Development of multidrug-resistant Escherichia coli in some Egyptian veterinary farms.

Background and Aim: Food of animal origin is considered a major source of foodborne diseases. In this context, multidrug-resistant (MDR) Escherichia coli pose a serious hazard to public health due to the consumption of food contaminated with antibiotics that are used for the treatment of various bacterial infections in farm animals. Therefore, this study aimed to determine the effect of the excessive use of antibiotics on the development of MDR E. coli strains in Egyptian poultry, dairy, and meat farms. Materials and Methods: A total of 1225 samples were randomly collected from poultry, dairy, and meat products intended for human consumption in different governorates. E. coli were isolated from the collected samples and subjected to biochemical identification and antibiotic sensitivity tests with antibiotics commonly used in human and veterinary medicine. Then, amoxicillin (AML)- and oxytetracycline (OT)-resistant E. coli isolates were subjected to a polymerase chain reaction test to detect the bla TEM and tetA genes, respectively. Results: E. coli were isolated from 132 out of 350, 148 out of 350, 177 out of 350, and 35 out of 175 poultry, milk, meat, and human samples, respectively. Most of the isolates expressed multidrug resistance, and resistance genes (bla TEM and tetA) were detected in all the tested AML- and OT-resistant E. coli isolates. Conclusion: Foods of animal origin may represent a source of MDR E. coli, which can be a major threat to public health.

First Report of Duck Hepatitis A Virus 3 from Duckling Flocks of Egypt.

Duck hepatitis A viruses (DHAV-1, DHAV-2, and DHAV-3) are the predominant causes of duck virus hepatitis (DVH), a disease of ducklings that leads to massive morbidities, mortalities, and economic losses. As a duck-producing country, Egypt suffered lately from several attacks of DVH, despite the regular vaccination of birds. Between Spring 2016 and Summer 2018, 54 duckling flocks in the Sharkia province of Egypt were tested using the reverse-transcription PCR (RT-PCR) based on the DHAV-3D targeting primers. Of them, 27.8% (15/54) were positive. Upon retesting of positive samples using RT-PCR and duck hepatitis A virus (DHAV)-3 VP1-based primers, 33.3% (5/15) contained DHAV-3 RNA. For further analysis at the molecular level, the VP1 and the 3D genes were sequenced using the same primer sets used earlier. The phylogenetic trees confirmed that study sequences belonged to DHAV-3. However, they were displayed as a separate cluster following a geographically dependent distribution. They were also completely unrelated to the Egyptian DHAV-1-based vaccine. This was further confirmed by low nucleotide and amino acid identities in relation to this vaccine. In addition, the VP1 and 3D genes had the same phylogenetic topography. The study VP1 sequences had three unique amino acid substitutions (L59, V208 only in one strain, and C219). As far as we know, this is the first report on DHAV-3 outside Asia, particularly in Egypt. Accordingly, the vaccination strategy against DHAV should be quickly updated to avoid further dissemination of the virus. The epidemiology, pathogenicity, and evolution of DHAV-3 should be carefully monitored in Egypt.

Use of molecular biology tools for rapid identification and characterization of Pasteurella spp.

AIM: This study aimed to create rapid characterization and genotyping of Pasteurella multocida (PM) protocol using modern molecular biology techniques. MATERIALS AND METHODS: Thirty bacterial isolates were characterized by capsular and somatic identification using conventional procedure followed by multiplex polymerase chain reaction (PCR), restriction endonucleases analysis (REA), and finally confirmed by sequence analysis. Two local vaccine strains and two field isolates were identified as PM Type A and B. RESULTS: A total of 30 isolates were found positive for PM either morphologically and biochemically; however, multiplex PCR technique identified only 22 isolates as Pasteurella species using universal primers while 8 isolates were found negative for PM. 12 of 22 isolates (54%) were characterized at the same reaction into PM Type A, five isolates (23%) were Type B and the rest five isolates (23%) of tested isolates were negative for Types A, B, and D. Hemorrhagic septicemia Type B: 2 or B: 5 could be identified somatically within PM capsular serogroup B using PCR technique. Somatic characterization of PM was done using REA that could identify all PM Type A into A:1 and all PM Type B into B: 2. These protocols were verified for its accuracy and reliability by sequence analysis of two vaccine strains of PM Type A and B that were characterized previously by biochemical and serological methods as well as two selected isolates from the 22 positive isolates representing PM Type A and B. CONCLUSION: PCR and REA could confirm the identity of PM and provide a rapid and reliable characterization in comparison with biochemical analysis and conventional serotyping that may take up to 2 weeks. Hence, they can reduce the time needed for polyvalent vaccine production and when the reference antisera are unavailable. Moreover, the identity of Omp-H for vaccine and field strains may provide better data to control Pasteurellosis in Egypt.

Polymerase chain reaction detection of genes responsible for multiple antibiotic resistance Staphylococcus aureus isolated from food of animal origin in Egypt.

AIM: The aim of our study was polymerase chain reaction (PCR) detection of the genes responsible for the multiple antibiotic resistance S. aureus isolated from food of animal origin in Egypt. MATERIALS AND METHODS: A total of 125 samples were randomly collected from milk, meat, and their products from Giza and Beni-Suef Governorates markets. The S. aureus isolates were subjected to antimicrobial sensitivity tests using four antibacterial disks (Oxoid), and then the polymerase chain reaction (PCR) was performed for detection of antibiotic resistance genes. RESULTS: Out of 125 samples, 19 S. aureus isolates were detected. All detected isolates were multiple drug resistance (MDR). The penicillin-, erythromycin-, kanamycin-, and tetracycline-resistant isolates were examined by PCR for resistance genes blaZ, (msrA, ermB, and ermC), aac(6')aph (2"), and tetK. The isolates harbored these resistance genes with percentage of 100% (100%, 0%, and 100%), 62.5%, and 100%, respectively. CONCLUSION: Contaminated foods of animal origin may represent a source of MDR S. aureus that can be a major threat to public health.

Matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry as a reliable proteomic method for characterization of Escherichia coli and Salmonella isolates.

AIM: Identification of pathogenic clinical bacterial isolates is mainly dependent on phenotypic and genotypic characteristics of the microorganisms. These conventional methods are costive, time-consuming, and need special skills and training. An alternative, mass spectral (proteomics) analysis method for identification of clinical bacterial isolates has been recognized as a rapid, reliable, and economical method for identification. This study was aimed to evaluate and compare the performance, sensitivity and reliability of traditional bacteriology, phenotypic methods and matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry (MALDI-TOF MS) in the identification of clinical Escherichia coli and Salmonella isolates recovered from chickens. MATERIALS AND METHODS: A total of 110 samples (cloacal, liver, spleen, and/or gall bladder) were collected from apparently healthy and diseased chickens showing clinical signs as white chalky diarrhea, pasty vent, and decrease egg production as well as freshly dead chickens which showing postmortem lesions as enlarged liver with congestion and enlarged gall bladder from different poultry farms. RESULTS: Depending on colonial characteristics and morphological characteristics, E. coli and Salmonella isolates were recovered and detected in only 42 and 35 samples, respectively. Biochemical identification using API 20E identification system revealed that the suspected E. coli isolates were 33 out of 42 of colonial and morphological identified E. coli isolates where Salmonella isolates were represented by 26 out of 35 of colonial and morphological identified Salmonella isolates. Serological identification of isolates revealed that the most predominant E. coli serotypes were O1 and O78 while the most predominant Salmonella serotype of Salmonella was Salmonella Pullorum. All E. coli and Salmonella isolates were examined using MALDI-TOF MS. In agreement with traditional identification, MADI-TOF MS identified all clinical bacterial samples with valid scores as E. coli and Salmonella isolates except two E. coli isolates recovered from apparently healthy and diseased birds, respectively, with recovery rate of 93.9% and 2 Salmonella isolates recovered from apparently healthy and dead birds, respectively, with recovery rate of 92.3%. CONCLUSION: Our study demonstrated that Bruker MALDI-TOF MS Biotyper is a reliable rapid and economic tool for the identification of Gram-negative bacteria especially E. coli and Salmonella which could be used as an alternative diagnostic tool for routine identification and differentiation of clinical isolates in the bacteriological laboratory. MALDI-TOF MS need more validation and verification and more study on the performance of direct colony and extraction methods to detect the most sensitive one and also need using more samples to detect sensitivity, reliability, and performance of this type of bacterial identification.

Using real-time polymerase chain reaction as an alternative rapid method for enumeration of colony count in live Brucella vaccines.

AIM: Brucellosis is a major bacterial zoonosis of global importance affecting a range of animal species and man worldwide. It has economic, public health, and bio-risk importance. Control and prevention of animal brucellosis mainly depend on accurate diagnostic tools and implementation of effective and safe animal vaccination program. There are three types of animal Brucella live vaccines - Brucella melitensis Rev-1 vaccine, Brucella abortus S19, and B. abortus RB51. Evaluation of these vaccines depends mainly on enumeration of Brucella viable count. At present, used colony count method is time consuming, costly and requires especial skills. Hence, the aim of this study is to use and standardize real-time polymerase chain reaction (RT-PCR) as an alternative, quantitative, sensitive, and rapid method to detect the colony count of Brucella in live Brucella vaccine. MATERIALS AND METHODS: Four batches of different live Brucella vaccines were evaluated using of conventional bacterial count and RT-quantitative PCR (RT-qPCR) using BSCP31 gene specific primers and probe. Standard curve was generated from DNA template extracted from 10-fold serial dilution of living B. abortus RB51 vaccine to evaluate the sensitivity of RT-qPCR. RESULTS: Results revealed that three batches of living Brucella vaccines were acceptable for Brucella colony count when traditional bacterial enumeration method was used. Results of RT-qPCR were identical to that of conventional bacterial count. CONCLUSIONS: Results concluded that RT-qPCR was relatively sensitive compared to traditional bacterial colony count of these vaccines.

Studies on theileria and babesia infecting live and slaughtered animals in Al Arish and El Hasanah, North Sinai Governorate, Egypt.

During the year 2001, a total of 475 sheep, 200 goats, 135 cattle and 190 camels in El Arich city and El Hassanah center were examined for Babesia ovis and Theileria ovis. Blood films were taken from the vein of the ear. Meanwhile, the animals were examined for tick infestations. B. ovis and Th. ovis were detected in 13 (2.7%), and 14 (2.9%) sheep, 14 (7.0%), and 15 (7.5%) goats, 13 (9.6%), and 11 (8.1%) cattle and 18 (9.5%), and 24 (12.6%) camels respectively. On the other hand, double infection was found in 114 (24%) sheep, 51 (25%) goats, 27 (20%) cattle and 66 (34.7%) camels. Adult ticks were identified as Rhipicephalus appendiculatus, R. bursa, R. turanicus and Haemaphysalis parva on sheep, Hyalomma anatolicum excavatum and Haemaphysalis sulcata on goats, Hyalomma lusitanicum on cattle and Hyalomma dromedarii, H. impeltatum, H. marginitum and H. a. anatolicum on camels. Babesia ovis and/or Theileria ovis were recorded in ticks gut and/or salivary glands in R. appendiculatus (20.%), R. bursa (16.7%), R. turanicus (10%), Haemaphysalis parva (10%), H. a. excavaium (30.%), H. dromedarii (18%), and H. a. anatolicum (6.7%).

ALERT-India 1981-89: nine years' experience of leprosy control in the slums of Bombay.

Bombay has a population of about 8 million people, one-half of whom live in slums. In 1981, ALERT-India started its first leprosy control project in N, S and T Wards of Greater Bombay Municipal Corporation covering an area of 122 sq km in the north-eastern suburbs of Vidhyavihar, Ghatkopar, Vikhroli, Kanjurmarg, Bhandup and Mulund, with a total population of 1,100,000 according to the 1981 census. In the 9 years of operation, over 12,000 patients have been registered and treated and of these 7425 have been released from treatment, having satisfactorily completed courses of chemotherapy. However, over 1000 cases are still identified every year by house-to-house or school surveys, or by self-reporting, including a considerable percentage in children. The origin, development, staff structure, operational procedure, administration and recording system of ALERT-India are described in detail, with emphasis on what has been accomplished with purely outpatient facilities, using paramedical workers, all of whom have received inservice training from Government recognized training centres for their specific tasks. The account includes a brief description of an expansion of the organization's work into townships in New Bombay, where preliminary surveys in 1988 confirmed the presence of leprosy cases and the need for treatment facilities. The discussion addresses: 1, the better use of the large volume of statistical information which has been collected by ALERT-India during the past 9 years, with emphasis on its value in assessing the impact on the control programme and modifying future policy; 2, the need to radically examine the present policy of survey, versus an 'education campaign approach' with regard to increasing early case-detection and self-reporting; 3, the establishment of a central coordinating body for leprosy control in Bombay to exchange information, coordinate efforts and formulate a future plan of action, the latter in association with the National Leprosy Eradication Programme; and 4, the development of a health education resource centre in association with the Bombay Municipal Corporation.