Bovine piroplasmosis‐anaplasmosis and clinical signs of tropical theileriosis in the plains of Djurdjura (north Algeria)

Abstract The study was conducted during tick activity season over a period of 5 years in the Djurdjura Plains, Algeria. A total of 299 cattle (Holstein, Montbeliard, Fleckvieh and crossbred animals) with clinical signs were included in this study. A total of 171 animals were found positive for at least one pathogen by Giemsa‐stained blood smears examination Theileria annulata (136/299, 45.5%), Babesia bovis (14/299, 4.7%), B. bigemina (3/299, 1.0%) and Anaplasma marginale (12/299, 4.0%) were identified. Six animals were co‐infected by T. annulata and A. marginale. Although no ticks were collected from diseased animals, clinical signs in cattle were hyperthermia (120/136, 88.3%), gluttony followed by anorexia (113/136, 83.1%), lymph node enlargement (99/136, 72.8%), anaemia (82/136, 60.3%), icterus (58/136, 42.6%) and haemoglobinuria (36/136, 26.5%). Gluttony followed by anorexia was considered highly suggestive of an incubation of tropical theileriosis as shown by a higher receptivity index (IR = 0.89–1). This clinical sign is evident in young Montbeliard and young Holstein males with anaemia (IR = 1) and icterus (IR = 0.78–0.81) which is earlier than haemoglobinuria (IR = 0.51–0.54). The incidence of T. annulata was maximum in July (n = 57), as well as B. bovis (n = 6) and A. marginale (n = 13). These results highlight the preponderance of tropical theileriosis in north‐central Algeria, where gluttony followed by anorexia is probably a prodromal symptom during the incubation period of the disease.

In Algeria, tropical theileriosis is the most prevalent infection in cattle, followed by anaplasmosis and babesiosis, with incidences of 57.2%, 6.2% and 3.8%, respectively (Ziam et al., 2016;Ziam & Benaouf, 2004). Of these three enzootic diseases, tropical theileriosis remains by far the dominant summer disease in Algeria.
In epidemiological studies of piroplasmosis and anaplasmosis, serology and PCR are excellent diagnostic tools with good sensitivity and specificity. However, PCR could not discriminate carrier from diseased animals and serology might not be positive during the disease course particularly in early clinical cases. That is why these tests could not be used for confirmation of disease cases (Ait-Hamou et al., 2012;Bilgic et al., 2016;El Haj et al., 2002;Ziam et al., 2015).
Nevertheless, suspicion of the disease is based on the association of epidemiological elements (period of specific tick activity, the type of livestock, state of the stables in the case of tropical theileriosis presence of cracks and crevices) and clinical signs associated, confirmed by positive Giemsa stained blood or lymph node smears (Ziam, Saidani, & Aissi, 2017). Unfortunately, the remoteness and/or the lack of well-equipped veterinary laboratories complicate correct diagnosis of piroplasmosis and anaplasmosis.
As differential diagnosis between these three tick-borne diseases might be difficult, especially in relapsing tropical theileriosis cases or in some reinfection cases with a new genotype which are associated with a transient lymph node enlargement. Identification of prodromal symptoms of the disease may help clinicians to better identify the disease outcome and initiate early treatment before the onset of hyperthermia and generalized swelling of lymph nodes, severe anaemia, icterus and petechia, decreasing the probability of the animal recovery. In Algeria, tropical theileriosis is the dominant cattle summer disease (Ayadi, Gharbi, & Benchikh-Elfegoun, 2017;Rouina, 1984;Ziam et al., 2017). The objective of this work was to highlight the clinical incidence of bovine anaplasmosis and piroplasmosis, especially tropical theileriosis, and associated early clinical signs in cattle of exotic and local breeds in the plains of Djurdjura, Algeria.

| Study area
The study was conducted in the plains of Djurdjura located in the North Central Algeria. It is a vast area, of 5,144,2 km 2 , between longitudes 7°08' to 8°37' E and latitude 36°43' to 37°7' N, this region has a landscape interspersed with valleys and mountainous regions ( Figure 1).

| Climate and vegetation
Djurdjura region has a Mediterranean climate. Its annual rainfall varies between 600 in the south and 1.200 mm in north with a relative humidity of 65%-75% during the summer season. The region is sub-humid to humid with hot and dry summers and cool and rainy winters. Temperature reaches 35°C in summer and drops to 5°C in winter, the northwest winds generate heavy rainfall accompanied by cold waves (Amroun, Bensidhoum, Delattre, & Gaubert, 2013;Meddour, Meddour-Sahar, Derridj, & Gehu, 2010). According to Meddour et al. (2010), cedar forests of the Atlas and cork oak F I G U R E 1 Geographic localization of the region of Djurdjura, North Algeria, triangle shows the localities where bovines were sampled represents 42% of the total area of the region, and more than 3,800 plant species of plants were recorded. Natural grasslands constitute the bulk of the diet of cattle (Meddour et al., 2010).

| Animals
This study was carried out between 2004 and 2008, during tick activity period, from May to September (Gharbi et al., 2020). Moreover, anaplasmosis is also transmitted mechanically by haematophagous vectors (Tabanus, Stomoxys…). Suspected cattle with piroplasmosis or anaplamosis, presented to the veterinary clinics (n = 7), were sampled in the Djurdjura area from 270 farms having a herd size of more than 15 animals. Cattle were reared under a semi-intensive system and consisted of exotic breed Holstein, Montbeliard, Fleckvieh and crossbred. Grass, hay, straw and crop residues are the main diet of the animals, supplemented with concentrates.

| Diagnosis and treatment
A total of 299 cattle showing clinical signs of either piroplasmosis or anaplasmosis, such as hyperthermia, lymph node enlargement, agalactia, haemoglobinuria, icterus and gluttony followed by pronounced anorexia were included in the study. Clinical examination, Giemsastained blood smears, pathogen identification and treatment of animals were done as described previously (Ziam et al., 2017). Briefly, blood smears were made from the ear vein of each suspected sick animal, fixed in methanol, stained with Giemsa and examined under a microscope using an oil-immersion ×100 objective. At least, 100 microscopic fields were carefully examined for blood forms of Anaplasma spp., Theileria spp. and Babesia spp. An animal was considered positive when showing the presence of one or more of these pathogens.
Theileria annulata positive animals were treated with buparvaquone (ButalexTM, Schering-Plough) at a dose of 2.5 mg/kg; whereas imidocarb dipropionate (Imizol®, Merck) at a dose of 6.6 mg/kg was used to treat babesiosis and anaplasmosis. After treatment, there was no further follow-up for pathogen presence.

| Index of receptivity (IR)
A correlation between an observed symptom and the number of animals truly affected by T. annulata, B. bovis, B. bigemina and A. marginale was evaluated using the index of receptivity (IR) as proposed by Ziam et al., (2016). The IR (varies from 0 to 1) was calculated by relating the total frequency of a clinical sign to the number of animals actually affected by T. annulata, B. bovis, B. bigemina or A. marginale showing this sign. The IR is more important when it rises or reaches 1 and less important when it falls to zero.

| Statistical analysis
Mixed-effect logistic regression was used to evaluate the influence of age, sex and breed on the incidence of various diseases and to compare their incidences. The comparisons of the frequencies of clinical signs were performed using the chi-square test. Prioritization of clinical signs was performed according to the method of classification tree and regression (Dahms, 2004). B. bigemina and A. marginale were low and therefore the frequency of these specific clinical signs was high (Table 1). In dairy cows, a total of 97 lactating cows showed sudden agalactia. The presence of dead ticks was always confirmed through inspection of the tickfixation sites like ear conch, baleen, ano-genital region, legs and the inguinal region.
These pathogens were identified in 171 ( (Table 2) There was no statistically difference between infection rates by B. bigemina in the three breeds Montbeliard, Fleckvieh and crossbred (p > .05). The incidence of infection by each pathogen was higher in adults than in young animals as well as in females (p < .001) compared to males (

| Chronology of different pathogens
The chronological distribution of clinical cases for each pathogen during the study period (May to September) is shown in Figure 2.
The overall incidences for each pathogen were significantly different (p < .001). Tropical theileriosis increased linearly from May to June with an incidence of 12 to 39 new clinical cases, respectively, and reaching a peak of 57 cases in July. Thereafter, the disease decreased towards 26 cases in August and dropped to two cases in September. Statistical analysis indicated that the rate of tropical theileriosis in July was significantly higher (p < .001) than those of May, June, August and September and the incidence during June was also significant (p < .001), compared to those of May, August and September and between August and September (p < .001). The number of clinical cases due to A. marginale and B. bovis (p < .05-.001) was higher in July than during the other months of the summer season ( Figure 2). Only three animals were infected by B. bigemina during May, July and August.

| Index of receptivity
The IR of the various clinical signs, for each identified pathogen, in piroplasmosis and anaplasmosis suspected cattle are presented in T. annulata as a specific clinical sign in all breeds when the classification tree and regression was used (IR = 0.89) ( Table 3). The IR of icterus was also high in the investigated breeds (IR = 0.84) (Table 3) infected by T. annulata. This symptom associated with gluttony followed by anorexia is mostly seen in tropical theileriosis. The IR of haemoglobinuria was 0.51 (Table 3) (Table 3).
Gluttony followed by anorexia is a specific and common symptom in young Holstein, Montbeliard and Fleckvieh as well as in Holstein, (Table 4). There is no difference in such symptoms between males and females of Montbeliard and crossbred animals (IR = 0.90-1) (

| D ISCUSS I ON
Theileria annulata is an intracellular parasite diverting cellular biochemical signalling pathways to ensure its development in the host cell. They infect B-cells and other monocytes transforming them into lymphoblast and monoblast cells (Dobbelaere & Rottenberg, 2003). Theileria-transformed cells act like cancer cells and are known to consume a lot of glucose (Haidar, Echebli, Ding, Kamau, & Langsley, 2015;Haidar, Whitworth, et al., 2015).
It has been shown that cows with a starting T. annulata infection increase their food intake, particularly high-energy foods (Ziam et al., 2016). This study indicated that 122 animals out of 299 suspected cases showed gluttony for up to 24 hr, and a large proportion of these animals were naturally infected with T. annulata (Table 4). This gluttony seems to be the expression of an infectious state in which the parasite interferes with its host cell metabolism to its advantage. Thus, these animals need external supply of energy to satisfy the high glucose uptake by Theileria-transformed cells (Haidar, Echebli, et al., 2015;Haidar, Whitworth, et al., 2015).
These results confirm our preliminary observations and indicate that gluttony in cattle might be a pathognomonic symptom for early diagnosis of tropical theileriosis. Similar observation was reported in India (Narladkar et al., 2005).
Our data pointed out that the gluttony followed by anorexia was associated with other symptoms related to the evolution of the disease. Among them, hyperthermia, lymph node enlargement, anaemia, icterus and haemoglobinuria, similar to previously reported results (Darghouth, Kilani, & Bouattour, 2003;Rouina, 1984;Ziam et al., 2017). Abbreviations: Gluttony/anorexia, gluttony followed by anorexia; IR, Index of receptivity corresponding to the ratio of overall frequency of clinical sign/ number of animals positive for blood smear.

TA B L E 4 Receptivity index according to age and sex of cattle with tropical theileriosis from the plains of Djurdjura, North Algeria
Epidemiological parameters Hyperthermia Adenitis Anaemia
Icterus was seen in the early stages of tropical theileriosis as confirmed by an IR = 0.84 and haemoglobinuria appears towards the final phase of the disease (IR = 0.51) similar to the results reported by Darghouth et al. (2003). This symptom is the result of haemolysis, induced by merozoïtes of T. annulata, followed by excretion of haemoglobin in urines (Gharbi et al., 2012). In this survey, young animals were not infected with Babesia spp.
According to Figueroa et al. (2010), this might be due to a natural resistance in calves aged 6 to 9 months, the absence of exposure to exophilic ticks associated with low infestation rates in calves and finally due to colostral immunity (Figueroa et al., 2010).
The high rates of tropical theileriosis in this study are similar to those previously observed in the wilayates of Annaba and El Tarf in eastern Algeria (Ziam et al., 2016). The high prevalence of clinical cases due to tropical theileriosis in adult exotic breeds suggests endemic instability or relapsing disease due to stress. By studying this phenomenon in dairy cattle over four summers, (Darghouth et al., 2003) argued that stress due to milk production, pregnancy and calving are essentially implicated in the susceptibility to infection and subsequently in the expression of clinical symptoms.
Even though no live ticks were found on the animals during our study period, lesions testifying earlier infestations by ticks were found in the genito-anal, inguinal region and auricular conch. Only dry ticks, probably dead following acaricidal treatments, were found on animals. Our attempts to collect these ticks were unsuccessful.

ACK N OWLED G EM ENTS
This work received financial support from the Algerian Ministry of Higher Education and Scientific Research. We are grateful to Dr. Youcef TOUDERT, a practitioner in Ben M'hidi in El Tarf, for reporting the gluttony of cattle incubating tropical theileriosis. We