Subtypes and phylogenetic analysis of Blastocystis sp. isolates from West Ismailia, Egypt.

In Egypt, Blastocystis sp. is not yet on the diagnostic list of parasitology reports, and information about its subtypes (STs) is scarce. This study investigated its prevalence and its STs/alleles, performed phylogenetic analysis, and considered the distribution of risk factors associated with Blastocystis sp. infections in West Ismailia, Ismailia governorate. Sociodemographic data, exposure factors, and previous parasitic infection status were recorded for symptomatic and asymptomatic individuals. Microscopy, polymerase chain reaction, sequencing, and phylogenetic analysis for Blastocystis sp. isolated from fecal samples were performed. Eighty Blastocystis sp.-infected individuals (15.3%) were examined. The age of the individuals ranged between 0.60 and 85.0 (mean 17.10 ± 15.70), the male/female ratio was 33/47, and the asymptomatic/symptomatic ratio was 55/25. The findings demonstrate clear evidence of direct contact with animals, poor water quality, and previous parasitic infections. Eleven samples yielded three Blastocystis STs (ST1: allele 4, ST2: alleles 9 and 12, and ST3: allele 34), with ST3 (45.5%) representing the most common subtype. Phylogenetic analysis with a robust bootstrap revealed three distinct clades for isolates of each subtype. This study updates the epidemiological knowledge of the distribution of Blastocystis sp. STs in Egypt and expands the current understanding of the prevalence, risk factor frequencies, and genetic diversity of this protist in the studied area.

Virulence and antimicrobial-resistance of shiga toxin-producing E. coli (STEC) Isolated from edible shellfish and its public health significance.

Shiga toxin-producing E. coli (STEC) are an important cause of foodborne illness in humans with infections ranging from mild non-bloody diarrhea to bloody diarrhea (BD) and hemolytic uremic syndrome (HUS). This study aimed to investigate the distribution of STEC in shellfish from coastal shores of Lake Timsah in Ismailia Governorate, Egypt and its probable hazard to seafood consumers. Samples from the external surface and tissues of shrimp (n = 45), crabs (n = 45), and oysters (n = 45) batches were examined bacteriologically for the presence of STEC and tested for their antibiotic sensitivity. Moreover, occurrence of virulence genes was determined via detection of stx1, stx2 and eaeA genes using PCR. Overall, E. coli and presumptive STEC isolates (from CHROMagar) were identified from the surface (55.6 and 5.9%) and tissues (42.2 and 8.9%) of the examined shellfish batches, respectively. Five STEC isolates had been confirmed and found belonging to O26:H11, O125:H6, O146:H21, and O159 serogroups, those were 4 isolates from tissues of the three shellfish species and one isolate from the crab surface. The STEC isolates were multi-drug resistant, showing complete resistance to; penicillins, amoxycillin/clavulanic acid, colistin, fosfomycin, ceftriaxone, ciprofloxacin, and tetracycline, however, they were sensitive to gentamycin except O159 serogroup. The current study revealed low level of contamination of shellfish from coastal shores of Lake Timsah with STEC, however, it also highlights the extreme level of antimicrobial resistance exhibited by the presumptive and confirmed STEC isolates which is very hazardous for seafood consumers in the study area.

Role of dietary Spirulina platensis and betaine supplementation on growth, hematological, serum biochemical parameters, antioxidant status, immune responses, and disease resistance in Nile tilapia.

Spirulina platensis is, a freshwater microalga, broadly used worldwide. It not only stimulates the immune systems of aquatic organisms but also provides a protein-rich diet and commonly used in the manufacture of aquafeeds. This study was planned to evaluate the growth performance, hepato-renal, and immune response biomarkers of Spirulina and Betaine on Nile tilapia (Oreochromis niloticus) and their protective effect against infection with Aeromonas hydrophila. O. niloticus juveniles (20.22 ± 0.86 g) were divided into four groups (n = 10 per replicate). For 8 weeks, the first and second groups (TS&TB) were fed with 0.5% and 0.3% concentrations of Spirulina and Betaine supplemented diets, respectively; the third group (TSB) was fed with a Spirulina and Betaine mixed diet; the fourth group was fed with a basal diet (without supplementation, T0), which served as control. Dietary inclusion of Spirulina and Betaine significantly improved (P ˂ 0.05) the weight gain, final weight, and food conversion ratio, especially in the TS group. The activities of hepatic malonaldehyde were unchanged in TS & TSB groups and the muscular significantly decreased (P ˂ 0.05) in the same groups, while both increased in the TB group; meanwhile, levels of glutathione reductase were significantly upregulated in all treated groups. Serum interleukins, TNF- alpha, and IL-10 levels were also significantly reduced in all treatment groups. A significant protective power against pathogenic Aeromonas infection was evidenced in all treated groups. Findings in this study highlight the reputation of Spirulina and Betaine as immunostimulants and protective agents against A. hydrophila infection in O. niloticus.

Characterization of enterohemorrhagic Escherichia coli from diarrhoeic patients with particular reference to production of Shiga-like toxin.

Enterohemorrhagic Escherichia coli (EHEC) is a subtype of pathogenic E. coli that causes diarrhea or hemorrhagic colitis in humans, which can progresses to hemolytic uremic syndrome (HUS), a leading cause of acute renal failure in children, and morbidity and mortality in adults. Stool samples (n = 273) of patients (1 day-40 years old) suffered from bloody diarrhea and abdominal cramps, were examined bacteriologically and molecularly for the presence and pathogenicity of EHEC with phylogenetic analysis of the obtained stx1, stx2, and eaeA virulence genes' sequences. Overall, 71 (26.01%) E. coli isolates were identified as EHEC with the following serogroupes: O1:H11 (3), O128:H2 (9), O26:H11 (6), O157:H7 (3), O25:H2 (7), O145:H328 (2), O125:H6 (9), O86:H8 (5), O18:H15 (11) and untypable (16). The highest isolation rate were in samples belonged to infants below two years old (42.25%). Antimicrobial susceptibility testing showed that all isolates were highly sensitive to ciprofloxacin, nitrofurantoin, gentamycin, imipenem and vancomycin (100% each), however, they were resistant to ampicillin, cephalexin, penicillin and tetracycline (100% each). In-vitro pathogenicity testing of the isolates revealed that 67 (94.37%) isolates were positive for Congo red test, 47 (66.20%) isolates possessed P fimbriae (MRHA) and 17 (23.94%) possessed type 1 fimbriae (MSHA). Moreover, 46 (64.79%) isolates exhibited hemolysis and 42 (59.15%) isolates showed distinct cytopathic effect to Vero cells. Molecular detection of enterohemorrhagic E. coli (EHEC) pathotype virulence genes, confirmed the presence of stx1 gene in O157:H7 (MA2), O26:H11, O145:H328 and O125:H6 serogroups; stx2 gene in (O157:H7 (MA1), O128:H2 and O25:H2; while all serogroups except (O125:H6) carried the eaeA intimin virulence gene. A phylogenetic tree, based on the nucleotide sequences of toxin-encoding genes, demonstrates that Shiga toxin E. coli (STEC) isolates have considerable genetic variation and belong to various phylogenetic groupings.

Pathways of Methicillin-Resistant Staphylococcus aureus in Animal Model: New Insights Regarding Public Health.

BACKGROUND: Staphylococcus aureus is considered one of the major threats regarding food safety worldwide. Methicillin-resistant S. aureus (MRSA) strains in livestock, companion animals, and wild animals continue to be a potential risk to people working with them. AIM: The current research aims to investigate the potential pathways of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) strains in the body after oral infection using the experimental mouse model. METHODS: Seven groups of SPF male mice were purchased and housed. On day 1, six groups of mice were infected orally by the sterile gastric probe using 100 µL/mice of LA-MRSA bacterial suspension (1 × 108 colony-forming units (CFU)/mL). The remaining group was kept as negative controls. Over 15 days, these animals have been monitored. Fresh fecal samples were screened for LA-MRSA at day 0, day 7 and day 14 following oral administration of MRSA strains. All animals were sacrificed at day 15, and internal organs (liver, lung, kidney, and intestine) were harvested aseptically and divided into two sections. The first part was histopathologically investigated, while the other half has been tested for LA-MRSA re-isolation. RESULT: The oral challenge of mice by MRSA strains showed that MRSA was re-isolated from feces and intestines of all inoculated mice groups and from internal organs (liver, lung, kidney and intestine) of most mice. Results were confirmed by the detection of the bacteria in gram-stained tissue sections and changes in H&E-stained histopathological tissue sections from these organs. CONCLUSION: Data from the present study indicate the possible colonization of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in internal organs following oral infection and thus posing a risk for food-borne infection of MRSA. Infected animals could pass LA-MRSA through feces again, resulting in increased dispersion and environmental contamination.

Inhibitory activity of chitosan nanoparticles against Cryptosporidium parvum oocysts.

Cryptosporidium is a ubiquitous harsh protozoan parasite that resists many disinfectants. It remains viable and infective for a long time in water and food causing global outbreaks. Chitosan (the deacetylated chitin molecule) was used in its nanosuspension form to evaluate its effect against Cryptosporidium parvum. The experiments were performed in vitro in serial concentrations and confirmed in mice in vivo infectivity assay. Chitosan nanoparticles (Cs NPs) were toxic to Cryptosporidium oocysts. The effect appeared to decrease the number of Cryptosporidium oocysts and altered their content. The destruction rate of oocysts was dependent on the dose of chitosan and the time of exposure (P < 0.05). Higher doses of Cs NPs over a prolonged period exhibited a significantly higher destruction rate. Using staining and light microscopy, remarkable destructive changes were observed in the oocysts' morphology. The minimal lethal dose for > 90% of oocysts was 3000 µg/ml, no mice infections in vivo were observed. The results in this study elucidate Cs NPs as an effective anti-cryptosporidial agent.

Detection of Dirofilaria immitis antigen and antibodies against Anaplasma phagocytophilum, Borrelia burgdorferi and Ehrlichia canis in dogs from ten provinces of China.

Despite the fact vector-borne diseases (VBDs) have been increasingly reported in dogs worldwide, there are only limited reports on VBDs in dogs in China with most being based on molecular detection of active infections. To provide further data on the exposure of dogs in China to VBD agents, we used commercial immunochromatographic assays to test plasma from 637 apparently healthy indoor and breeding colony dogs from 21 veterinary clinics in 10 provinces in China and a commercial dog breeding facility for circulating antigen of Dirofilaria immitis, and for circulating antibodies against Ehrlichia spp., Anaplasma spp., and Borrelia burgdorferi. Overall, we found only low levels of exposure to Ehrlichia spp. (4.7%; 30/637), Anaplasma spp. (1.4%; 9/637), B. burgdorferi (0.9%; 6/637) and D. immitis (0.2%; 1/637) with most of the positive animals coming from the commercial breeding colony (26/103; 25.2%) where ectoparasites were most commonly noted. At least one vector-borne agent was found in dogs from 6 of the 10 provinces investigated. Our results confirm exposure of dogs from around China to a variety of VBDs, even indoor pets seldom observed to harbor ectoparasites.

Serological and molecular evidence of Coxiella burnetii in samples from humans and animals in China.

INTRODUCTION: Coxiella burnetii is the agent of Q fever, a worldwide zoonosis. To add to the available knowledge of the disease in China, C. burnetti infections were investigated in convenience samples from five animal species and humans from Yangzhou, Jiangsu province, eastern China. MATERIALS AND METHODS: Commercial ELISA kits were used to detect antibodies to phase I and II C. burnetii. A FRET-qPCR targeting the outer membrane protein com1 gene was also developed to detect C. burnetii DNA in blood samples from animals and humans, and bovine milk samples. RESULTS: Seropositive cattle (44/150; 29%), goats (33/150; 22%), humans (45/180; 25%) and pigs (4/130; 3%) were found, while dogs (0/136; 0%) and cats (0/140; 0%) were seronegative. Seropositivity in humans was associated with increasing age, but there was no gender difference. DNA was amplified from two milk samples (2/150, 1.3%), while none of the blood samples were positive. The sequences of the obtained amplicons were identical to those of the com1 gene of the universal C. burnetii RSA 493 strain and other stains from China. CONCLUSIONS: The findings indicaten that C. burnetii is endemic in Yangzhou, China, and therefore human and animal health workers should be aware of the possibility of infections and the occurrence of outbreaks of Q fever.

High seroprevalence of Coxiella burnetii in dairy cattle in China.

Coxiella burnetii is the agent of Q fever, a zoonosis which occurs worldwide. As there is little reliable data on the organism in China, we investigated C. burnetii infections in dairy cattle herds around the country. Opportunistic whole blood samples were collected from 1140 dairy cattle in 19 herds, and antibodies to phase I and II C. burnetii antigens were detected using commercial ELISA kits. Seropositive cattle (381/1140, 33 %) were detected in 13 of the 15 surveyed provinces and in 16 of the 19 herds (84 %) studied. Our data indicates C. burnetii is widespread in China and that animal and human health workers should be aware of the possibility of Q fever infection in their patients.

Molecular detection of vector-borne agents in dogs from ten provinces of China.

BACKGROUND: Although many vector-borne agents are potential zoonoses and cause substantial morbidity and mortality in dogs worldwide, there are limited data on these organisms in dogs of China. METHODS: Quantitative PCRs for vector-borne agents were performed to investigate their prevalences in convenience whole blood samples obtained from 1114 dogs from 21 veterinary clinics and a commercial dog breeding facility in ten provinces of China. In addition, the PCRs were performed on 146 Rhipicephalus sanguineus senso lato and 37 Linognathus setosus collected from dogs in the commercial dog breeding facility. RESULTS: DNAs of Babesia gibsoni and B. vogeli (1.2 %), Ehrlichia canis (1.3 %), Hepatozoon canis (1.8 %) and Theileria orientalis (0.1 %) or a closely related organism were detected in the bloods of the dogs studied, and Babesia vogeli (3.4 %) and Ehrlichia canis (4.1 %) in R. sanguineus senso lato. The qPCRs for Anaplasma spp., Dirofilaria immitis and Leishmania spp. were negative for all blood samples, ticks and lice. At least one vector-borne agent was found in dogs from 5 of the 10 provinces investigated in this study. Overall, 4.4 % (49/1117) of the dogs studied were positive for at least one vector-borne agent with the prevalence being highest in the commercial breeding colony (24/97; 24.7 %). CONCLUSIONS: Our study confirms that B. vogeli, B. gibsoni, H. canis, and E. canis occur in China. Also, we present evidence that T. orientalis or a closely related organism can infect dogs.

A pan-Theileria FRET-qPCR survey for Theileria spp. in ruminants from nine provinces of China.

BACKGROUND: Theileria spp. are tick transmitted protozoa that can infect large and small ruminants causing disease and economic losses. Diagnosis of infections is often challenging, as parasites can be difficult to detect and identify microscopically and serology is unreliable. While there are PCR assays which can identify certain Theileria spp., there is no one PCR that has been designed to identify all recognized species that occur in ruminants and which will greatly simplify the laboratory diagnoses of infections. METHODS: Primers and probes for a genus-specific pan-Theileria FRET-qPCR were selected by comparing sequences of recognized Theileria spp. in GenBank and the test validated using reference organisms. The assay was also tested on whole blood samples from large and small ruminants from nine provinces in China. RESULTS: The pan-Theileria FRET-qPCR detected all recognized species but none of the closely related protozoa. In whole blood samples from animals in China, Theileria spp. DNA was detected in 53.2% of the sheep tested (59/111), 44.4% of the goats (120/270) and 30.8% of the cattle (380/1,235). Water buffaloes (n = 29) were negative. Sequencing of some of the PCR products showed cattle in China were infected with T. orientalis/T. sergenti/T. buffeli group while T. ovis and T. luwenshuni were found in sheep and T. luwenshuni in goats. The prevalence of Theileria DNA was significantly higher in Bos p. indicus than in Bos p. taurus (77.7% vs. 18.3%) and copy numbers were also significantly higher (10(4.88) vs. 10(3.00) Theileria 18S rRNA gene copies/per ml whole blood). CONCLUSIONS: The pan-Theileria FRET-qPCR can detect all recognized Theileria spp. of ruminants in a single reaction. Large and small ruminants in China are commonly infected with a variety of Theileria spp.

First report of Babesia gibsoni in Central America and survey for vector-borne infections in dogs from Nicaragua.

BACKGROUND: Although many vector-borne diseases are important causes of morbidity and mortality in dogs in tropical areas and potential zoonoses, there is little information on these conditions in Central America. METHODS: Seven qPCRs for vector-borne pathogens were performed on a Roche LightCycler PCR Instrument to investigate their prevalence in a convenience sample of whole blood samples from apparently healthy dogs in Nicaragua. Also, a qPCR targeting the canine hydroxymethylbilane synthase (HMBS) gene was used as an endogenous internal control and verified the quality and quantity of DNA in the samples was appropriate for the study. RESULTS: We found DNA of Rickettsia felis (5%), Babesia spp. (26%), Hepatozoon canis (51%), Anaplasma platys (13%) and Ehrlichia canis (56%) in the 39 dogs studied. The qPCRs for Coxiella burnetii and Dirofilaria immitis were negative. Of the 30 (80%) dogs that were positive by qPCR, 12 (31%) were positive for one agent, 11 (28%) for two, 3 (8%) for three, and 4 (10%) for four agents. CONCLUSIONS: This is the first report of B. gibsoni in dogs from Central America and the first recording of vector-borne agents in dogs from Nicaragua. Dogs in Nicaragua are commonly infected with a variety of vector-borne pathogens, some of which may also infect people.

Molecular detection of Dirofilaria immitis, Hepatozoon canis, Babesia spp., Anaplasma platys and Ehrlichia canis in dogs on Costa Rica.

Although vector-borne diseases are important causes of morbidity and mortality in dogs in tropical areas, there is little information on these conditions in Costa Rica. In PCRs of blood from dogs in Costa Rica, we did not detect DNAs of Rickettsia (R.) felis and Coxiella (C.) burnetii but we did find evidence of infection with Dirofilaria (D.) immitis (9/40, 22.5%), Hepatozoon (H.) canis (15/40, 37.5%), Babesia spp. (10/40, 25%; 2 with B. gibsoni and 8 with B. vogeli), Anaplasma (A.) platys (3/40, 7.5%) and Ehrlichia (E.) canis (20/40, 50%). Nine dogs (22.5%) were free of any vector-borne pathogens while 14 (35%) were infected with a single pathogen, 11 (27.5%) with two, 4 (10%) with three, 1 (2.5%) with four, and 1 (2.5%) with five pathogens. Dogs in Costa Rica are commonly infected with vector-borne agents.