Isolation and propagation of an Egyptian Theileria annulata infected cell line and evaluation of its use as a vaccine to protect cattle against field challenge.

Tropical theileriosis is an important protozoan tick-borne disease in cattle. Vaccination using attenuated schizont-infected cell lines is one of the methods used for controlling the disease. This study describes the production of attenuated schizont-infected cell lines from Egypt and an evaluation of its use as a vaccine to protect calves against clinical disease upon field challenge. Two groups of exotic and crossbred male calves were divided into vaccinated and control groups. The vaccinated groups were inoculated with 4 ml (1 × 106 cells/ml) of the attenuated cell line. Three weeks after vaccination, calves of both groups were transported to the New Valley Governorate (Egyptian oasis) where they were kept under field conditions and exposed to the natural Theileria annulata challenge. All animals in the control group showed severe clinical signs and died despite treatment with buparvaquone, which was administered after two days of persistent fever due to a severe drop in packed cell volume (PCV). Animals in the vaccinated group became seropositive without developing severe clinical signs other than transient fever. Post-mortem examinations revealed enlarged and fragile lymph nodes, spleen, and liver with necrosis and hemorrhages. These findings indicate that the Egyptian attenuated cell line was successful in protecting both exotic and crossbred animals against tropical theileriosis under field conditions.

Excretion Dynamics of Arboviruses in Mosquitoes and the Potential Use in Vector Competence Studies and Arbovirus Surveillance.

The increasing threat of arboviruses such as West Nile virus (WNV) and Usutu virus (USUV) requires the fast and efficient surveillance of these viruses. The examination of mosquitoes takes up an important part; however, these investigations are usually very time-consuming. An alternative sample type for arbovirus surveillance might be mosquito excreta. In order to determine the excretion dynamics under laboratory conditions, laboratory colonies of Aedes vexans and Culex pipiens biotype molestus were infected with WNV, USUV or tick-borne encephalitis virus (TBEV). After infection, the excreta were sampled and investigated for viral RNA. Excretion of viral RNA together with infectious blood meal could be detected up to five days after infection. Further excretion seemed to correlate with a disseminated infection in mosquitoes, at least after USUV infection. In addition, it could be determined that the amount of viral RNA in the excretions correlated positively with the viral load in the mosquito bodies. Overall, this study shows that the usage of mosquito excreta as a sample type for surveillance enables the detection of endemic viruses (WNV, USUV) as well as non-mosquito-borne viruses (TBEV). In addition, examination of viral shedding during vector competence studies can provide insights into the course of infection without sacrificing animals.

Vector Competence of German Aedes punctor (Kirby, 1837) for West Nile Virus Lineages 1 and 2.

West Nile virus (WNV) is a zoonotic flavivirus transmitted by mosquitoes as a biological vector. Because of its biting behavior, the widespread snow-melt mosquito Aedes punctor could be a potential bridge vector for WNV to humans and nonhuman mammals. However, little is known on its role in transmission of WNV. The aim of this study was to determine the vector competence of German Ae. punctor for WNV lineages 1 and 2. Field-collected larvae and pupae were reared to adults and offered infectious blood containing either an Italian WNV lineage 1 or a German WNV lineage 2 strain via cotton stick feeding. Engorged females were incubated for 14/15 or 21 days at 18 °C. After incubation; surviving mosquitoes were dissected and forced to salivate. Mosquito bodies with abdomens, thoraces and heads, legs plus wings and saliva samples were investigated for WNV RNA by RT-qPCR. Altogether, 2/70 (2.86%) and 5/85 (5.88%) mosquito bodies were found infected with WNV lineage 1 or 2, respectively. In two mosquitoes, viral RNA was also detected in legs and wings. No saliva sample contained viral RNA. Based on these results, we conclude that Ae. punctor does not play an important role in WNV transmission in Germany.

Phylogenetic study of Theileria ovis and Theileria lestoquardi in sheep from Egypt: Molecular evidence and genetic characterization.

BACKGROUND AND AIM: Ovine theileriosis caused by Theileria ovis and Theileria lestoquardi is an important infectious disease affecting small ruminants in regions of the tropic and subtropic zones. There is limited studies about ovine theileriosis in Egypt; so the present study aims to assess the occurrence of ovine theileriosis in Egypt at the molecular level. MATERIALS AND METHODS: Blood samples were collected from 115 randomly selected sheep, which were apparently healthy; the ages of the sampled sheep ranged from 1 to 5 years old, from a local breed (barkae and balade), and showed no symptoms indicating infection with Theileria spp. The study was conducted in three governorates representing Lower Egypt (Menoufia and Beheira) and Upper Egypt (El-Wady El-Geded). All blood samples were subjected to polymerase chain reaction (PCR) and semi-nested PCR to target Theileria spp. 18S rRNA genes. Positive samples were sequenced, and these sequences were analyzed using nucleotidebasic local alignment search tool (BLAST). RESULTS: Six animals (5.22%) were PCR-positive carriers for ovine theileriosis. Nucleotide BLAST and phylogenetic analyses of the six obtained sequences showed that T. ovis was present in five animals (4.37%) in Menoufia (n=2) and El-Wady El-Geded (n=3), whereas T. lestoquardi was detected in 1 animal (0.87%) in El-Wady El-Geded. CONCLUSION: This study is the first to provide molecular evidence, genetic characterization, and phylogenetic analysis of ovine Theileria spp. in Egypt. Specifically, T. lestoquardi and T. ovis carrier statuses of sheep were confirmed. These results highlight the importance of developing an effective control strategy against ovine theileriosis carriers that might develop and/or spread theileriosis.

Tick species identification and molecular detection of tick-borne pathogens in blood and ticks collected from cattle in Egypt.

To address the lack of information on ticks infesting cattle in Egypt and the pathogens that they transmit, the current study aimed to (i) provide insight into tick species found on cattle in Egypt, (ii) identify the pathogens in ticks and their cattle hosts and (iii) detect pathogen associations in ticks and cattle. Tick samples and blood from their bovine hosts were collected from three different areas in Egypt (EL-Faiyum Oasis, Assiut Governorate and EL-Kharga Oasis). Tick species were identified by morphology and by sequence analysis of the cytochrome C oxidase subunit 1 (cox1) gene. Tick pools and blood samples from cattle were screened by the Reverse Line Blot hybridization (RLB) assay for the simultaneous detection of tick-borne pathogens, including Babesia, Theileria, Anaplasma, Ehrlichia, and Rickettsia spp., as well as the tick endosymbiont Midichloria mitochondrii. The RLB results were confirmed with specific conventional and semi-nested PCRs followed by sequencing. In total, 570 ticks (males, females and nymphs) were collected from 41 heads of cattle. Altogether 398 ticks belonged to the genus Hyalomma (397 Hyalomma excavatum and one Hyalomma scupense) while 172 ticks were identified as Rhipicephalus annulatus. Pooled H. excavatum ticks tested positive for several protozoa and bacteria with different minimum infection rates (MIRs): Theileria annulata (18.1 %), Babesia occultans (1.8 %), Anaplasma marginale (28.5 %), Anaplasma platys (0.25 %), Midichloria mitochondrii (11.6 %), Ehrlichia chaffeensis-like (1.8 %) and Ehrlichia minasensis (1 %). In R. annulatus, several agents were identified at different MIRs: T. annulata (2.3 %), B. bovis (0.6 %), A. marginale (18.0 %), A. platys (1.2 %), M. mitochondrii (2.9 %), E. minasensis (0.6 %). Pathogens co-detection in tick pools revealed A. marginale and T. annulata in 13.3 % samples followed by the co-detection of A. marginale and M. mitochondrii (8.4 %). In addition, triple co-detection with A. marginale, T. annulata and M. mitochondrii were found in 5.3 % of the tick pools. In cattle, the most common coinfection was with A. marginale and T. annulata (82.9 %) followed by the coinfection between A. marginale, T. annulata and B. bovis (4.9 %), A. marginale and B. bigemina (2.4 %) and finally the coinfection between T. annulata and B. occultans (2.4 %). Anaplasma platys, Babesia occultans, and E. minasensis were detected for the first time in Egypt in both cattle and ticks. These findings should be taken in consideration regarding human and animal wellbeing by the public health and veterinary authorities in Egypt.

Knowledge and perception on ticks and tick-borne diseases among veterinary medicine students from the North African countries of Algeria, Egypt, and Tunisia.

Ticks are important vectors of both animal and human pathogens. The epidemiology of tick-borne diseases (TBDs) has dramatically changed in several regions in the world. As parasitology is a continuously growing field, assessing the knowledge of veterinary medicine students provides useful indicators and information on the level of intervention required to adapt parasitological courses to meet the demands in a changing world. This study aimed to assess, in three North African veterinary education establishments, the basic parasitology knowledge of veterinary medicine students. Such a study is essential to build up core competencies regarding ticks and TBDs, and to suggest suitable adjustment measures to parasitology courses. The present study was based on a self-administered and anonymous questionnaire on ticks and TBDs basic knowledge and perception. The survey was completed by 558 veterinary medicine students in Algeria, Egypt, and Tunisia in 2018. The students were divided into two groups: the "before" group - students who had not yet completed the parasitology course, and the "after" group - students who had already completed it. In all studied countries, the "after" students' group gave significantly more correct answers (83.16%) than the "before" students' group (16.84%) (p < 0.001). Similarly, the percentage of "no answer" was higher in the "before" students' group (51.02%) compared to the "after" students' group (48.98%) (p < 0.001). The most frequent false answers provided by the "after" students' group regarded the number of tick species present in their own countries (5.14% of correct answers), and the most common tick species in their countries (18.11% of correct answers). Almost 58.38% (216/370) of the "after" students' group knew that ticks transmit zoonotic pathogens to humans; among them, only 63 (29.17%) gave the correct names of the zoonotic diseases in their country. Among the three countries, more than 80% of the "after" students' group thought that climate has an influences on ticks. According to this group, the most frequent factor that has influences on ticks' abundance is heat (53.02%). As North African countries share several similitudes, we suggest creating a network of parasitological teachers where common teaching sources and resources could be developed for both teachers and students in the region. This network would allow the exchange of teaching approaches and materials to introduce harmonization into veterinary parasitological courses across North African countries. This is particularly important when considering the increasing incidence of ticks and TBDs in the region.

Epidemiology and genotyping of Anaplasma marginale and co-infection with piroplasms and other Anaplasmataceae in cattle and buffaloes from Egypt.

BACKGROUND: Anaplasma marginale is an obligate intracellular bacterium and the main cause of bovine anaplasmosis in tropical and subtropical regions. In Egypt, data regarding the prevalence of A. marginale in ruminant hosts and of the circulating genotypes is lacking. This study therefore aimed to (i) investigate the presence, epidemiology and genotypes of A. marginale in cattle and buffaloes in Egypt, (ii) to evaluate suitable diagnostic tools and (iii) to identify co-infections of A. marginale with other selected tick-borne pathogens. METHODS: Blood samples were collected from 394 animals (309 cattle and 85 buffaloes) from three different areas in Egypt. For the detection of A. marginale infection, several tests were compared for their sensitivity and specificity: blood smear analysis, enzyme-linked immunosorbent assay (ELISA), PCR, real-time PCR and reverse line blot (RLB) assay. Co-infections with A. marginale, piroplasms and other Anaplasmataceae were surveyed by RLB while A. marginale genotypes were identified by amplifying and sequencing the partial msp1α gene. RESULTS: Anaplasma marginale DNA was amplified by qPCR in 68.3% of cattle and 29.4% of buffaloes. RLB showed infection with A. marginale in 50.2% of cattle and 42.5% of buffaloes. Blood smear analysis detected this agent in 16.2% of cattle and 2.4% of buffaloes. ELISA showed specific antibodies against A. marginale in 54.9% of cattle. Anaplasma marginale was associated, in cattle and buffaloes, with several tick-borne pathogens (Theileria annulata, Babesia bovis, Babesia bigemina, Babesia occultans and Anaplasma platys). A significant difference of A. marginale infection level was noticed in cattle, where animals between 3-5-years-old had a higher prevalence (79.2%) compared to those older than 5 years (36.4%) and younger than 3 years (59.7%) and one year (64.5%), respectively (P = 0.002281). Microsatellite analysis identified 15 different genotypes. CONCLUSIONS: The epidemiological findings revealed high prevalence of A. marginale in cattle and buffaloes in all the investigated areas. The circulation of diverse genotypes was observed, most of these A. marginale genotypes being specific for Egypt. The qPCR assay was confirmed to be the most sensitive tool for detection of A. marginale in cattle and buffaloes even in the carrier state, highlighting the importance of using suitable diagnostic tests.

Prevalence of enterotoxins and other virulence genes of Staphylococcus aureus caused subclinical mastitis in dairy cows.

BACKGROUND AND AIM: Milk production is one of the main props for the national economy. One of the crucial problems in this industry is subclinical mastitis, which harms this industry that considered the backbone of the economy. It is an infectious and zoonotic disease; the infection can spread between dairy animals through milkers' hands, and milking machines, while the human infection occurs due to the consumption of apparently hygienic milk. Staphylococcus aureus is one of the main causative agents of clinical and subclinical mastitis. It is also considered one of the bacteria incriminated in food intoxication of humans due to its virulence factors as enterotoxins and toxic shock syndrome. The current study was designed to assess the prevalence of S. aureus and its enterotoxins, as well as, its other virulence factors in milk collected from cows that suffer from subclinical mastitis. MATERIALS AND METHODS: Sixty cows were collected from different dairy farms located in Assiut Governorate, Egypt. These cows were subjected to the clinical examination of the udder and its lymph nodes before sampling. Milk samples were collected from clinically healthy udders. All the milk samples were examined by California mastitis test (CMT), polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA) for confirmation subclinical mastitis, presence of S. aureus and its enterotoxins genes and other virulence factors in the examined milk samples. RESULTS: The cows included in the current study had healthy udders. The sixty collected milk samples were tested by CMT. 48/60 (80.0%) were positive samples; from the 48 positive samples, 46 (95.83%) samples were confirmed positive by S. aureus 16s rRNA PCR assay. Multiplex PCRs confirmed the presence of staphylococcus enterotoxin gene C (sec) in one sample, staphylococcus enterotoxin gene D (sed) in 23 samples, while ELISA assay confirmed the presence of the same enterotoxin in only two samples. On the other hand, other groups of genes responsible for some other virulence factors of S. aureus like the extracellular thermostable nuclease (nuc) gene were found in 33 samples, while toxic shock syndrome (tsst) gene and methicillin restraint S. aureus (mecA) gene were not detected in this study. CONCLUSION: Subclinical mastitis is one of the hidden factors that adversely affect the health of both animals and humans. The milk is usually appeared good and may be consumed by humans especially children; however, it causes severe public health problems. In addition, the infected animals with this form of mastitis can spread the infection to other dairy animals and may be turned to a clinical case of contagious mastitis that may be ended by animal culling or death. S. aureus is one of the main causes of subclinical mastitis in cattle. In addition to extracellular thermostable nuclease (nuc) gene, staphylococcus enterotoxin gene C (sec) and staphylococcus enterotoxin gene D (sed) are the most common virulence genes confirmed in subclinical mastitis milk. These results highlighted the need to apply more hygienic measures in the dairy farms to avoid spreading the infection between animals to ensure the production of safe and healthy food to humans.

Current status of tropical theileriosis in Northern Africa: A review of recent epidemiological investigations and implications for control.

Tropical theileriosis caused by the apicomplexan hemoparasite Theileria annulata is a tick-borne disease that constraints livestock production in parts of Europe, Asia and Africa. Four Hyalomma tick species transmit T. annulata in at least eight Africa countries (Mauritania, Morocco, Algeria, Tunisia, Egypt, Sudan, South Sudan and Ethiopia). The two dominant T. annulata vector ticks present in Africa, H. scupense and H. anatolicum, underlie two different patterns of transmission, which in turn greatly influence the epidemiology of tropical theileriosis. H. dromedarii and H. lusitanicum are also capable of transmitting T. annulata in North Africa, but their roles are associated with specific production systems and agro-ecological contexts. The emergence of resistance to the most widely used theilericidal compound, buparvaquone, continues to limit the effectiveness of chemotherapy. In addition, acaricide use is increasingly becoming unsustainable. Deployable T. annulata attenuated live vaccines established from local strains in Tunisia, Sudan and Egypt are available, and recent work has indicated that these vaccines can be protective under conditions of natural transmission. However, vaccination programmes may vary over space and time due to differences in the prevalence of disease amongst cattle populations, as well seasonal variation in vector activity. We review recent descriptive and analytical surveys on the epidemiology of T. annulata infection with reference to (a) demographic aspects such as breeds and ages of cattle herds previously exposed to distinct T. annulata infection pressures and (b) seasonal dynamics of tick activity and disease transmission. We then discuss how the wider endemic patterns that we delineate can underpin the development and execution of future vaccination programmes. We also outline options for integrated control measures targeting tick vectors and husbandry practices.

Serum-free in vitro cultivation of Theileria annulata and Theileria parva schizont-infected lymphocytes.

Theileriosis is a tick-borne disease caused by intracellular protozoa of the genus Theileria. The most important species in cattle are Theileria annulata and Theileria parva. Both species transform leucocyte host cells, resulting in their uncontrolled proliferation and immortalization. Vaccination with attenuated T. annulata-infected cell lines is currently the only practical means of inducing immunity in cattle. Culture media for Theileria spp. typically contain 10%-20% foetal bovine serum (FBS). The use of FBS is associated with several disadvantages, such as batch-to-batch variation, safety and ethical concerns. In this study, the suitability of serum-free media for the cultivation of Theileria-transformed cell lines was examined. Three commercial serum-free media (HL-1, ISF-1 and Hybridomed DIF 1000) were evaluated for their ability to support growth of the T. annulata A288 cell line. The generation doubling times were recorded for each medium and compared with those obtained with conventional FBS-containing RPMI-1640 medium. ISF-1 gave the shortest generation doubling time, averaging 35.4 ± 2.8 hr, significantly shorter than the 52.2 ± 14.9 hr recorded for the conventional medium (p = .0011). ISF-1 was subsequently tested with additional T. annulata strains. The doubling time of a Moroccan strain was significantly increased (65.4 ± 15.9 hr) compared with the control (47.7 ± 7.5 hr, p = .0004), whereas an Egyptian strain grew significantly faster in ISF-1 medium (43.4 ± 6.5 hr vs. 89.3 ± 24.8 hr, p = .0001). The latter strain also showed an improved generation doubling time of 73.7 ± 21.9 hr in an animal origin-free, serum-free, protein-free medium (PFHM II) compared with the control. Out of four South African T. parva strains and a Theileria strain isolated from roan antelope (Hippotragus equinus), only one T. parva strain could be propagated in ISF-1 medium. The use of serum-free medium may thus be suitable for some Theileria cell cultures and needs to be evaluated on a case-by-case basis. The relevance of Theileria cultivation in serum-free media for applications such as vaccine development requires further examination.

Phylogenetic analysis of Salmonella species isolated from cows, buffaloes, and humans based on gyrB gene sequences.

This study aimed to investigate the role of dairy cows and buffaloes as reservoirs of nontyphoidal salmonelloses (NTS), to reveal the occurrence of NTS among dairy workers and children with acute diarrhea and to study the gyrB gene phylogenetic relations of the obtained Salmonella strains, 300 samples were chosen randomly from clinically infected animals, including 100 feces and 50 raw milk from buffaloes and cows. Five hundred samples were chosen randomly from healthy animals, including 150 feces and 100 raw milk from buffaloes and cows. A total of 160 stool samples were randomly chosen from healthy workers (60) and children with acute diarrhea (100). Salmonella species were isolated from the examined samples and identified by polymerase chain reaction. Sequencing and phylogenetic analyses of gyrB gene were also performed. S. enteritidis and S.typhimurium were isolated from 0.5% (2/400) of the cows and buffaloes, respectively. Dairy workers were found to be at greater risk of exposure to Salmonella infection (5%) than children (1%). S. enteritidis was isolated from 1.7% (1/60) of dairy workers. S. typhimurium was isolated from 3.33% (2/60) and 1% (1/100) of dairy workers and children, respectively. Phylogenetic analysis of Salmonella species gyrB gene sequences from both animals and humans falls inside one clade, and all of them were closely related to each other with less significant genetic distance (99.9:100). In conclusion, cows and buffaloes act as reservoirs of Salmonella infection in dairy farms in Egypt and contribute a risk of zoonotic transmission to human.

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.

Co-infection with different serotypes of FMDV in vaccinated cattle in Southern Egypt.

During 2015-2016 period, an outbreak of foot-and-mouth disease virus (FMDV) was observed in cattle in four governorates of the upper of Egypt. The infection was extended to the vaccinated cattle. A total of 54 mouth swabs and serum samples were collected from vaccinated cattle for serological and virological investigation. The typical clinical signs of FMDV infection were observed in all cattle under investigation. All samples were positive for FMDV using molecular methods, while the serological method showed 85% positive of tested samples. Typing of FMDV-positive samples using serotype-specific primers showed that 51.8% of samples were serotype O, 9.2% were serotype A, and 18.5% were SAT 2. Surprisingly, co-infections of serotypes A/SAT 2 (12.9%) and O/SAT 2 (7.4%) were also detected. By geographical location, the 3 serotypes A, O, and SAT2 were detected in all four governorates. The phylogenetic assessment of the detected viruses showed that two distinct groups of FMDV serotype O of East Africa-3 (EA-3) topotype were most closely related to circulating viruses in Sudan, as well as FMDV strains belonging to the topotype VII of serotype SAT 2. The detected SAT 2 strains clustered in separate clades in topotype VII, indicating new incursions. The VP1 signatures and protein sequences of some characterized viruses were analyzed. Multiple mutations were detected in VP1. Therefore, to enhance the control of FMD in Egypt, we recommend establishing an active surveillance system to characterize newly emerging virus strains/serotypes and subsequently updating vaccine strains.

Epidemiological study on tropical theileriosis (Theileria annulata infection) in the Egyptian Oases with special reference to the molecular characterization of Theileria spp.

Theileria annulata infection is a tick-borne disease known as Egyptian fever since 1947. It is a destructive obstacle for the livestock production in the Egyptian Oases (EL-Wady EL-Geded Province). The present study was conducted on 1068 cattle, ranged from below one year to more than eight years old; belonged to different farms and villages in EL-Wady EL-Geded Province. The infection was confirmed by blood smears, Tams-1 target based polymerase chain reaction (Tams-1 PCR), 18Ss rRNA polymerase chain reaction and semi nested-polymerase chain reaction (nPCR) followed by DNA sequencing and phylogenetic analyses, in addition to tick identification. Molecular techniques confirmed the infection in 63.6% (679/1068) of the examined animals while Giemsa-stained blood smears confirmed it in 36.8% (393/1062). Male and female animals showed molecular confirmed infection rates of 64.5 and 62.7%, respectively. Animals less than one year old were more infected (83.33%, 400/480) followed by animals less than three years (57.31%, 149/260) and animals less than five years (42.45%, 90/212), respectively. On the other hand, animal of five years old or above were less infected and the infection rate in this group was estimated to be 34.48% (40/116). Two tick species were identified during the present study: Hyalomma anatolicum and Rhipicephalus annulatus. Theileria annulata was the only Theileria species found in the Egyptian oases in respect to phylogenetic analysis of the obtained sequences.

Comparison between conventional and molecular methods for diagnosis of bovine babesiosis (Babesia bovis infection) in tick infested cattle in upper Egypt.

Ticks and tick-borne diseases are the main problems affecting the livestock production in Egypt. Bovine babesiosis has adverse effects on the animal health and production. A comparison of Giemsa stained blood smears, polymerase chain reaction (PCR) and nested PCR (nPCR) assays for detection of Babesia bovis infection in Egyptian Baladi cattle (Bos taurus) in reference to reverse line blot was carried out. The sensitivity of PCR and nested PCR (nPCR) assays were 65 and 100 % respectively. Giemsa stained blood smears showed the lowest sensitivity (30 %). According to these results using of PCR and nPCR target for B. bovis, [BBOV-IV005650 (BV5650)] gene are suitable for diagnosis of B. bovis infection. The 18Ss rRNA partial sequence confirmed that all the positive samples were Babesia bovis and all of them were deposited in the GenBank databases (Accession No: KM455548, KM455549 and KM455550).

Assessment of the First Commercial ELISA Kit for the Diagnosis of Theileria annulata.

The present study assesses the efficacy of SVANOVIR Theileria annulata-Ab, the first commercial ELISA kit for the diagnosis of Theileria annulata infection in cattle based on a recombinant protein known as T. annulata surface protein (TaSp). As a reference test, a polymerase chain reaction (PCR) assay depending on T. annulata merozoite surface antigen (Tams-1) was applied. A total of 468 blood samples as well as serum samples were randomly collected from cattle and tested in the PCR as well as in the ELISA developed in this study. Moreover, all samples were also analyzed by conventional Giemsa-stained blood smear. The results of this study revealed a good correlation between the results obtained by PCR and the ELISA, whereas all PCR positive samples scored correctly positive in the ELISA and 73 of the 125 PCR negative samples scored correctly negative. Taken together, a sensitivity of 91.25% and a specificity of 78.4% were recorded, when compared to the PCR data. In conclusion, the SVANOVIR Theileria annulata-Ab is a suitable diagnostic assay for use in the diagnosis and epidemiological surveys of Theileria annulata infection in chronic and carrier animals.

Microscopical and serological studies on Sarcocystis infection with first report of S. cruzi in buffaloes (Bubalus bubalis) in Assiut, Egypt.

This study was performed for the purpose of investigating the prevalence and the species composition of Sarcocystis spp. in buffaloes in Assiut province, Egypt. Macroscopically we reported the infection of buffaloes with Sarcocystis fusiformis, while microscopically three Sarcocystis species (Sarcocystis cruzi, Sarcocystis levinei and Sarcocystis hominis) cysts were recognized, and were differentiated by their morphological features using both histopathological sections and electron microscope scanning. Regarding the prevalence of Sarcocystis species among buffaloes in Assiut province, we reported that, using gross examination of 90 buffaloes' esophagus, only 23 samples out of 90 (25.5 %) were found to be infected; on the other hand, by using microscopical examination, the prevalence was 27.7 % (25 samples out of 90 samples were found to be infected). Using ELISA, 85 samples out of 90 (94.4 %) were found positive, an overall prevalence of 94.4 %. In this work we concluded that customary meat inspection methods in abattoirs in Egypt are insufficient for detecting Sarcocystis infection. Due to the presence of hidden or microscopic cysts, we strongly recommend the use of combined microscopical examination and ELISA for Sarcocystis diagnosis, to avoid human infection of such zoonotic parasite and to control the consequent disease. In addition, this study introduced the first report of S. cruzi in buffaloes in Egypt, and proved the hypothesis that S. cruzi is able to use animals such as water buffalo as intermediate hosts.