Natural and Experimental Persistence of Highly Pathogenic H5 Influenza Viruses in Slurry of Domestic Ducks, with or without Lime Treatment.

Infections by A/H5 and A/H7 avian influenza viruses (AIVs) can cause acute disease and are therefore notifiable in poultry and wild birds. During winter 2015-2016, several cases of infection caused by highly pathogenic (HP) AIVs belonging to the A/H5N1, A/H5N2, and A/H5N9 subtypes were detected in southwestern France. Throughout winter 2016-2017, several cases of infections caused mainly by A/H5N8 HP AIV (A/goose/GD/1/1996, clade 2.3.4.4) were detected across Europe. On both occasions, the viruses were widely detected on palmiped farms in France. This study was designed to evaluate the persistence of A/H5 HP AIV in slurry from various duck productions. This was achieved (i) in the laboratory setting by artificially spiking four AIV-free slurry samples with known amounts of A/H5N9 HP AIV and monitoring virus infectivity, with or without lime treatment to achieve pH 10 or pH 12, and (ii) by sampling slurry tanks on five naturally A/H5N8 HP-contaminated farms. Experimental results in artificially spiked slurry suggested virus survival for 4 weeks in slurry from Muscovy or Pekin duck breeders and for 2 weeks in slurry from ducks for foie gras production during the assisted-feeding period, without lime treatment. Persistence of infectious A/H5N9 HP AIV in all slurry samples after lime treatment at pH 10 or pH 12 was less than 1 week. The A/H5N8 HP AIV persisted in naturally contaminated untreated slurry for 7 weeks. The results obtained provide experimental support for the 60-day storage period without treatment or the 7-day interval after lime treatment defined in French regulations for slurry sanitization.IMPORTANCE From November 2015 to July 2017, two successive episodes of H5 highly pathogenic avian influenza viruses (HP AIVs) infections occurred on poultry farms in France, mostly in domestic ducks raised for foie gras production in southwestern France. During the two epizootics, epidemiological investigations were carried out on infected farms and control and biosafety measures were implemented in association with surveillance in order to stop the spread of the viruses. Effluents are known to be an important factor in environmental dissemination of viruses, and suitable effluent management is needed to help prevent the spread of epizootics to other farms or pathogen persistence at the farm level. The present study was therefore designed to assess how long infectious A/H5 HP AIVs can persist in naturally or experimentally contaminated fecal slurry samples from ducks, with or without sanitization by lime treatment.

Detection of Enteric Viruses and Bacterial Indicators in a Sewage Treatment Center and Shallow Water Bay.

The incidence of enteric viruses in treated wastewater and their potential release into the environment or use for agriculture are very critical matters in public health. In our study, PCR (polymerase chain reaction) analysis of enteric viruses was performed on 59 samples of influents and effluents collected from Tubli wastewater treatment plant (Water Pollution Control Center (WPCC)) and Tubli Bay, where the effluents were discharged, in Kingdom of Bahrain during two sampling periods. Four clinically essential waterborne enteric viruses were examined: enterovirus (EV), hepatitis A virus (HAV), astroviruses (AV), and rotaviruses (RV) and compared to standard bacterial and bacteriophages indicators of fecal pollution. Detection rates of EV, AV, HAV, and RV in the influent samples were 100%, 75%, 12.5%, and 12.5%, respectively, while 50% of the effluent samples from Tubli WPCC contained only EV RNA. None of the tested enteric viruses could be detected in any of the samples collected directly from Tubli Bay. Effluent samples from Tubli plant did not show significant seasonal differences. Since detection of enteric viruses genome does not necessarily indicate infectivity, the infectivity of these viruses was evaluated through isolation and growth of indictor bacteria and bacteriophages. High concentration of fecal bacteriological indicators was detected in all effluents samples (100%): 3.20 × 103 cfu/mL for E. coli, 1.32 × 103 cfu/mL for Salmonella spp., and 1.92 × 103 cfu/mL for Shigella spp. E. coli and Salmonella specific bacteriophages were also detected in the effluent samples in high titers. The combined results of PCR and bacterial enumeration point to a probable public health risk via the use of these wastewaters in agriculture or their discharge into the sea. Continuous surveillance of viral and bacterial prevalence and their resistance to sewage disinfection procedures could contribute to a better control of risks associated with the recycling of effluent wastewater and its release into the environment.

Isolation and characterization of a potentially novel Siphoviridae phage (vB_SsapS-104) with lytic activity against Staphylococcus saprophyticus isolated from urinary tract infection.

Antibiotic resistance is increasing among Staphylococcus saprophyticus strains isolated from urinary tract infection. This necessitates alternative therapies. For this, a lytic phage (vB_SsapS-104) against S. saprophyticus, which formed round and clear plaques on bacterial culture plates, was isolated from hospital wastewater and characterized. Microscopy analysis showed that it had a small head (about 50 nm), tail (about 80 nm), and a collar (about 22 nm in length and 19 nm in width) indicating to be a phage within Siphoviridae family. Phage vB_SsapS-104 showed a large latency period of about 40 min, rapid adsorption rate that was significantly enhanced by MgCl2 and CaCl2, and high stability to a wide range of temperatures and pH values. Restriction analyses demonstrated that phage consists of a double-stranded DNA with an approximate genome size of 40 Kb. BLAST results did not show high similarity (megablast) with other previously identified phages. But, in Blastn, similarity with Staphylococcus phages was observed. Phage vB_SsapS-104 represented high anti-bacterial activity against S. saprophyticus isolates in vitro as it was able to lyse 8 of the 9 clinical isolates (%88.8) obtained from a hospital in Gorgan, Iran. It was a S. saprophyticus-specific phage because no lytic activity was observed on some other pathogenic bacteria tested. Therefore, phage vB_SsapS-104 can be considered as a specific virulent phage against of S. saprophyitcus isolated from urinary tract infection. This study provided the partial genomic characterization of S. saprophyticus phage and its application against urinary tract infection associated with S. saprophyticus. This phage also can be considered as a good candidate for a therapeutic alternative in the future.

Efficacy of lytic Staphylococcus aureus bacteriophage against multidrug-resistant Staphylococcus aureus in mice.

INTRODUCTION: The use of bacteriophages as an alternative treatment method against multidrug-resistant bacteria has not been explored in Kenya. This study sought to determine the efficacy of environmentally obtained lytic bacteriophage against multidrug-resistant Staphylococcus aureus (MDRSA) bacterium in mice. METHODOLOGY: Staphylococcus aureus bacterium and S. aureus-specific lytic phage were isolated from sewage and wastewater collected within Nairobi County, Kenya. Thirty mice were randomly assigned into three groups: MDRSA infection group (n = 20), phage-infection group (n = 5), and non-infection group (n = 5). The MDRSA infection group was further subdivided into three groups: clindamycin treatment (8 mg/kg; n = 5), lytic phage treatment (108 PFU/mL (n = 5), and a combination treatment of clindamycin and lytic phage (n = 5). Treatments were done at either 24 or 72 hours post-infection (p.i), and data on efficacy, bacterial load, and animal physical health were collected. RESULTS: Treatment with phage was more effective (100%) than with clindamycin (62.25% at 24 hours p.i and 87.5% at 72 hours p.i.) or combination treatment (75% at 24 hours p.i. and 90% at 72 hours p.i.) (p < 0.001). CONCLUSIONS: The results show that the environmentally obtained S. aureus lytic bacteriophage has therapeutic potential against MDRSA bacterium in mice.