A high-performance polymer composite column for coronavirus nucleic acid purification.

Here, we report the development of a novel polymer composite (PC) purification column and kit. The performance of the PC columns was compared to conventional silica gel (SG) columns for the purification of nucleic acids from coronaviruses, including SARS-CoV-2, in 82 clinical samples. The results shows that PC-based purification outperforms silica gel (SG)-based purification by enabling a higher sensitivity (94%), accuracy (97%), and by eliminating false positives (100% specificity). The high specificity is critical for efficient patient triage and resource management during pandemics. Furthermore, PC-based purification exhibits three times higher analytical precision than a commonly used SG-based nucleic acid purification thereby enabling a more accurate quantification of viral loads and higher reproducibility.

A High-Performance Polymer Composite Column for Coronavirus Nucleic Acid Purification.

Here, we report the development of a novel polymer composite (PC) purification column and kit. The performance of the PC columns was compared to conventional silica gel (SG) columns for the purification of nucleic acids from coronaviruses, including SARS-CoV-2, in 82 clinical samples. The results shows that PC-based purification outperforms silica gel (SG)-based purification by enabling a higher sensitivity (94%), accuracy (97%), and by eliminating false positives (100% selectivity). The high selectivity is critical for efficient patient triage and resource management during pandemics. Furthermore, PC-based purification exhibits three times higher analytical precision than a commonly used SG-based nucleic acid purification thereby enabling a more accurate quantification of viral loads and higher reproducibility.

In vitro virucidal activity of a commercial disinfectant against viruses of domestic animals and poultry.

Outbreaks of viral diseases in animals are a cause of concern for animal welfare and economics of animal production. One way to disrupt the cycle of infection is by combating viruses in the environment and prohibiting them from being transmitted to a new host. Viral contamination of the environment can be reduced using well-tested and efficacious disinfectants. Duplalim is a commercially available disinfectant consisting of 12% glutaraldehyde and 10% quaternary ammonium compounds. We evaluated this disinfectant for its efficacy against several viruses in poultry (n = 3), pigs (n = 5), dogs (n = 2), and cattle (n = 4). In suspension tests, 1:100 dilution of Duplalim was found to inactivate more than 99% of these 14 viruses in 15 min or less. The titers of a majority of these viruses decreased by ≥99.99% in <60 min of contact time. In conclusion, the ingredient combination in Duplalim is very effective in inactivating common viruses of domestic animals and poultry.

Durable nanocomposite face masks with high particulate filtration and rapid inactivation of coronaviruses.

The COVID-19 pandemic presents a unique challenge to the healthcare community due to the high infectivity rate and need for effective personal protective equipment. Zinc oxide nanoparticles have shown promising antimicrobial properties and are recognized as a safe additive in many food and cosmetic products. This work presents a novel nanocomposite synthesis approach, which allows zinc oxide nanoparticles to be grown within textile and face mask materials, including melt-blown polypropylene and nylon-cotton. The resulting nanocomposite achieves greater than 3 log10 reduction (≥ 99.9%) in coronavirus titer within a contact time of 10 min, by disintegrating the viral envelope. The new nanocomposite textile retains activity even after 100 laundry cycles and has been dermatologist tested as non-irritant and hypoallergenic. Various face mask designs were tested to improve filtration efficiency and breathability while offering antiviral protection, with Claros' design reporting higher filtration efficiency than surgical masks (> 50%) for particles ranged 200 nm to 5 µm in size.

Comparison of samplers collecting airborne influenza viruses: 1. Primarily impingers and cyclones.

Researchers must be able to measure concentrations, sizes, and infectivity of virus-containing particles in animal agriculture facilities to know how far infectious virus-containing particles may travel through air, where they may deposit in the human or animal respiratory tract, and the most effective ways to limit exposures to them. The objective of this study was to evaluate a variety of impinger and cyclone aerosol or bioaerosol samplers to determine approaches most suitable for detecting and measuring concentrations of virus-containing particles in air. Six impinger/cyclone air samplers, a filter-based sampler, and a cascade impactor were used in separate tests to collect artificially generated aerosols of MS2 bacteriophage and swine and avian influenza viruses. Quantification of infectious MS2 coliphage was carried out using a double agar layer procedure. The influenza viruses were titrated in cell cultures to determine quantities of infectious virus. Viral RNA was extracted and used for quantitative real time RT-PCR, to provide total virus concentrations for all three viruses. The amounts of virus recovered and the measured airborne virus concentrations were calculated and compared among the samplers. Not surprisingly, high flow rate samplers generally collected greater quantities of virus than low flow samplers. However, low flow rate samplers generally measured higher, and likely more accurate, airborne concentrations of Infectious virus and viral RNA than high flow samplers. To assess airborne viruses in the field, a two-sampler approach may work well. A suitable high flow sampler may provide low limits of detection to determine if any virus is present in the air. If virus is detected, a suitable lower flow sampler may measure airborne virus concentrations accurately.

Greater than 3-Log Reduction in Viable Coronavirus Aerosol Concentration in Ducted Ultraviolet-C (UV-C) Systems.

Control technologies to inactivate airborne viruses effectively are needed during the ongoing SARS-CoV-2 pandemic, and to guard against airborne transmitted diseases. We demonstrate that sealed UV-C flow reactors operating with fluences near 253 ± 1 nm of 13.9-49.6 mJ cm-2 efficiently inactivate coronaviruses in an aerosol. For measurements, porcine respiratory coronavirus (PRCV) was nebulized in a custom-built, 3.86 m wind tunnel housed in a biosafety level class II facility. The single pass log10 reduction of active coronavirus was in excess of 2.2 at a flow rate of 2439 L min-1 (13.9 mJ cm-2) and in excess of 3.7 (99.98% removal efficiency) at 684 L min-1 (49.6 mJ cm-2). Because virus titers resulting from sampling downstream of the UV-C reactor were below the limit of detection, the true log reduction is likely even higher than measured. Comparison of virus titration results to reverse transcriptase quantitative PCR and measurement of fluorescein concentrations (doped into the nebulized aerosol) reveals that the reduction in viable PRCV is primarily due to UV-C based inactivation, as opposed to physical collection of virus. The results confirm that UV-C flow reactors can efficiently inactivate coronaviruses through incorporation into HVAC ducts or recirculating air purifiers.

Stability of SARS-CoV-2 and other coronaviruses in the environment and on common touch surfaces and the influence of climatic conditions: A review.

Although the unprecedented efforts the world has been taking to control the spread of the human coronavirus disease (COVID-19) and its causative aetiology [severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)], the number of confirmed cases has been increasing drastically. Therefore, there is an urgent need for devising more efficient preventive measures, to limit the spread of the infection until an effective treatment or vaccine is available. The preventive measures depend mainly on the understanding of the transmission routes of this virus, its environmental stability, and its persistence on common touch surfaces. Due to the very limited knowledge about SARS-CoV-2, we can speculate its stability in the light of previous studies conducted on other human and animal coronaviruses. In this review, we present the available data on the stability of coronaviruses (CoVs), including SARS-CoV-2, from previous reports to help understand its environmental survival. According to available data, possible airborne transmission of SARS-CoV-2 has been suggested. SARS-CoV-2 and other human and animal CoVs have remarkably short persistence on copper, latex and surfaces with low porosity as compared to other surfaces like stainless steel, plastics, glass and highly porous fabrics. It has also been reported that SARS-CoV-2 is associated with diarrhoea and that it is shed in the faeces of COVID-19 patients. Some CoVs show persistence in human excrement, sewage and waters for a few days. These findings suggest a possible risk of faecal-oral, foodborne and waterborne transmission of SARS-CoV-2 in developing countries that often use sewage-polluted waters in irrigation and have poor water treatment systems. CoVs survive longer in the environment at lower temperatures and lower relative humidity. It has been suggested that large numbers of COVID-19 cases are associated with cold and dry climates in temperate regions of the world and that seasonality of the virus spread is suspected.

Genetic Diversity of Ornithobacterium rhinotracheale Isolated from Chickens and Turkeys in the United States.

Ornithobacterium rhinotracheale (ORT) is an important bacterial pathogen of great economic significance to poultry production. This bacterium causes severe disease in chickens and turkeys worldwide. The objective of this study was to characterize ORT isolates from two different geographic locations in the United States by multilocus sequence typing (MLST). A total of 60 isolates were included in this study; 36 from California and 24 from Minnesota. All 60 isolates were confirmed to be ORT by PCR that targeted the 16S rRNA gene. The results of MLST revealed eight different sequence types (ST) of ORT. Out of these, four were novel and were assigned numbers ST-32, ST-33, ST-34, and ST-35. ST-1 was the predominant sequence type among all isolates followed by ST-9 and ST-8. Only one isolate was identified as ST-2. No significant variation was seen in STs in ORT isolated from different years. In turkeys, 76.3% (29/38) of isolates belonged to ST-1 and 7.9% (3/38) to ST-8. Of the chicken isolates, 72.2% (13/18) belonged to ST-1 and 16.6% (3/18) to ST-9. Isolates from both states showed low genetic variability. Of the 32 isolates from California, 24 (75%) were identified as ST-1 and 4 (12.5%) were identified as ST-9. The most prevalent sequence type was ST-1 (17/24) followed by ST-8 (3/24) in Minnesota. Three isolates from turkeys in Minnesota belonged to the same ST (ST-8) as the already known ORT strain RefO, which isolated from a rook in Germany in 2000. Whether this sequence type had evolved from wild birds could not be ascertained in this study.

Virulence factors and antibiograms of Escherichia coli isolated from diarrheic calves of Egyptian cattle and water buffaloes.

Diarrhea caused by Escherichia coli in calves is an important problem in terms of survivability, productivity and treatment costs. In this study, 88 of 150 diarrheic animals tested positive for E. coli. Of these, 54 samples had mixed infection with other bacterial and/or parasitic agents. There are several diarrheagenic E. coli pathotypes including enteropathogenic E. coli (EPEC), Shiga-toxin producing E. coli (STEC), enterotoxigenic E. coli (ETEC) and necrotoxigenic E. coli (NTEC). Molecular detection of virulence factors Stx2, Cdt3, Eae, CNF2, F5, Hly, Stx1, and ST revealed their presence at 39.7, 27.2, 19.3, 15.9, 13.6, 9.0, 3.4, and 3.4 percent, respectively. As many as 13.6% of the isolates lacked virulence genes and none of the isolate had LT or CNF1 toxin gene. The odds of isolating ETEC from male calves was 3.6 times (95% CI: 1.1, 12.4; P value = 0.042) that of female calves, whereas the odds of isolating NTEC from male calves was 72.9% lower (95% CI: 91.3% lower, 15.7% lower; P value = 0.024) than that in females. The odds of isolating STEC in winter was 3.3 times (95% CI: 1.1, 10.3; P value = 0.037) that of spring. Antibiograms showed 48 (54.5%) of the isolates to be multi-drug resistant. The percent resistance to tetracycline, streptomycin, ampicillin, and trimethoprim-sulfamethoxazole was 79.5, 67.0, 54.5, and 43.0, respectively. Ceftazidime (14.8%), amoxicillin-clavulanic acid (13.6%) and aztreonam (11.3%) showed the lowest resistance, and none of the isolates was resistant to imipenem. The results of this study can help improve our understanding of the epidemiological aspects of E. coli infection and to devise strategies for protection against it. The prevalence of E. coli pathotypes can help potential buyers of calves to avoid infected premises. The antibiograms in this study emphasizes the risks associated with the random use of antibiotics.

Bactericidal Efficacy of a Two-Dimensional Array of Integrated, Coaxial, Microhollow, Dielectric Barrier Discharge Plasma Against Salmonella enterica Serovar Heidelberg.

We studied the efficacy of cold atmospheric-pressure plasma (CAP), generated by a two-dimensional array of integrated, coaxial, microhollow, dielectric barrier discharge plasma, against Salmonella enterica serovar Heidelberg (SH) on stainless steel, romaine lettuce, and chicken breast. Exposure of SH to CAP on a dry stainless steel surface had low bactericidal efficacy; only 2.5 log10 colony-forming units (CFUs) were inactivated after 10 min of exposure. On the other hand, the presence of moisture led to decontamination of ∼6.5 log10 CFUs after only 3 min. Although complete decontamination was not achieved on lettuce and chicken breast samples after 10 min of exposure, SH counts were reduced by ∼4.5 and 3.7 log10 CFUs, respectively. A partial suppression of bactericidal effects was observed on steel surfaces when it was coated with bovine serum albumin before spiking with bacteria and exposure to plasma, indicating that the proteinaceous nature of chicken meat may be partially responsible for lower efficacy of CAP on chicken muscles. The initial bacterial load was also found to affect the anti-SH efficacy; at high (∼6.5 log CFUs) and low (∼3.5 CFUs) initial counts, the time required for complete decontamination on stainless steel and lettuce decreased from 3 to 0.5 min and >10 to 1 min, respectively. However, the analysis of inactivation kinetics showed that effects of initial loads of contamination on the rate of bacterial inactivation were not statistically significant. This is consistent with other findings for conditions where both bacterial loads were under the multilayering threshold that might have affected the rate of killing.

Ìn situ inactivation of human norovirus GII.4 by cold plasma: Ethidium monoazide (EMA)-coupled RT-qPCR underestimates virus reduction and fecal material suppresses inactivation.

Cold atmospheric-gaseous plasma (CAP) is an emerging non-thermal technology for decontamination of foodborne bacterial and viral pathogens. We obtained a >5 log10 reduction in the titer (TCID50) of feline calicivirus (FCV) on stainless steel discs and Romaine lettuce leaves after 3 min wet exposure to air plasma generated by a two-dimensional array of integrated coaxial-microhollow dielectric barrier discharge (2D-AICM-DBD). However, when human norovirus (HuNoV GII.4) was treated for 5 min under the same conditions, ~2.6 log10 (>99.5%) reduction in genome copy number was observed as measured by ethidium monoazide-coupled RT-qPCR (EMA-RT-qPCR). To assess this discrepancy, we studied CAP's effect on FCV by the cell culture method and by the EMA-coupled RT-qPCR method. It was found that the molecular titration method (EMA-RT-qPCR) underestimates the level of virus reduction by CAP. Additionally, the fecal matter present in HuNoV samples partially suppressed virucidal activity of CAP. Assuming that the lower virus reduction measured by EMA-RT-qPCR method compared to cell culture method for FCV is the same as for HuNoV, we can conclude that FCV may be used as a surrogate for HuNoV to assess the virucidal effect of CAP. CAP is able to inactivate 3.5 Log10 units of HuNoV at low titers after 2 min of exposure.

Involvement of Egyptian Foods in Foodborne Viral Illnesses: The Burden on Public Health and Related Environmental Risk Factors: An Overview.

Foodborne viral diseases are a major public health threat and pose a huge burden on the economies of both developed and developing countries. Enteric viruses are the causative agents of most foodborne illnesses and outbreaks. Egypt is classified by WHO among the regions with intermediate to high endemicity for various enteric viruses. This is manifested by the high prevalence rates of different enteric virus infections among Egyptian population such as Hepatitis A and E viruses, human rotaviruses, human noroviruses, human astroviruses, and human adenovirus. Recently, a number of foodborne gastroenteritis and acute hepatitis outbreaks have occurred in the US, Canada, Australia, and the European Union countries. Some of these outbreaks were attributed to the consumption of minimally processed foods imported from Egypt indicating the possibility that Egyptian foods may also be partially responsible for high prevalence of enteric virus infections among Egyptian population. In the absence of official foodborne-pathogen surveillance systems, evaluating the virological safety of Egyptian foods is a difficult task. In this review, we aim to provide a preliminary evaluation of the virological safety of Egyptian foods. A comprehensive review of prevalence studies on enteric virus infections shows hyperendemicity of several enteric viruses in Egypt and provides strong evidence of implication of Egyptian foods in these infections. We also address possible environmental risk factors that may lead to the contamination of Egyptian foods with enteric viruses. In addition, we describe potential obstacles to any plan that might be considered for improving the virological safety of Egyptian foods.

Cold argon-oxygen plasma species oxidize and disintegrate capsid protein of feline calicivirus.

Possible mechanisms that lead to inactivation of feline calicivirus (FCV) by cold atmospheric-pressure plasma (CAP) generated in 99% argon-1% O2 admixture were studied. We evaluated the impact of CAP exposure on the FCV viral capsid protein and RNA employing several cultural, molecular, proteomic and morphologic characteristics techniques. In the case of long exposure (2 min) to CAP, the reactive species of CAP strongly oxidized the major domains of the viral capsid protein (VP1) leading to disintegration of a majority of viral capsids. In the case of short exposure (15 s), some of the virus particles retained their capsid structure undamaged but failed to infect the host cells in vitro. In the latter virus particles, CAP exposure led to the oxidation of specific amino acids located in functional peptide residues in the P2 subdomain of the protrusion (P) domain, the dimeric interface region of VP1 dimers, and the movable hinge region linking the S and P domains. These regions of the capsid are known to play an essential role in the attachment and entry of the virus to the host cell. These observations suggest that the oxidative effect of CAP species inactivates the virus by hindering virus attachment and entry into the host cell. Furthermore, we found that the oxidative impact of plasma species led to oxidation and damage of viral RNA once it becomes unpacked due to capsid destruction. The latter effect most likely plays a secondary role in virus inactivation since the intact FCV genome is infectious even after damage to the capsid.

In Vitro Antiviral Activity of Clove and Ginger Aqueous Extracts against Feline Calicivirus, a Surrogate for Human Norovirus.

Foodborne viruses, particularly human norovirus, are a concern for public health, especially in fresh vegetables and other minimally processed foods that may not undergo sufficient decontamination. It is necessary to explore novel nonthermal techniques for preventing foodborne viral contamination. In this study, aqueous extracts of six raw food materials (flower buds of clove, fenugreek seeds, garlic and onion bulbs, ginger rhizomes, and jalapeño peppers) were tested for antiviral activity against feline calicivirus (FCV) as a surrogate for human norovirus. The antiviral assay was performed using dilutions of the extracts below the maximum nontoxic concentrations of the extracts to the host cells of FCV, Crandell-Reese feline kidney (CRFK) cells. No antiviral effect was seen when the host cells were pretreated with any of the extracts. However, pretreatment of FCV with nondiluted clove and ginger extracts inactivated 6.0 and 2.7 log of the initial titer of the virus, respectively. Also, significant dosedependent inactivation of FCV was seen when host cells were treated with clove and ginger extracts at the time of infection or postinfection at concentrations equal to or lower than the maximum nontoxic concentrations. By comprehensive two-dimensional gas chromatography-mass spectrometry analysis, eugenol (29.5%) and R-(-)-1,2-propanediol (10.7%) were identified as the major components of clove and ginger extracts, respectively. The antiviral effect of the pure eugenol itself was tested; it showed antiviral activity similar to that of clove extract, albeit at a lower level, which indicates that some other clove extract constituents, along with eugenol, are responsible for inactivation of FCV. These results showed that the aqueous extracts of clove and ginger hold promise for prevention of foodborne viral contamination.

Identification of the biologically active liquid chemistry induced by a nonthermal atmospheric pressure plasma jet.

The mechanism of interaction of cold nonequilibrium plasma jets with mammalian cells in physiologic liquid is reported. The major biological active species produced by an argon RF plasma jet responsible for cell viability reduction are analyzed by experimental results obtained through physical, biological, and chemical diagnostics. This is complemented with chemical kinetics modeling of the plasma source to assess the dominant reactive gas phase species. Different plasma chemistries are obtained by changing the feed gas composition of the cold argon based RF plasma jet from argon, humidified argon (0.27%), to argon/oxygen (1%) and argon/air (1%) at constant power. A minimal consensus physiologic liquid was used, providing isotonic and isohydric conditions and nutrients but is devoid of scavengers or serum constituents. While argon and humidified argon plasma led to the creation of hydrogen peroxide dominated action on the mammalian cells, argon-oxygen and argon-air plasma created a very different biological action and was characterized by trace amounts of hydrogen peroxide only. In particular, for the argon-oxygen (1%), the authors observed a strong negative effect on mammalian cell proliferation and metabolism. This effect was distance dependent and showed a half life time of 30 min in a scavenger free physiologic buffer. Neither catalase and mannitol nor superoxide dismutase could rescue the cell proliferation rate. The strong distance dependency of the effect as well as the low water solubility rules out a major role for ozone and singlet oxygen but suggests a dominant role of atomic oxygen. Experimental results suggest that O reacts with chloride, yielding Cl2(-) or ClO(-). These chlorine species have a limited lifetime under physiologic conditions and therefore show a strong time dependent biological activity. The outcomes are compared with an argon MHz plasma jet (kinpen) to assess the differences between these (at least seemingly) similar plasma sources.

Virucidal effect of cold atmospheric gaseous plasma on feline calicivirus, a surrogate for human norovirus.

Minimal food-processing methods are not effective against foodborne viruses, such as human norovirus (NV). It is important, therefore, to explore novel nonthermal technologies for decontamination of foods eaten fresh, minimally processed and ready-to-eat foods, and food contact surfaces. We studied the in vitro virucidal activity of cold atmospheric gaseous plasma (CGP) against feline calicivirus (FCV), a surrogate of NV. Factors affecting the virucidal activity of CGP (a so-called radio frequency atmospheric pressure plasma jet) were the plasma generation power, the exposure time and distance, the plasma feed gas mixture, and the virus suspension medium. Exposure to 2.5-W argon (Ar) plasma caused a 5.55 log10 unit reduction in the FCV titer within 120 s. The reduction in the virus titer increased with increasing exposure time and decreasing exposure distance. Of the four plasma gas mixtures studied (Ar, Ar plus 1% O2, Ar plus 1% dry air, and Ar plus 0.27% water), Ar plus 1% O2 plasma treatment had the highest virucidal effect: more than 6.0 log10 units of the virus after 15 s of exposure. The lowest virus reduction was observed with Ar plus 0.27% water plasma treatment (5 log10 unit reduction after 120 s). The highest reduction in titer was observed when the virus was suspended in distilled water. Changes in temperature and pH and formation of H2O2 were not responsible for the virucidal effect of plasma. The oxidation of viral capsid proteins by plasma-produced reactive oxygen and nitrogen species in the solution was thought to be responsible for the virucidal effect. In conclusion, CGP exhibits virucidal activity in vitro and has the potential to combat viral contamination in foods and on food preparation surfaces.

Antiviral Effects of Lactococcus lactis on Feline Calicivirus, A Human Norovirus Surrogate.

Foodborne viruses, particularly human norovirus (NV) and hepatitis virus type A, are a cause of concern for public health making it necessary to explore novel and effective techniques for prevention of foodborne viral contamination, especially in minimally processed and ready-to-eat foods. This study aimed to determine the antiviral activity of a probiotic lactic acid bacterium (LAB) against feline calicivirus (FCV), a surrogate of human NV. Bacterial growth medium filtrate (BGMF) of Lactococcus lactis subsp. lactis LM0230 and its bacterial cell suspension (BCS) were evaluated separately for their antiviral activity against FCV grown in Crandell-Reese feline kidney (CRFK) cells. No significant antiviral effect was seen when CRFK cells were pre-treated with either BGMF (raw or pH 7-adjusted BGMF) or BCS. However, pre-treatment of FCV with BGMF and BCS resulted in a reduction in virus titers of 1.3 log10 tissue culture infectious dose (TCID)50 and 1.8 log10 TCID50, respectively. The highest reductions in FCV infectivity were obtained when CRFK cells were co-treated with FCV and pH 7-adjusted BGMF or with FCV and BCS (7.5 log10 TCID50 and 6.0 log10 TCID50, respectively). These preliminary results are encouraging and indicate the need for continued studies on the role of probiotics and LAB on inactivation of viruses in various types of foods.

Some factors affecting tannase production by Aspergillus niger Van Tieghem.

One variable at a time procedure was used to evaluate the effect of qualitative variables on the production of tannase from Aspergillus niger Van Tieghem. These variables including: fermentation technique, agitation condition, tannins source, adding carbohydrates incorporation with tannic acid, nitrogen source type and divalent cations. Submerged fermentation under intermittent shaking gave the highest total tannase activity. Maximum extracellular tannase activity (305 units/50 mL) was attained in medium containing tannic acid as tannins source and sodium nitrate as nitrogen source at 30 °C for 96 h. All added carbohydrates showed significant adverse effects on the production of tannase. All tested divalent cations significantly decreased tannase production. Moreover, split plot design was carried out to study the effect of fermentation temperature and fermentation time on tannase production. The results indicated maximum tannase production (312.7 units/50 mL) at 35 °C for 96 h. In other words, increasing fermentation temperature from 30 °C to 35 °C resulted in increasing tannase production.

Some factors affecting tannase production by Aspergillus niger Van Tieghem

One variable at a time procedure was used to evaluate the effect of qualitative variables on the production of tannase from Aspergillus niger Van Tieghem. These variables including: fermentation technique, agitation condition, tannins source, adding carbohydrates incorporation with tannic acid, nitrogen source type and divalent cations. Submerged fermentation under intermittent shaking gave the highest total tannase activity. Maximum extracellular tannase activity (305 units/ 50 mL) was attained in medium containing tannic acid as tannins source and sodium nitrate as nitrogen source at 30 ºC for 96 h. All added carbohydrates showed significant adverse effects on the production of tannase. All tested divalent cations significantly decreased tannase production. Moreover, split plot design was carried out to study the effect of fermentation temperature and fermentation time on tannase production. The results indicated maximum tannase production (312.7 units/50 mL) at 35 ºC for 96 h. In other words, increasing fermentation temperature from 30 ºC to 35 ºC resulted in increasing tannase production.