Ebrahim Rahimi

Molecular assay to fraud identification of meat products

Detection of species fraud in meat products is important for consumer protection and food industries. A molecular technique such as PCR method for detection of beef, sheep, pork, chicken, donkey, and horse meats in food products was established. The purpose of this study was to identification of fraud and adulteration in industrial meat products by PCR-RFLP assay in Iran. In present study, 224 meat products include 68 sausages, 48 frankfurters, 55 hamburgers, 33 hams and 20 cold cut meats were collected from different companies and food markets in Iran. Genomic DNA was extracted and PCR was performed for gene amplification of meat species using specific oligonucleotid primers. Raw meat samples are served as the positive control. For differentiation between donkey’s and horse’s meat, the mitochondrial DNA segment (cytochrome-b gene) was amplified and products were digested with AluI restriction enzyme. Results showed that 6 of 68 fermented sausages (8.82%), 4 of 48 frankfurters (8.33%), 4 of 55 hamburgers (7.27%), 2 of 33 hams (6.6%), and 1 of 20 cold cut meat (5%) were found to contain Haram (unlawful or prohibited) meat. These results indicate that 7.58% of the total samples were not containing Halal (lawful or permitted) meat and have another meat. These findings showed that molecular methods such as PCR and PCR-RFLP are potentially reliable techniques for detection of meat type in meat products for Halal authentication.

Detection of Toxoplasma gondii in raw caprine, ovine, buffalo, bovine, and camel milk using cell cultivation, cat bioassay, capture ELISA, and PCR methods in Iran.

This study was conducted to determine the presence of Toxoplasma gondii in animal milk samples in Iran. From a total of 395 dairy herds in three provinces of Iran, 66 bovine, 58 ovine, 54 caprine, 33 buffalo, and 30 camel herds were studied, and from these parts of Iran, 200 bovine, 185 ovine, 180 caprine, 164 buffalo, and 160 camel milk samples were collected from various seasons. Samples were tested for Toxoplasma gondii by cell line culture, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) technique. Only the results of cell line cultivation were confirmed by bioassay in cat. Results indicated that all herds were infected with Toxoplasma gondii. The culture method showed that 51 out of 889 milk samples (5.73%) were positive for Toxoplasma gondii, and all 51 positive culture results were positive with bioassay in cat. The Fars province had the highest prevalence of Toxoplasma gondii (6.84%). The ELISA test showed that 41 milk samples (4.61%) were positive for the presence of Toxoplasma gondii, while the PCR showed that 46 milk samples were positive for Toxoplasma gondii. The results showed higher sensitivity of PCR and higher specificity of ELISA. Caprine had the highest (10%) and camel had the lowest (3.12%) prevalence rate of parasite. The summer season had the highest (76.47%) but winter (3.92) had the lowest incidence of Toxoplasma gondii. This study is the first prevalence report of direct detection of Toxoplasma gondii in animal milk samples in Iran.

Lead and cadmium concentrations in goat, cow, sheep, and buffalo milks from different regions of Iran.

In total, 137 goat, cow, sheep, and buffalo milk samples were collected in different regions of Iran and analysed to determine concentrations of lead and cadmium by a graphite furnace atomic absorption spectrometric method. The mean recovery of the analytical method was 96.3% and 104% for cadmium and lead, respectively. The mean lead and cadmium contents obtained from 137 samples were 1.93 ± 1.48 (range: 0.18-6.11 ng/ml) and 9.51 ± 4.93 ng/ml (range: 1.84 ng/ml-30.50 ng/ml), respectively. Lead concentration in 8.1% of sheep and 1.9% of cow milk samples was higher than the newly established Codex standard. The mean concentrations of cadmium and lead in animals aged ≤ 3 years (n=80; 1.40 ± 1.05 ng/ml and 7.91 ± 3.60 ng/ml, respectively) were lower than in animals aged >3 years (n=58; 2.69 ± 1.67 ng/ml and 11.8 ± 5.71 ng/ml, respectively).

Detection of Coxiella burnetii by Nested PCR in Bulk Milk Samples from Dairy Bovine, Ovine, and Caprine Herds in Iran

The epidemiology of Q‐fever in Iran is essentially unknown. This study was conducted to determine the prevalence rate of Coxiella burnetii in bulk milk samples from dairy bovine, ovine, and caprine herds in Chaharmahal va Bakhtiari province, Iran. In this study, 376 bulk milk samples from 79 dairy bovine, ovine, and caprine herds were tested for C. burnetii using a nested PCR assay. The animals whose milk samples collected for this study were clinically healthy. In total, 13 of 210 (6.2%) bovine milk samples were positive; the positive samples originated from 5 of 28 (17.9%) commercial dairy herds. All 110 ovine bulk milk samples from 31 sheep breeding farms were negative and only 1 of 56 (1.8%) caprine bulk milk samples from 20 goat breeding farms was positive for C. burnetii. Although no extensive prevalence study was undertaken, the results of this study indicate that clinically healthy cattle are important sources of C. burnetii infection in Iran. To our knowledge, this study is the first report of direct identification of C. burnetii by PCR in bulk milk samples from dairy bovine and caprine herds in Iran. Further intensive prevalence studies on Coxiella infection among farmers, milk‐processing workers, veterinarians, and slaughterhouse workers and on possible dangers of dairy products will be needed to elucidate the epidemiology of Q fever in Iran.

Incidence of Shiga toxin-producing Escherichia coli serogroups in ruminant's meat☆

To assess the presences of Escherichia coli, its serogroups, virulence factors and antibiotic resistance properties in ruminants meat, a total of 820 raw meat samples were collected and then evaluated using culture, PCR and disk diffusion methods. Totally, 238 (29.02%) samples were positive for presence of Escherichia coli. All of the isolates had more than one virulence gene including Stx1, Stx2, eaeA and ehly. All investigated serogroups were found in beef and sheep and all except O145, O121 and O128 were found in goat. The O91, O113, O111, O103, O26 and O157 serogroups were found in camel. Totally, aadA1-blaSHV combination was the most predominant antibiotic resistance gene. The highest resistance of STEC strains was seen against penicillin while resistance to nitrofurantoin and ciprofloxacin was minimal. These findings showed that health care and meat inspection should be reconsidered in Iranian slaughterhouses and butchers.

Determination of mercury, cadmium and lead in human milk in Iran

Breast milk contains both essential and nonessential trace elements. Mercury, cadmium and lead are nonessential, potentially toxic heavy metals with hematotoxic, neurotoxic and nephrotoxic properties even at very low concentrations. The objectives of this study were to determine the concentrations of mercury, cadmium and lead in the breast milk of healthy lactating women who were living in Isfahan, Iran. Concentrations of mercury, cadmium and lead were determined by graphite furnace atomic absorption spectrometry in 37 milk samples from healthy lactating women collected on first to sixth postpartum week. Accuracy of the analysis was checked by various methods including the use of reference material. The mean ± SD of the concentrations of mercury, cadmium and lead in human milk were 0.92 ± 0.54 μg/L (range 0.0–2.07 μg/L), 1.92 ± 1.04 μg/L (range 0.45–5.87 μg/L) and 7.11 ± 3.96 μg/L (range 3.06–19.47 μg/L), respectively. The results of this study showed that the concentrations of mercury, lead and cadmium in the milk samples from lactating women in Isfahan were high, which makes a major public health hazard for the inhabitants, especially neonatal and children, of the industrial locations. The results of the present study indicate a need for establishing safe intake values of heavy metals in human milk.

Prevalence and Antimicrobial Resistance of Campylobacter Species Isolated from Raw Camel, Beef, Lamb, and Goat Meat in Iran

Campylobacter spp. are one of the most common causes of acute bacterial gastroenteritis in human beings which are transmitted mostly via food originating from animals. This study was conducted to determine the prevalence and antimicrobial resistance of Campylobacter spp. isolated from retail raw meats in Iran. From June 2008 to June 2009, a total of 722 raw meat samples from camel (n = 107), beef (n = 190), lamb (n = 225), and goat (n = 180) were purchased from randomly selected retail outlets in Isfahan and Yazd, Iran, and were evaluated for the presence of Campylobacter spp. In this study, 50 of the 722 meat samples (6.9%) were contaminated with Campylobacter spp. The highest prevalence of Campylobacter spp. was found in lamb meat (12.0%), followed by goat meat (9.4%), beef meat (2.4%), and camel meat (0.9%). The most prevalent Campylobacter spp. isolated from the meat samples was Campylobacter jejuni (84.0%); the remaining isolates were Campylobacter coli (16.0%). Susceptibilities of 50 Campylobacter isolates were determined for 10 antimicrobial drugs using the disk-diffusion assay. Resistance to tetracycline was the most common finding (68.0%), followed by resistance to ciprofloxacin (46.0%) and nalidixic acid (40.0%). All of the isolates were susceptible to erythromycin, gentamicin, and chloramphenicol. Significantly higher prevalence rates of Campylobacter spp. (p < 0.05) were found in the lamb meat samples taken in spring (20.0%) and summer (18.9%). To our knowledge, this study is the first report of the isolation of Campylobacter spp. from raw camel, lamb, and goat meat in Iran.

Molecular Characterization of Shiga Toxin-Producing Escherichia coli Isolated from Ruminant and Donkey Raw Milk Samples and Traditional Dairy Products in Iran

The aims of the current study were to detect the virulence factors and antibiotic resistance of Shiga toxin-producing E. coli, in animal milk and dairy products in Iran. After E. coli dentification with culture method, PCR assay were developed for detection of pathogenic genes, serotypes and antibiotic resistance genes of E. coli. Results showed that out of 719 samples, 102 (14.18%) were confirmed to be positive for E. coli and out of 102 positive samples, 17.64% were O26 and 13.72% were O157 and 1.96% were O91 and 1.96% were O145 serotypes. Totally, the prevalence of stx1 and papA genes were the highest while the prevalence of sfaS and fyuA were the lowest in the positive samples. PCR results showed that tetA, tetB were the highest (64.70%) and aac(3)-IV were the lowest (27.45%) antibiotic resistant genes in E. coli positive samples. Our study indicated that the isolated E. coli trains in these regions had a highest antibiotic resistance to tetracycline (58.82%) and the lowest to nitrofurantoin (3.92%). tetA gene and E. coli O157 serotype had highest and aac(3)-IV gene, and E. coli O145 serotype had a lowest frequency rates of antibiotics resistance genes, in the region.

Detection of Helicobacter pylori in Bovine, Buffalo, Camel, Ovine, and Caprine Milk in Iran

Helicobacter pylori infection in humans is one of the most common infections worldwide. However, the origin and transmission of this bacterium has not been clearly explained. One of the suggested theories is transmission via raw milk from animals to human beings. This study was conducted to determine the prevalence rate of H. pylori in bulk milk samples from dairy bovine, buffalo, camel, ovine, and caprine herds in Iran. In the present study, 447 bulk milk samples from 230 dairy bovine, buffalo, camel, ovine, and caprine herds were collected in four provinces and tested for H. pylori by cultural method and polymerase chain reaction (PCR) for the detection of the ureC (glmM) gene. The animals whose milk samples collected for this study were clinically healthy. Using the cultural method, three of 447 milk samples (0.67%), including two sheep (2.2%) and one buffalo (1.6%) milk samples, were found to be contaminated with H. pylori. H. pylori ureC gene was detected in 56 (12.5%) of milk samples, including 19 cow (14.1%), 11 sheep (12.2%), nine goat (8.7%), two camel (3.6%), and 15 buffalo (23.4%) milk samples. Using PCR method, there were significant differences (p<0.05) in the level of contamination with H. pylori between milk samples collected from different species. The present study is the first report of the isolation of H. pylori from raw sheep and buffalo milk in Iran and the first demonstration of H. pylori DNA in camel and buffalo milk.

Prevalence of Clostridium difficile in raw beef, cow, sheep, goat, camel and buffalo meat in Iran

BackgroundClostridium difficile has been shown to be a nosocomial pathogen associated with diarrhoea and pseudomembranous colitis in hospitalised patients and the infection is believed to be acquired nosocomially. Recent studies have shown the occurrence of C. difficile in food animals which may act as a source of infection to humans.The aim of this study was to determine the occurrence of C. difficile in retail raw beef, cow, sheep, goat, camel and buffalo meat in Iran.MethodFrom April to October 2012, a total of 660 raw meat samples from beef, cow, sheep, goat, camel and buffalo were purchased from 49 butcheries in Isfahan and Khuzestan provinces, Iran, and were evaluated for the presence of C. difficile using a method including selective enrichment in C. difficile broth, subsequent alcohol shock-treatment and plating onto C. difficile selective medium. C. difficile isolates were tested for the presence of toxin genes and were typed using PCR ribotyping.ResultsIn this study, 13 of 660 meat samples (2%) were contaminated with C. difficile. The highest prevalence of C. difficile was found in buffalo meat (9%), followed by goat meat (3.3%), beef meat (1.7%), cow (0.94%) and sheep meat (0.9%). Seven of the 13C. difficile strains (53.9%) were positive for tcdA, tcdB and cdtB toxin genes and were classified as ribotype 078. Four strains (30.8%) were positive tcdA, and tcdB, and one strain (7.7%) was possessed only tcdB. The remaining isolate was non-toxigenic. Susceptibilities of 13C. difficile isolates were determined for 11 antimicrobial drugs using the disk diffusion assay. Resistance to clindamycin, gentamycin, and nalidixic acid was the most common finding.ConclusionsTo our knowledge, the present study is the first report of the isolation of C. difficile from raw buffalo meat. This study indicates the potential importance of food, including buffalo meat, as a source of transmission of C. difficile to humans.