FIRST REPORT OF TRYPANOSOMA EVANSI INFECTION (SURRA) IN A PUMA (FELIS CONCOLOR) OF LAHORE ZOO, PAKISTAN.

The blood protozoan Trypanosoma evansi, which is transmitted by biting flies, is frequently neglected due to subclinical infections. This report describes a case of trypanosomiasis due to T. evansi in a 9-yr-old male puma (Felis concolor) housed at the Lahore Zoo in Pakistan. Early in January 2015, this male puma presented with chronic lethargy, weight loss, incoordination, hyperthermia, anorexia, sunken eyes, and unthriftiness. Microscopic examination of Giemsa-stained blood smears showed numerous Trypanosoma parasites. The puma was treated with diminazene aceturate subcutaneously twice. A few days later, a blood smear examination showed absence of trypanosomes. Five months later the cat presented with acute epistaxis and died. Postmortem examination showed emaciation, pale liver and kidneys, and hemorrhages on the spleen. Examination of a blood smear taken at the time of death showed numerous Trypanosoma parasites. PCR testing confirmed the presence of Trypanosoma DNA. DNA sequencing of two amplicons confirmed the presence of Trypanosoma in the blood smears with a 98-99% identity with the previously identified GenBank sequences. A phylogenetic tree was then constructed. Further studies are needed to improve our knowledge about the epidemiology and pathogenesis of T. evansi infection in wild animal species.

Molecular prevalence and genetic characterization of piroplasms in dogs from Tunisia.

In this study, the prevalence of piroplasms in dogs was assessed using polymerase chain reaction (PCR) to identify Babesia and Theileria species in 200 dogs from Northern and Central Tunisia between spring and autumn 2014. The overall molecular prevalence for piroplasms was 14·5% ± 0·05 (29/200); PCR detected 2 species, namely Babesia vogeli and Theileria annulata with an overall prevalence of 12·5 ± 0·04 and 2% ± 0·02, respectively. No differences in the molecular prevalences of B. vogeli were revealed for age and sex (P > 0·05). The molecular prevalence of B. vogeli was significantly higher in central Tunisia (26·5% ± 0·01) compared with the North (9·6% ± 0·04) (P 0·05). Comparison of the partial sequences of 18S rRNA and Tams 1 genes confirmed the presence of 2 novel B. vogeli and T. annulata genotypes. This is the first molecular detection of T. annulata and genetic characterization of dogs' piroplasms in Tunisia. Further studies are needed to better assess the epidemiological feature of piroplasms infection in North Africa.

High Toxoplasma gondii frequency of infection in the genital tract of ewes previously positive for the parasite DNA in meat, in Sidi Bouzid, Tunisia.

The aim of the present study was to estimate the molecular prevalence of T. gondii along various segments of the genital tract of confirmed chronically infected ewes. Genital tracts were collected from 42 ewes; meat samples from the same ewes were previously confirmed positive for T. gondii DNA. The whole DNA was extracted from 4 parts of the genital tract (ovary, horns, body of the uterus and vagina). PCR was used to amplify a 114 base-pairs of T. gondii B1 gene. For all studied samples, 95.2 % had at least one infected genital part. Toxoplasma gondii infection was confirmed by sequencing 20 amplicons randomly chosen. The majority of infected animals has 4 T. gondii-infected genital anatomical parts. Principal component analysis (PCA) revealed a cluster of adult animals (>24 months) with positive PCR in the ovaries and the vagina and another cluster of Barbarine animals having positive PCR in the horns and body of the uterus. General linear model confirmed PCA results and showed a significant higher prevalence of T. gondii in the ovaries and vagina of older animals (p = 0.001 and p = 0.02, respectively) and a higher prevalence of T. gondii in the horns of the uterus of Barbarine animals (p = 0.03). Toxoplasma gondii seems to highly persist along the various segments of the ewe's genital tract but further investigations are necessary to link such prevalence with the pathological implications.

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.

Increased number of large non-atretic follicles and co-dominance effects account for high litter sizes in Bonga sheep.

To understand the ovarian basis for prolificacy of Bonga sheep, a total of 31 ewes were selected based on litter size (LS) records and divided into two groups: High Prolificacy (HP) (n = 20) with LS ≥ 2 and Low Prolificacy (LP) (n = 11) with LS = 1. At a synchronized estrus, follicular dynamics were determined using transrectal ultrasonography. Plasma estradiol concentrations were also monitored. In total 27 ewes were observed in estrus being 9/11 LP (82%) and 18/20 HP (90%). On the day of estrus (day 0), the mean number of large follicles was higher (p < .05) in HP (1.78 ± 0.19) than in LP (1.0 ± 0.28) ewes. Prior to estrus, more (p < .05) medium follicles were visible for HP compared to LP ewes. Plasma estradiol concentrations were higher in HP compared to LP ewes (18.91 ± 0.41 vs. 14.51 ± 0.65 pg/ml; p < .05) and similarly was ovulation number (2.3 ± 0.15 vs. 1.28 ± 0. 14; p < .05). Higher ovulation rates and litter size in Bonga sheep are evidenced by the previous presence of more large follicles and the existence of co-dominance effects as most likely medium follicles are selected to ovulate.

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.

Neospora caninum Natural Infection in Tunisian Rams: Serological Study and Molecular Identification of Infection in Semen.

PURPOSE: The aim of this study was to estimate the seroprevalence and molecular prevalence and perform a molecular identification of Neospora caninum in semen of Tunisian rams. METHODS: A total of 92 blood samples were collected from four farms located in four Tunisian governorates (Jendouba, Kairouan, Zaghouan and Ben Arous) and samples were screened with a commercial ELISA kit for N. caninum antibodies. For the same rams, semen samples were collected and tested for the presence of N. caninum ITS1 gene using PCR. Five amplicons were randomly selected for sequencing. A phylogenetic tree was constructed to compare the partial sequences of the ITS1 gene with sequences deposited in GenBank. RESULTS: The seroprevalence of N. caninum infection was 25% (23/92) and PCR revealed that the molecular infection prevalence in semen was 11.95% (11/92). Kappa test showed an average agreement between seroprevalence and parasite prevalence in semen (κ = 0.44). The highest molecular prevalence was for rams that accomplished more than two mating seasons (21.0 ± 12.1%) compared to those performed less than two mating seasons and yearling individuals (4.0 ± 5.5%) (P = 0.01). There were no differences in N. caninum molecular prevalence according to either breed or locality. Comparison of the partial sequences of the ITS1 gene revealed 99-100% similarity with those deposited in GenBank. CONCLUSION: To the best of our knowledge, this is the first detection and molecular identification of N. caninum in semen from rams in North Africa. Our findings indicate that N. caninum infection rate was high in rams.

Detection of Toxoplasma gondii infection in semen of rams used for natural mating in commercial sheep farms in Tunisia.

Toxoplasmosis, a parasitic infection, disrupts sexual functions resulting in decline in reproductive and economic performance in sheep. Lacking discernible symptoms, toxoplasmosis is difficult to detect and diagnose in infected animals. Here, to estimate the prevalence of natural infection by Toxoplasma gondii, we used PCR to amplify and detect T. gondii DNA in semen from 92 rams of three breeds from four regions in Tunisia and seroprevalence was determined using a commercial ELISA kit. From the PCR amplifications, 51.09 ±â€¯10.21% of the rams were tested positive for T. gondii with an overall seroprevalence of 39.13 ±â€¯9.97%. Risk factors including ram location and number of accomplished mating seasons significantly (p < .05) affected the sero- and molecular prevalence of T. gondii in semen but, there was a fair concordance between sero- and molecular prevalence (Kappa = 0.33). Sequences of T. gondii from five positive samples were 100% identical (same haplotype). Comparison of these sequences with those archived at the GenBank showed a sequence similarity range between 95 and 100%. The haplotype defining the five Tunisian sequences was similar to the one observed in chicken, cats, European pole cat and humans from Brazil, St Kitts and Nevis, Great Britain and Tunisia, respectively. This indicates its wide geographic distribution and non-species specificity. Our findings suggest a high prevalence of toxoplasmosis in Tunisian matting rams; further studies concerning its venereal transmission capacity are needed prior to recommending a systematic screening of T. gondii DNA in rams' semen used for both natural breeding and artificial insemination.

Toxoplasma gondii infection and toxoplasmosis in North Africa: a review.

Toxoplasmosis is an important zoonosis caused by an obligate intracellular parasitic protozoan, Toxoplasma gondii. The disease is distributed worldwide and can affect all warm-blooded vertebrates, including humans. The present review aimed to collect, compile and summarize the data on the prevalence of T. gondii infection in humans and animals in the five North African countries (Morocco, Algeria, Tunisia, Libya and Egypt). Published data from national and international databases were used. Distribution patterns and risk factors for T. gondii infection are discussed, focusing on biotic and abiotic factors. This review is a comprehensive epidemiological analysis of T. gondii infection in North Africa and will therefore be a useful tool for researchers. It can also be used to propose or enhance appropriate national toxoplasmosis control programs.

Molecular prevalence and phylogenetic analysis of Theileria annulata and Trypanosoma evansi in cattle in Northern Tunisia.

The present study aimed to estimate the molecular prevalence of Theileria annulata and Trypanosoma evansi infection in cattle in Northern Tunisia. A total number of 96 cattle from five farms were evaluated. T. annulata and T. evansi prevalences were 61% [56/66] and 10% [7/13], respectively, at a confidence interval (CI) of 95%, while co-infection was present in 6% [4/8] of the tested animals at a CI of 95%. There was a significant correlation between age and the prevalence of T. annulata infection, whereas, there was no significant association shown with the age of cattle and T. evansi infection. Sequence and phylogenetic analyses showed that the T. annulata Tams1 gene and T. evansi ITS1 rDNA gene were highly conserved with 97.1-100% and 98.3-100% sequence identity, respectively.

Molecular detection of Toxoplasma gondii infection in slaughtered ruminants (sheep, goats and cattle) in Northwest Tunisia.

The present study aimed to estimate the molecular prevalence of T. gondii infection in meat from slaughtered sheep, goats and cattle in Northwest Tunisia (Béja district). PCRs were performed on genomic DNA extracted from 420 meat samples (150 ewes, 120 goats and 150 cows). The overall molecular prevalence of T. gondii in sheep, goats and cattle were 33.3 (50/150), 32.5 (39/120) and 19.3% (29/150), respectively. Toxoplasma gondii molecular prevalences in the three meat ruminant species were significantly higher in adults compared to young animals (p<0.001). The infection prevalence differed significantly within localities in sheep (p<0.001), goats (p<0.001) and cattle (p=0.019). These results provide important information about exposure of humans to T. gondii through the consumption of raw or undercooked ruminants' meat. Extension programmes should be implemented to decrease the risk of infection related to sheep, goats and cattle meat manipulation and raw or undercooked meat consumption.

Molecular detection and phylogenetic analyses of Toxoplasma gondii from naturally infected sheep in Northern and Central Tunisia.

Toxoplasmosis is a worldwide zoonosis caused by the parasitic protozoan Toxoplasma gondii. It can infect all warm-blooded vertebrate species and causes abortions and birth defects in pregnant women and pregnant ewes. The objective of this study was to estimate the prevalence of infection with T. gondii in sheep meat in the region of Sidi Bouzid (central Tunisia) and Beja (northern Tunisia), the realization of a descriptive study of risk factors and the phylogenetic analyses of T. gondii. Neck muscle samples were obtained from 174 ewes and ewe lamb slaughtered in Sidi Bouzid and 150 lambs slaughtered in Beja. DNA was extracted from the samples using the Wizard® genomic DNA purification kit. A nested PCR using two pairs of primers (NN 1 and NN2, Tg-NP1 and Tg-NP2) were used to detect infection with T. gondii, which was then confirmed by sequencing. Eight T. gondii amplicons were sequenced (accession number KT896498) and deposited in GenBank. The T. gondii amplicons showed 97-100% identities with GenBank sequences. A phylogenetic tree was then constructed. The nested PCR detected T. gondii DNA in 31% of animals tested in Sidi Bouzid and 32% of lambs tested in Beja. No significant difference in the prevalence of T. gondii infection was established between the two tested regions. In both regions, no significant variation of the infection depending on age, breed and locality was found.

First molecular detection and characterization of Sarcocystis species in slaughtered cattle in North-West Tunisia.

Sarcocystis spp. is one of the most prevalent foodborne parasites infecting both animals and humans. Consumption of raw or undercooked infected meat is a risk factor of human intestinal sarcocystosis. The present study aimed to estimate the prevalence of Sarcocystis species infecting slaughtered Tunisian cattle in North-West Tunisia (Béja governorate). DNA was extracted from 150 beef meat samples and a PCR-restriction fragment length polymorphism was used for identification. The overall infection prevalence of Sarcocystis spp. was 38% (57/150). Two species were identified, namely S. hominis (25%; 39/150) and S. cruzi (12%; 18/150). For both species, the highest prevalence was in Thibar locality (52.9 and 17.6% for S. hominis and S. cruzi, respectively). The molecular prevalence of S. cruzi was significantly higher in animals aged between two and eight years (19.2%; 10/52). This is the first molecular identification of Sarcocystis species in Tunisian cattle. Further studies in both human and animal Tunisian populations are needed to rank this parasitic disease among others.

Effect of the infection with the nematode Haemonchus contortus (Strongylida: Trichostrongylidae) on the haematological, biochemical, clinical and reproductive traits in rams.

This study aimed to investigate the effect of Haemonchus contortus infection on rams' haematological, biochemical and clinical parameters and reproductive performances. A total number of 12 Barbarine rams (control and infected) were included in the experiment. The infected group received 30 000 H. contortus third-stage larvae orally. Each ram's ejaculate was immediately evaluated for volume, sperm cell concentration and mortality rate. At the end of the experiment (day 82 post-infection), which lasted 89 days, serial blood samples were collected in order to assess plasma testosterone and luteinising hormone (LH) concentrations. There was an effect of time, infection and their interaction on haematological parameters (p < 0.001). In infected rams, haematocrit, red blood cell count and haemoglobin started to decrease from 21 days post-infection. There was an effect of time and infection for albumin. For total protein, only infection had a statistically significant effect. For glucose, only time had a statistically significant effect. Concentrations were significantly lower in infected rams compared to control animals. A significant effect of infection and time on sperm concentrations and sperm mortality was observed. The effect of infection appears in time for sperm concentrations at days 69 and 76 post-infection. Sperm mortality rate was significantly higher in infected animals at day 46 post-infection when compared to control group (p < 0.05). Finally, plasma testosterone traits (average concentration, cumulated levels during the sampling period and pulse frequency) were depressed in infected rams when compared to control counterparts; none of these endocrine traits were affected for plasma LH.

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.

Infestation of the spur-thighed tortoise (Testudo graeca) by Hyalomma aegyptium in Tunisia.

We examined 210 spur-thighed tortoises (Testudo graeca) for the presence of ticks in Tunisia during May 2014. A total number of 602 adult ticks were collected and identified leading to the estimation of parasitological indicators. All the ticks belonged to a single species: Hyalomma aegyptium. The mean infestation prevalence was 66.2%, mean overall infestation intensity and abundance were 4.33 and 2.86 ticks/tortoise respectively. Our survey showed that tortoises were significantly more infested by male ticks than females (p<0.001). The ticks were mainly present in the posterior limbs compared to other body regions (p<0.05). There was no significance variation of length and weight of tortoises according to sex (p<0.05). There was a significant correlation between the tortoises' size (length and weight) and tick infestation. This study showed high tick burdens of spur-thighed tortoises in Tunisia; further investigations are needed to determine exactly the role of this tick species in the transmission of different zoonotic pathogens.