Conservation agriculture has no significant impact on sheep digestive parasitism.

Conservation agriculture (CONS A) is a sustainable agriculture system based on crop rotation with no tillage. It has various environmental advantages compared to conventional agriculture (CONV A): decreased water evaporation, erosion, and CO2 emissions. In this first study of its kind, we aim to evaluate the impact of this type of agriculture on sheep gastrointestinal parasites. Two lamb groups aged between 5 and 10 months were randomly included to graze separately on CONS A and CONV A pastures. Each group was composed of two batches of three lambs, and these were followed up for two rearing months. Liveweight, hematological parameter variation, and digestive parasites were studied. At the end of the study period, lambs were slaughtered the carcass yield was determined, and a helminthological autopsy was performed on the digestive tracts of the animals to estimate different parasitological indicators. There was no difference between lambs reared on CONS A and those reared on CONV A for all parasite indicators (infestation intensity, abundance, and prevalence). The same trend was also obtained for hematological parameters, liveweight evolution, and carcass yield. These results prove that there is no impact of CONS A on the sheep's digestive parasitism. Further studies are needed to support these findings on larger animal samples and to investigate the impact of conservation agriculture on other parasite species. Similar studies could also be conducted on ruminant species.

First morphometric and molecular characterization of Fasciola spp. in Northwest Tunisia.

The aim of this study was to characterize the Tunisian Fasciola spp. flukes by morphometric and molecular analyses. Flukes were collected from livers of sheep slaughtered in Sejnane slaughterhouses (Bizerte gouvernorate, Northwest Tunisia) between January and March 2021.Five morphometric parameters were determined for all the liver flukes, as follows: (i) total body length (BL), (ii) distance between ventral sucker and the tail (VS-T), (iii) distance between oral sucker and ventral sucker (OS-VS), (iv) abdomen diameter (AD), (v) tail diameter (TD) and the body length to width ratio (BL/BW). Molecular identification of the fluke specimens was carried out by polymerase chain reaction, restriction fragment polymorphism (PCR-RFLP) of a 680 bp sequence of the internal transcribes spacer 1 (ITS1) gene and by amplification, sequencing, and phylogenetic analysis of a 500 bp sequence of the ITS2 gene. Morphometric measurements showed that the mean of the total body length of the adult flukes was 21.1 ± 2.7 mm with minimum and maximum lengths of 13 and 31 mm, respectively. The PCR-RFLP analysis revealed a single profile consisting of three bands of approximately 370, 100, and 60 bp. Fasciola sequences described in the present study (GenBank numbers: OQ457027 and OQ457028) showed 99.58-100% identity to Fasciola hepatica. In conclusion, the results of this study show that molecular and phylogenetic analyses confirm the presence of a single species of F. hepatica in the Sejnane region Northwest of Tunisia. However, further studies are needed to identify the occurrence of Fasciola species in other Tunisian regions.

Genome variation in tick infestation and cryptic divergence in Tunisian indigenous sheep.

BACKGROUND: Ticks are obligate haematophagous ectoparasites considered second to mosquitos as vectors and reservoirs of multiple pathogens of global concern. Individual variation in tick infestation has been reported in indigenous sheep, but its genetic control remains unknown. RESULTS: Here, we report 397 genome-wide signatures of selection overlapping 991 genes from the analysis, using ROH, LR-GWAS, XP-EHH, and FST, of 600 K SNP genotype data from 165 Tunisian sheep showing high and low levels of tick infestations and piroplasm infections. We consider 45 signatures that are detected by consensus results of at least two methods as high-confidence selection regions. These spanned 104 genes which included immune system function genes, solute carriers and chemokine receptor. One region spanned STX5, that has been associated with tick resistance in cattle, implicating it as a prime candidate in sheep. We also observed RAB6B and TF in a high confidence candidate region that has been associated with growth traits suggesting natural selection is enhancing growth and developmental stability under tick challenge. The analysis also revealed fine-scale genome structure indicative of cryptic divergence in Tunisian sheep. CONCLUSIONS: Our findings provide a genomic reference that can enhance the understanding of the genetic architecture of tick resistance and cryptic divergence in indigenous African sheep.

Presence of antibodies to Crimean Congo haemorrhagic fever virus in sheep in Tunisia, North Africa.

Crimean-Congo haemorrhagic fever (CCHF) is an emerging tick-borne disease causing severe and fatal haemorrhagic syndrome in humans. Hyalomma spp. ticks are the primary vectors and sheep are important CCHF virus (CCHFV)-amplifying hosts. In this study, blood samples and ticks collected in October 2019 from 270 sheep from 15 farms across Tunisia constituted the main research material. Moreover, the sera of the same animals taken at different periods between 2018 and 2019 were also used to obtain comparative results. To investigate the presence of anti-CCHFV antibodies in sheep, all sera were tested using ELISA. Reactive sera were further characterised by a virus neutralisation test (VNT). Overall, one out of the 270 tested sheep was both ELISA- and strongly VNT-positive to CCHFV. Another two sheep were borderline ELISA-positive but did not exhibit neutralising antibodies. Ninety-one ticks were collected from all sampled sheep, of which 34 (37.4%) belonged to Hyalomma spp. This is the first report of anti-CCHFV antibodies in sheep from Tunisia. Both the results of this study and the recent CCHFV detection in ticks collected from camels in southern Tunisia indicate that further studies are needed to determine the competent tick vector in the country and to characterise the epidemiological cycle of CCHFV.

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

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.

Tick Infestation and Piroplasm Infection in Barbarine and Queue Fine de l'Ouest Autochthonous Sheep Breeds in Tunisia, North Africa.

As ticks and tick-borne pathogens affect the productivity of livestock, searching for genetically resistant breeds to infestation by ticks may represent an alternative to the overuse of chemical drugs. The aim of this study was to assess if there is a difference in tick infestation among the main sheep breeds in Tunisia. The study was carried out between April 2018 and January 2020 in 17 small to middle-sized sheep flocks from 3 regions across Tunisia. Four hundred and thirty-nine ear-tagged ewes from Barbarine (n = 288, 65.6%) and Queue Fine de l'Ouest (QFO) (n = 151, 34.4%) breeds were examined and sampled each trimester. Ticks were identified to the species level, and piroplasms were detected using PCR that targets a common sequence ARNr18S to both Babesia and Theileria genera using catch-all primers. Totally, 707 adult ticks were collected from animals; 91.4% (646/707) of them were Rhipicephalus sanguineus s.l. Queue Fine de l'Ouest animals were markedly less infested by ticks, and no one of them was infected by piroplasms compared to the Barbarine breed. Indeed, during the first four seasons, 21 animals, all from the Barbarine breed, were detected positive for piroplasms. This is the first study in Tunisia about the low susceptibility of QFO ewes to infestation by ticks and to infection by piroplasms. The QFO sheep breed could be raised preferably at high-risk areas of tick occurrence and could be considered in concrete control strategies, including a breeding program.

Epidemiological investigation of Crimean-Congo haemorrhagic fever virus infection among the one-humped camels (Camelus dromedarius) in southern Tunisia.

Crimean-Congo haemorrhagic fever is a viral tick-borne zoonotic disease caused by a Nairovirus, Crimean-Congo haemorrhagic fever virus (CCHFV). The present survey aimed to determine the exposure of one-humped camels (Camelus dromedarius) from southern Tunisia to CCHFV. A total of 273 sera from extensively reared camels were collected from Tataouine district, Tunisia, and tested by CCHFV-specific enzyme linked immunosorbent assays. By combining the results of three serological tests, the overall seroprevalence of CCHFV was estimated as 89.7% (245/273). No viral RNA was detected from camel sera using quantitative real-time PCR (RT-qPCR). A total of 165 ticks were collected from camels and tested with RT-qPCR, and only one Hyalomma impeltatum tick was positive for virus RNA.

Presence of antibodies against tick-borne encephalitis virus in sheep in Tunisia, North Africa.

BACKGROUND: Tick-borne encephalitis virus (TBEv) is a flavivirus that circulates in a complex cycle involving small mammals as amplifying hosts and ticks as vectors and reservoirs. The current study aimed to investigate the presence of TBEv in Tunisian sheep. A sample of 263 adult sheep were selected from 6 localities where Ixodes ricinus is well established. Sera were screened using ELISA for TBEv IgG detection, then the doubtful and positive sera were tested by the seroneutralisation test (SNT) and screened for West Nile Virus (WNv) IgG for cross-reaction assessment. RESULTS: The ELISA for TBEv IgG detected one positive serum and 17 borderlines. The SNT showed one positive serum among the 18 tested, giving an overall antibody prevalence of 0.38% (95% CI = 0.07-2.12%). All but one serum tested negative to WNv ELISA. None of the sheep farmers reported neurological signs among sheep or humans in their households. CONCLUSIONS: The results may indicate the circulation of TBEv for the first time in Tunisia and in North Africa. Further studies based on either virus isolation or RNA detection, are needed to confirm the presence of TBEv in North Africa.

Individual variability among autochthonous sheep in Northern Tunisia to infection by abomasum nematodes and Babesia/Theileria parasites.

In Tunisia, livestock plays an important role in the economy; unfortunately, Tunisian sheep population faces several health challenges. The aim of this trial was to study phenotypic variability of four local sheep breeds and strains to abomasum nematodes infection and to Babesia/Theileria parasites. Faeces, blood and abomasum contents were collected from 310 sheep slaughtered in eight commercial slaughterhouses across North Tunisia. Haematological and biochemical parameters were assessed. DNA was extracted and catch-all primers were used to detect both Theileria spp. and Babesia spp. DNA. Faecal egg counts (FEC) was quantitatively assessed using simple flotation technique followed by McMaster technique. Male and female worms were collected from all abomasum contents and counted under a stereomicroscope. The percentage of faeces samples positive for GIN's eggs was 30.82%. After worms' recovery, the infection prevalence was estimated to 75.90%. The overall infection prevalence by Babesia spp. and Theileria spp. was 4.21%. The dispersion of observations plots obtained by principal component analysis (PCA) showed two clusters of individuals. The first cluster contains animals having positive Babesia/Theileria PCR, presence of nematodes in the abomasum contents and relatively low total worm count (TWC < 500) expect one animal which was found bearing high TWC (>500). In this same group, with a suspected form of resistance, animals showed normal values of albumin and normal haematological parameters (red blood cell count [RBC], haemoglobin [Hb] and packed cell volume [PCV]). The second cluster represents all the other observations in which subgroups of animals were distinguished on the basis of their potential resistance to abomasum nematodes. Multiple correlations showed significant positive correlations between RBC/Hb, RBC/PCV, PCV/Hb and FEC/TWC. Significant negative correlations were observed between TWC/RBC and TWC/Hb. It is concluded that the phenotypic variability among local sheep breeds is essential for more advanced genetic and genomic studies.

Seroprevalence of anti-Mycobacterium avium subsp. paratuberculosis antibodies in female sheep in Tunisia.

Paratuberculosis (Ptb) is a widespread chronic infection caused by Mycobacterium avium subsp. paratuberculosis (Map) that affects both domestic and wild ruminants. Most of the studies focused on cattle while, the prevalence of Ptb in sheep in different regions of the world is not well investigated. This study aimed to address this gap of knowledge by screening adult female sheep for paratuberculosis antibodies in different geographical regions of Tunisia. A total number of 338 female sheep from 15 small to middle-sized, extensively managed sheep farms in six regions across Tunisia were sampled. Animals were clinically examined before blood sampling. Sera were tested for the presence of anti-Mycobacterium avium subsp. paratuberculosis antibodies using a commercial ELISA kit. Six farms out of 15 comprised at least one seropositive animal and 11 female sheep out of 338 tested animals (3.25%; 95% CI = [1.83-5.73]) were seropositive to M. avium subsp. paratuberculosis. The seroprevalence was significantly lower in 5-year-old females (p = .04) and animals that do not graze (p = .02). Due to its huge economic and social impacts, paratuberculosis represents a health problem in Tunisia and several other countries Further investigations are needed to rank sheep Ptb in Tunisia among other diseases and to assess the main risk factors using a larger nation-wide survey.

Molecular prevalence of Toxoplasma gondii DNA in goats' milk and seroprevalence in Northwest Tunisia.

Toxoplasmosis is a worldwide zoonosis with high impact on human and animal health. Consumption of unpasteurized milk is a risk factor of human toxoplasmosis. The aim of this study was to estimate the seroprevalence and molecular prevalence of T. gondii in goats' milk in Northwest of Tunisia (Jendouba Governorate). A total number of 77 blood samples were collected from six herds were screened with a commercial ELISA kit for T. gondii antibodies. For the same goats' samples, a nested PCR was performed to detect T. gondii DNA in milk. The seroprevalence of T. gondii infection was 31.2% (±0.05) while the molecular prevalence of this parasite in milk was estimated to 7.8% (±0.03). A very low value of kappa showed that there is not agreement between seroprevalence and parasite prevalence in milk. These results suggest that the consumption of raw milk from naturally infected goats is a potential source of human infection. An extension programme should be implemented to decrease related to goats' raw milk consumption.

Prevalence of piroplasms in small ruminants in North-West Tunisia and the first genetic characterisation of Babesia ovis in Africa.

In this study, the prevalence of piroplasms in sheep and goats was assessed with Giemsa-stained blood smear examination, PCR and nested PCR-restriction fragment length polymorphism (RFLP) to identify Babesia and Theileria species, respectively, in 338 small ruminants (172 sheep and 166 goats) from three sites in North-West Tunisia during the 2011 summer season. The overall infection prevalence of piroplasms in Giemsa-stained blood smears was 3.2% (11/338), with a parasitaemia ranging from 0.01 to 0.05%. PCR detected two species, namely Babesia ovis (in sheep and goats) and Theileria ovis (in sheep), with an overall prevalence of 16.3%. The molecular prevalence of B. ovis was significantly higher in sheep than in goats (17.4% and 9%, respectively, p = 0.034). The same trend was observed for T. ovis in sheep and goats (5.8% and 0%, respectively, p = 0.004). Comparison of the partial sequences of the 18S ssu rRNA gene revealed 100% similarity amongst Babesia from sheep and goats. The single Theileria sequence in this study showed 100% similarity to T. ovis. A high similarity with all the blasted genotypes was reported for Theileria and Babesia sequences. This is the first molecular detection of B. ovis and genetic characterisation of small ruminants' piroplasms in Africa.

Population dynamics of ticks infesting the one-humped camel (Camelus dromedarius) in central Tunisia.

A tick population was monitored on 30 camels (Camelus dromedarius) over one year in Kairouan region, Central Tunisia. A total of 1630 ticks was collected and identified resulting in an estimate of different parasitological indicators. The ticks belonged to 2 genera and 5 species: Hyalomma impeltatum (53%) and Hyalomma dromedarii (45%) were the dominant species followed by Hyalomma excavatum (1%), Hyalomma marginatum (0.5%), and Rhipicephalus turanicus (0.5%) (p<0.001). Mean infestation prevalence was 90.6%; all the animals were infested by at least one tick from May to September. The highest mean prevalence was observed in H. impeltatum (60%), the lowest was reported in R. turanicus (0.03%) (p<0.05). Mean overall intensity of infestation was 4.4 ticks/animal. The highest mean intensity was observed in H. impeltatum (2.7 ticks/animal). Overall mean abundance of ticks was 4.4 ticks/animal. Different abiotic factors, namely monthly mean minimum and monthly mean maximum temperatures and the number of sunny days were positively correlated with overall monthly tick burdens which were in turn negatively correlated with the monthly mean relative humidity. This is the first study on camel tick dynamics in Tunisia.

Hyalomma scupense (Acari, Ixodidae) in northeast Tunisia: seasonal population dynamics of nymphs and adults on field cattle.

Hyalomma scupense is a two-host tick infesting mainly cattle representing in North Africa the vector of tropical theileriosis (Theileria annulata infection), a major tick-borne disease affecting cattle. Any effective control programme of ticks requires a good knowledge of the biology of the target species. In the present study, three cattle farms in northeast Tunisia were surveyed during the activity seasons for adult and nymphs of Hyalomma scupense. Several indicators were studied, including chronological indicators, infestation prevalence, infestation intensity and feeding predilection sites of the ticks. The adult ticks were present from mid-June to late November. Nymphs were observed on animals from early September to late November. A large proportion of the ticks were attached in the posterior udder quarters: 41% and 64% of adult ticks and nymphs, respectively. The animals that were heavily infested by adult ticks were also heavily infested by nymphs. Moreover, 17% of adult ticks and 53% of nymphs were present on only 5% of cattle population. These data are important for the success of targeted acaricide application leading to a dramatic decrease of acaricide quantity needed for the treatment. When the preferential sites of attachment are known, the effectiveness of manual removal of ticks can be improved. The presence of highly infested animals is to be considered when any control programme is implemented, since these animals harbour a high proportion of the ticks.

Vaccination of calves with an attenuated cell line of Theileria annulata and the sporozoite antigen SPAG-1 produces a synergistic effect.

The sporozoite surface antigen, SPAG-1 and the attenuated schizont infected Tunisian line CL1 of Theileria annulata have been shown, in previous studies, to induce variable levels of protection against homologous and heterologous sporozoite challenge, respectively. We report here the result of a vaccination trial comparing the protection level induced by the SPAG-1 antigen (as a recombinant full length His tagged protein) and the attenuated cell line, used singly or in combination. The results, after challenge of immunised calves with a lethal dose of sporozoites, show that SPAG-1 provides limited protection (one out of seven calves surviving), while the attenuated cell line provides moderate protection (three out of seven calves recovered). The combination of SPAG-1 and the attenuated cell line induced the best protection as indicated by the survival of all the vaccinated calves. These results, together with a range of parasitological and clinical parameters, demonstrate the enhanced protection provided by combining sporozoite and schizont antigens in vaccination against tropical theileriosis.

Infection of calves with Theileria annulata in Tunisia: Economic analysis and evaluation of the potential benefit of vaccination.

A field study aiming to estimate the costs of tropical theileriosis was carried out in an endemic region of the North of Tunisia. Three farms were monitored for two successive summer seasons. A total number of 56 calves and 12 sentinel cows were monitored. Calves were ranked into four groups: non infected animals, diseased animals with clinical tropical theileriosis, sub-clinical Theileria annulata infected animals with anaemia and sub-clinical T. annulata infected animals without anaemia. The total costs due to disease and infection were estimated to be 15,115.058 TD (Euro 9388.20). A high proportion of these costs (50.81%) is accounted for by asymptomatic infection. Sub-clinical infections with anaemia showed the highest losses in live weight, while disease cases (prevalence 42.86%) are responsible for 23.64% of the losses with death as the most important element. A cost-benefit analysis of vaccination using a Tunisian attenuated cell line vaccine was also undertaken. Considering that the vaccine would cost 5 TD (Euro 3.10), the cost-benefit ratio of vaccination is 23.7. Sensitivity analysis of the vaccination costs shows that the indifference decision point is around 118 TD (Euro 73.29).