Detection of Virulence and ß-lactamase resistance genes of non-typhoidal Salmonella isolates from human and animal origin in Egypt "one health concern".

BACKGROUND: Non-typhoidal Salmonella (NTS) is a major foodborne zoonotic pathogen worldwide. In the current study, Various NTS strains were isolated from (cows, milk and dairy products in addition to humans) in New Valley and Assiut Governorate, Egypt. NTS were firstly serotyped and tested by antibiotic sensitivity test. Secondly, some virulence genes and Antibiotic resistance genes have been identified by using PCR. Finally, Phylogenesis was performed depending on the invA gene, for two S. typhimurium isolates (one of animal origin and the other of human origin for evaluating zoonotic potential). RESULTS: Out of 800 examined samples, the total number of isolates was 87 (10.88%), which were classified into 13 serotypes, with the most prevalent being S. Typhimurium and S. enteritidis. Both bovine and human isolates showed the highest resistance to clindamycin and streptomycin, with 90.80% of the tested isolates exhibiting MDR. The occurrence of the invA gene was 100%, while 72.22%, 30.56%, and 94.44% of the examined strains were positive for stn, spvC, and hilA genes, respectively. Additionally, blaOXA-2 was detected in 16.67% (6/ 36) of the tested isolates, while blaCMY-1 was detected in 30.56% (11of 36) of the tested isolates. Phylogenesis revealed a high degree of similarity between the two isolates. CONCLUSIONS: The high occurrence of MDR strains of NTS in both human and animal samples with high degree of genetic similarity, shows that cows, milk and milk product may be a valuable source of human infection with NTS and interfere with treatment procedures.

An investigative study on the zoonotic potential of Helicobacter pylori.

BACKGROUND: Helicobacter pylori is one of the most common bacterial infections and is widespread globally. It causes a variety of gastrointestinal disorders, though a great proportion of infections are asymptomatic. A total of 143 fresh stool samples were collected from apparently healthy farm and pet animals (43 cattle, 50 buffaloes, 50 sheep, 50 dogs, and 50 cats), in addition to 768 human stool samples. The samples were examined using stool antigen and rapid antibody tests, and further confirmation of glmM "human antigen-positive samples and animal milk samples" was conducted by polymerase chain reaction (PCR). RESULTS: The prevalence rates of H. pylori infection in animals were 22.2% and 16% in antibody and stool antigen tests, respectively. The detection rates were 28%, 24%, 12%, 10%, and 4.7% in cats, dogs, buffaloes, sheep, and cattle, respectively. On the other hand, the prevalence rate of H. pylori infection in human stool samples was 74.8%, and a statistically significant association was observed between prevalence and several factors, such as sex, age, and locality. PCR was performed to detect the glmM gene of H. pylori, and this gene was found in 21 of 27 human antigen-positive samples and 5 of 13 animal milk samples. CONCLUSIONS: H. pylori was detected in both human and animal samples. Furthermore, glmM was found in milk and human samples. Our findings suggest that pet and farm animals could transmit H. pylori infection to humans.

Occurrence of Listeria spp. in Soft Cheese and Ice Cream: Effect of Probiotic Bifidobacterium spp. on Survival of Listeria monocytogenes in Soft Cheese.

Listeria monocytogenes is one of the most important emerging foodborne pathogens. The objectives of this work were to investigate the incidence of Listeria spp. and L. monocytogenes in soft cheese and ice cream in Assiut city, Egypt, and to examine the effect of some probiotic Bifidobacterium spp. (Bifidobacterium breve, Bifidobacterium animalis, or a mixture of the two) on the viability of L. monocytogenes in soft cheese. The existence of Listeria spp. and L. monocytogenes was examined in 30 samples of soft cheese and 30 samples of ice cream. Bacteriological analyses and molecular identification (using 16S rRNA gene and hlyA gene for Listeria spp. and L. monocytogenes, respectively) were performed on those samples. Additionally, Bifidobacterium spp. were incorporated in the making of soft cheese to study their inhibitory impacts on L. monocytogenes. Out of 60 samples of soft cheese and ice cream, 25 samples showed Listeria spp., while L. monocytogenes was found in only 2 soft cheese samples. Approximately 37% of soft cheese samples (11 out of 30) had Listeria spp. with about 18.0% (2 out of 11) exhibiting L. monocytogenes. In ice cream samples, Listeria spp. was presented by 47% (14 out of 30), while L. monocytogenes was not exhibited. Moreover, the addition of B. animalis to soft cheese in a concentration of 5% or combined with B. breve with a concentration of 2.5% for each resulted in decreasing L. monocytogenes efficiently during the ripening of soft cheese for 28 d. Listeria spp. is widely found in milk products. Probiotic bacteria, such as Bifidobacterium spp., can be utilized as a natural antimicrobial to preserve food and dairy products.

Pooled-Testing for SARS-CoV-2 Reverse Transcription Polymerase Chain Reaction (RT-PCR) in asymptomatic healthcare workers in EL-Raghy isolation COVID-19 hospital, Assiut University.

COVID-19 pandemic is a substantial challenge for healthcare systems. Severe acute respiratory syndrome coronavirus (SARS-CoV-2) reverse transcription-polymerase chain reaction (RT-PCR) tests are considered the gold standard technique for diagnosis of symptomatic and asymptomatic infectious viral carriers and for screening special or at-risk populations. The pooled testing procedure is commonly used to reduce the cost of screening a large number of individuals for infectious diseases. This work was conducted to verify the accuracy of the standard SARS COV-2 RT- real-time PCR kit for detecting a single positive sample in a pool of negative samples. Kit verification using negative and positive samples was performed for the selection of the target pool sizes. RNA extracts from 443 healthcare workers, after 15 days' rotation in EL-Raghy Isolation COVID-19 Hospital, Assiut University during the first outbreak of COVID-19 pandemic (the period from June to September 2020) were obtained and tested. Sixty-three different pool sizes (2, 3, 4, 5, 6, 7, 8, 9, and 10) were tested for the presence of SARS-CoV-2 using RT-qPCR. Of these, 53 pools (84.1%) were negatives and 10 pools (15.9 %) tested positive. The individual number of SARS- COV 2 RT-PCR tests used in different pool sizes was 40 tests. The total number of SARS- COV 2 RT-PCR test used in this study was 110 tests instead of 443 tests which reflect a decrease in cost up to 75.16%. In conclusion, the suggested pooling strategy can reduce testing loads which enable substantial savings in reagent costs, technical burden, and time to generate laboratory results.

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.

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.

Occurrence of Toxoplasma gondii in raw goat, sheep, and camel milk in Upper Egypt.

BACKGROUND AND AIM: Toxoplasmosis is a worldwide zoonotic disease with harmful effects on animal and human health. Ingestion of contaminated raw milk has been suggested as a vehicle for transmission of Toxoplasma gondii to human. The present study was performed for the detection of T. gondii in raw milk samples of goat, sheep, and camel in Upper Egypt using two different techniques (enzyme-linked immunosorbent assay [ELISA] and quantitative polymerase chain reaction [qPCR]). MATERIALS AND METHODS: This study was conducted to determine the T. gondii prevalence using ELISA and qPCR in raw goat, sheep, and camels milk (30 samples for each) collected from different locations in Upper Egypt. RESULTS: T. gondii IgG antibodies were detected in 90.0, 60.0, and 3.33% of goat, sheep, and camel milk samples, respectively. From the positive samples of T. gondii IgG, the parasitic DNA was detected only in two examined milk samples, one of them was present in goat milk sample and another one was found in sheep milk sample. On the other hand, the parasite was not detected in camels' milk samples. CONCLUSION: These results concluded that the raw milk was contaminated by T. gondii tachyzoites which could be a source of human infection. Restricted hygienic programs should be implemented in the animal farms to decrease the risk of milk contamination by this parasite.

Species adulteration in raw milk samples using polymerase chain reaction-restriction fragment length polymorphism.

BACKGROUND AND AIM: Milk adulteration is pivotal because it leads to worse effects in public health as human adverse reactions with clinical signs ranged from gastrointestinal signs to anaphylactic shock. This study was carried out to estimate the prevalence of adulteration in buffalo's milk sold in Assiut City, Egypt. MATERIALS AND METHODS: A total of 50 raw buffalo's milk samples were collected and examined for adulteration by addition of cow's milk. The examination carried out by applying polymerase chain reaction-restriction fragment length polymorphism technique using cytochrome b (cyt b) gene primers and Hinf I enzymes. The size of target gene was 360 bp in both animal species and amplicon can be digested using Hinf I enzyme, this restriction enzyme divided the essential band to clear three bands at 360, 210, and 150 bp in cows' milk, while, the enzyme could not be cleaved the amplicon in buffalo's samples. RESULTS: The obtained results cleared that the incidence of adulteration of buffalo's milk very high percentage reaches 90%. CONCLUSION: It could be concluded that the raw buffalo's milk sold in Assiut City subject to fraudulent practice and thus can lead to public health hazards.