parasites/microscopic field), mild (1-5 parasites/microscopic field) and high (5 > parasites/ microscopic field).

| Statistical analyses
Infection prevalences were compared using either chi-square test or stratified Mantel-Haenszel chi-square test at 0.05% threshold with Epi info 2000 software (Schwartz, 1993).

| RE SULTS
Among the 25 visited farms, 13 contained at least one cattle infected by one haemopathogen, 11 farms contained both single and coinfection of haemopathogens and one farm had non-infected cattle. bovis-infected cattle were at least 1.5 years old and were not anaemic. Two out of the 5 B. bovis-infected cattle were infested by ticks.
Among the 32 clinically suspected animals of piroplasmosis and/ or anaplasmosis, the morbidity, lethality and mortality rates were 18.9% (32/169), 6% (2/32) and 1.2% (2/169), respectively. Number of cattle Symptoms infested by ticks which could because of the use of acaricides by the majority of cattle owners (16/25) and the long delay between tick fixation and clinical symptoms occurrence, generally, the bloodmeal duration of ticks is shorter than the incubation period, that's why, when the animal shows clinical symptoms, the tick that transmitted the parasite could not be found attached to the animal. However, farmers do not use acaricides properly (concentrations of acaricides and/or interval between two applications were not corrected). The low tick numbers and the presence of clinical cases indicate that the visited farms were in an endemic instability state for tropical theileriosis.

| D ISCUSS I ON
In endemic regions, carrier state prevalence is high, it can even reach 100%. Giemsa-stained blood smears lacks sensitivity because the parasitaemia in these animals is usually low (Alvarez et al., 2019;Gharbi et al., 2012).
Infection prevalence of Theileria annulata was higher than A. marginale and B. bovis. The same trend was reported by Ait Hamou et al. (2012) in Morocco.
However, there was no significant difference in infection prevalence according to breeds. Carrier local cattle breeds are sources of infection for other animals, mainly susceptible breeds (Moni et al., 2019).
The majority of A. marginale-infected cattle were carriers. This result is similar to that reported by M'ghirbi et al. (2016), who found in Tunisia that all A. marginale-infected cattle were carriers. This infection persisted for the whole life of cattle (Aubry & Geale, 2011;Kocan et al., 2010). All A. marginale-infected cattle were more than 1-year age, the similar trend was also reported in B. bovis-infected cattle.
According to Kocan et al. (2003), calves are less susceptible to A. marginale infection. This can be explained by the presence of maternal antibodies (Abdela et al., 2017) and the lower attractivity of young cattle to ticks (Gharbi et al., 2013). In our study, there was a positive correlation between infection prevalence and age. Indeed, T. annulata infection prevalence in cattle aged of 3 years and more was significantly higher (58%) than the prevalence in cattle aged of less than 3 years (42%; p =.04). This can be explained by the stress caused by lactation that induces immunodepression and the presence of higher tick burdens when compared with young animals (Gharbi et al., 2013).
According to Yessinou et al. (2018), calves are less infested than adult cattle by ticks. However AL-Hosary et al. (2018) reported that cattle aged less than 1 year were more susceptible to infection by T. annulata (83%; 400/480). According to AL-Hosary et al. (2018) cattle acquire immunity with age and after multiple infections by T. annulata.
In our study, cattle probably have not acquired immunity; this can be explained by the introduction of naive cattle in the farms and the low tick burdens because of acaricides application.
Infection prevalence by B. bovis (3%; 5/169) was slightly lower than that reported by Ziam et Benarouf (2004) in eastern Algeria (5,6%). According to Ziam et al. (2017), the low rate of B. bovis is because of the low tick burdens in the studied region and to the relatively low pasturing duration. In addition, it can be explained by the fact that Beni Hamidene has a sub-humid and semi-arid climate which is not suitable for development of the ticks vector Rhipicephalus (Boophilus) annulatus. According to Benchikh-Elfegoun et al. (2007), this tick species is adapted to humid climate. However, according to Calder et al. (1996) parasitaemia fluctuation in B. bovis chronicallyinfected cattle could explain that the low detection of parasites ranges from 1. 10 -5 to 1. 10 -7 . The prevalence found in the present study was lower than in Syria (15.46%; Terkawi et al. 2012), Mongolia (18%; Battsetseg et al. 2018) and in South Africa (35.3%; Terkawi et al., 2011). The difference can be explained by the diagnostic method (ELISA), which has a higher sensitivity than Giemsastained blood smears of carrier animals (Guswanto et al., 2017).
In the present study, the prevalence of co-infections by T. annulata and B. bovis (1.2%) was similar to that reported by Ziam et al. (2017) (1.1%). The prevalence of co-infections by T. annulata and A. marginale (7.69%) was slightly higher than that reported by Ziam et al. (2017) (1.9%) in north-central Algeria. This could be because of the difference in Anaplasma spp. vector ecology, particularly its biomass and typology in the two regions, which have, as far as we know, never been studied.
All B. bovis-infected cattle (5/5) were aged of 1 year and more.
Young cattle are more resistant to babesiosis than adult (Ekici & Sevinc, 2009;Goff et al., 2001). In addition, calves are less exposed to the vectors of Babesia because R. (Boophilus) annulatus are found in the pastures where generally older cattle grazes.
This study showed the presence of three haemopathogens in cattle in Beni Hamidene locality, district of Constantine, Algeria.
Among these haemopathogens, Theileria annulata was the most frequent. This study also reported the main clinical features of theileriosis. No Atlas Brown cattle showed symptoms, as a local breed, breeding Atlas Brown cattle must be encouraged in the farms where control of vector ticks is difficult to implement. In addition, extension programmes must be implemented for Algerian breeders to optimise the acaricide application.
Moreover, farmers must be sensitised on the importance of sustainable control options (cleaning, wall roughcasting and smoothing) to reduce the shelters for Hyalomma scupense off-host stages.

ACK N OWLED G EM ENTS
The authors thank all cattle farmers of Beni Hamidene who accepted to handle their animals.

CO N FLI C T O F I NTE R E S T
All the authors declare that they have no conflicts of interest with the work presented here.

Pe e r Rev iew
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/vms3.459.