Prevalence, Electron Microscopy and Molecular Characterization of Cryptosporidium species Infecting Sheep in Egypt

Cryptosporidium sp. is predominant universally and sheep are an imperative zoonotic supply of the disease. Owing to the little information presented with respect to Cryptosporidium sp. infecting sheep, this study was directed to survey the predominance and molecular characterization of Cryptosporidium sp. among sheep of different ages and sexes in Qalyubia governorate, Egypt. The fecal specimens were gathered from 432 sheep of various ages (≤1 to <6, 6-12 and >12 months) and sexes. The samples were microscopically examined after staining by modified Zeihl- Neelsen technique and the intestinal mucosa was scanned by electron microscopy. A nested PCR was connected to amplify a 830 bp of 18S rRNA sequence of Cryptosporidium. RFLP (restriction fragment length polymorphism) technique using SspI and VspI enzymes for digestion of the secondary product of PCR for species identification was applied. The total infection rate was 25.93%. The parasite was more prevalent in males than females of different age groups. Two zoonotic Cryptosporidium species were distinguished after RFLP-PCR sequencing: C. parvum and C. ubiquitum (identified previously as Cervine genotype). The finding recommends that sheep must be considered as a noteworthy potential source of human cryptosporidiosis. A strict reconnaissance of zoonotic cryptosporidiosis must be set up to counteract human infection and to assess forthcoming disease when applying control programs

Prevalence, Electron Microscopy and Molecular Characterization of Cryptosporidium species Infecting Sheep in Egypt

Cryptosporidium sp. is predominant universally and sheep are an imperative zoonotic supply of the disease. Owing to the little information presented with respect to Cryptosporidium sp. infecting sheep, this study was directed to survey the predominance and molecular characterization of Cryptosporidium sp. among sheep of different ages and sexes in Qalyubia governorate, Egypt. The fecal specimens were gathered from 432 sheep of various ages (≤1 to 12 months) and sexes. The samples were microscopically examined after staining by modified Zeihl- Neelsen technique and the intestinal mucosa was scanned by electron microscopy. A nested PCR was connected to amplify a 830 bp of 18S rRNA sequence of Cryptosporidium. RFLP (restriction fragment length polymorphism) technique using SspI and VspI enzymes for digestion of the secondary product of PCR for species identification was applied. The total infection rate was 25.93%. The parasite was more prevalent in males than females of different age groups. Two zoonotic Cryptosporidium species were distinguished after RFLP-PCR sequencing: C. parvum and C. ubiquitum (identified previously as Cervine genotype). The finding recommends that sheep must be considered as a noteworthy potential source of human cryptosporidiosis. A strict reconnaissance of zoonotic cryptosporidiosis must be set up to counteract human infection and to assess forthcoming disease when applying control programs

Molecular diagnosis and biochemical studies of tick-borne diseases (anaplasmosis and babesiosis) in Aberdeen Angus Cattle in New Valley, Egypt

Background and Aim: Anaplasmosis and babesiosis are tick-borne diseases that threaten livestock production with subsequent considerable economic losses. This study was conducted to diagnose Anaplasma and Babesia infection using molecular techniques in imported Aberdeen Angus cattle imported from Uruguay to El-Kharga Oasis in New Valley, Egypt, and to investigate the effects of disease on some serum biochemical and oxidative stress parameters. Materials and Methods: Blood samples were collected from 31 cattle, 21 diseased and ten apparently normal, of varying ages and sex. The blood was used for the preparation of blood smears, polymerase chain reaction assay, and separation of serum for biochemical investigation. The experimental production farm at the Faculty of Agriculture, New Valley University, was infested with ticks and variable clinical manifestations during the period from December 2017 to March 2018. One calf died of a suspected blood parasite infection. Results: The blood film examination revealed infection by blood parasites in 21 samples. Anaplasma marginale and Babesia bovis were identified in 12 and 14 samples, respectively. A total of 14 samples were examined by polymerase chain reaction (PCR) to make these identifications. Biochemical parameters showed significantly elevated serum alanine aminotransferase, aspartate aminotransferase, total bilirubin (T. Bil), and urea in blood from parasite-infected female cattle and male calves compared with controls. Increased serum total protein, globulin, and creatinine were recorded only in infected female cattle. The blood glucose level was significantly decreased in infected female cattle and male calves compared with controls. Furthermore, albumin and albumin/globulin ratio was significantly reduced in the infected female cattle. Oxidative stress profiles of infected animals showed a significant increase in serum nitric oxide and malondialdehyde, and both total antioxidant capacity and reduced glutathione (GSH) were significantly reduced in comparison with control animals. Conclusion: The incidence of A. marginale and B. bovis infection is high in imported Aberdeen Angus cattle in New Valley Province. PCR methods provide a short-term assessment of disease. An extensive epidemiological survey, employing serology together with molecular genetic methods, monitoring of abundance and distribution of tick vectors, availability of vaccination programs, and tracking of animal transport is also needed for control of blood parasites

Seroprevalence and molecular characterization of Mycobacterium bovis infection in camels (Camelus dromedarius) in the Delta region, Egypt

Aim: This study aimed to determine the prevalence rates of Mycobacterium infection in camel sera collected before slaughter and gross lesion tissue collected at postmortem (PM) using enzyme-linked immunosorbent assay (ELISA), bacteriological culture, and polymerase chain reaction (PCR). In addition, serum samples from humans who had occupational contact with camels were tested by ELISA and sputum sample by culture. Materials and Methods: ELISA was performed on serum samples antemortem. In addition, bacteriological culture and PCR were conducted after PM. Tuberculosis infection was identified in humans who had contact with camels using ELISA for serum samples and culture for sputum samples. Results: Tuberculous lesions were detected in 184 of 10,903 camels (1.7%). The ELISA results revealed that of the 184 examined camel serum samples, 124 (67.39%) were positive and all 20 camel serum samples that had no associated tuberculous lesions were negative. Moreover, only one of 48 (2.08%) human serum samples was positive by ELISA. Mycobacterial culture revealed 112 isolates from the 184 examined camel samples (60.87%), while human sputum sample cultures were all negative. PCR analysis identified the mpb70 gene in three of seven randomly tested samples. Conclusion: Gene sequencing was performed on two samples and the sequences were submitted to the National Center for Biotechnology Information GenBank (accession numbers MF990289 and MG59479). A phylogenetic tree was constructed based on the partial DNA sequences of the mpb70 gene; the similarity between the isolates was 98.1%. The similarities between the two isolates and the standard strains of Mycobacterium bovis in GenBank were 98.1% and 100%, respectively. Further investigation on the antemortem detection of M. bovis infection in camels is needed to decrease public risk

Molecular Detection, Serotyping, and Antibiotic Resistance of Shiga Toxigenic Escherichia coli Isolated from She-Camels and In-Contact Humans in Egypt

This study aims to determine the prevalence of STEC in she-camels suffering from mastitis in semi-arid regions by using traditional culture methods and then confirming it with Serological and molecular techniques in milk samples, camel feces, as well as human stool samples for human contacts. In addition, an antibiotic susceptibility profile for these isolates was investigation. Mastitic milk samples were taken after California Mastitis Test (CMT) procedure, and fecal samples were taken from she-camels and human stool samples, then cultured using traditional methods to isolate Escherichiacoli. These isolates were initially classified serologically, then an mPCR (Multiplex PCR) was used to determine virulence genes. Finally, both camel and human isolates were tested for antibiotic susceptibility. Out of a total of 180 she-camels, 34 (18.9%) were mastitic (8.3% clinical and 10.6% sub-clinical mastitis), where it was higher in camels bred with other animals. The total presence of E. coli was 21.9, 13.9, and 33.7% in milk, camel feces, and human stool, respectively, whereas the occurrence of STEC from the total E. coli isolates were 36, 16, and 31.4% for milk, camel feces, and stool, respectively. Among the camel isolates, stx1 was the most frequently detected virulence gene, while hlyA was not detected. The most detected virulence gene in human isolates was stx2 (45.5%), followed by stx1. Camel STEC showed resistance to Oxytetracycline only, while human STEC showed multiple drug resistance to Amoxicillin, Gentamycin, and Clindamycin with 81.8, 72.7, and 63.6%, respectively. Breeding camels in semi-arid areas separately from other animals may reduce the risk of infection with some bacteria, including E. coli; in contrast, mixed breeding with other animals contributes a significant risk factor for STEC emergence in camels

Molecular Characterization and Developing a Point-of-Need Molecular Test for Diagnosis of Bovine Papillomavirus (BPV) Type 1 in Cattle from Egypt

Bovine papillomatosis is a viral disease of cattle causing cutaneous warts. A diagnosis of this viral infection is very mandatory for combating the resulting economic losses. Given the limited data available about bovine papillomavirus (BPV) in Egypt, the present study involved the molecular diagnosis of bovine papillomavirus type-1 (BPV-1), -2, -4, -5, and -10 in cattle presenting cutaneous warts on the head and neck from New Valley Province, Egypt. The phylogenetic analysis of the detected types of BPV was also performed, followed by developing a point-of-need molecular assay for the rapid identification of identified BPV types. In this regard, a total of 308 cattle from private farms in Egypt were clinically examined, of which 13 animals presented cutaneous warts due to suspected BPV infection. The symptomatic animals were treated surgically, and biopsies from skin lesions were collected for BPV-1, -2, -4, -5, and -10 molecular identification using polymerase chain reaction (PCR). The presence of BPV-1 DNA was confirmed in 11 collected samples (84.6%), while BPV-2, -4, -5, and -10 were not detected. Sequencing of the PCR products suggested the Egyptian virus is closely related to BPV found in India. An isothermal nucleic acid amplification test (NAAT) with labeled primers specific for the BPV-1 L1 gene sequence, and based on recombinase polymerase amplification (RPA), in combination with a lateral flow strip assay for the detection of RPA products, was developed and tested. The point-of-need molecular assay demonstrated a diagnostic utility comparable to PCR-based testing. Taken together, the present study provides interesting molecular data related to the occurrence of BPV-1 in Egypt and reveals the genetic relatedness of the Egyptian BPV-1 with BPV-1 found in buffalo in India. In addition, a simple, low-cost combined test was also validated for diagnosis of the infection. The present study suggests the necessity of future investigations about the circulating strains of the virus among the cattle in Egypt to assess their genetic relatedness and better understand the epidemiological pattern of the disease