Combination of cell culture and quantitative PCR (cc–qPCR) to assess disinfectants efficacy on Cryptosporidium oocysts under standardized conditions

Oocysts of Cryptosporidium parvum are resistant to environmental conditions and many disinfectants. A combination of cell culture and quantitative real time PCR (cc–qPCR) is established for evaluation of anticoccidial disinfectants against C. parvum. C. parvum oocysts were treated with disinfectants, washed and oocysts were incubated with HCT-8 cell monolayers in the presence of excystation medium for 3 h. Subsequently, unbound parasites were removed by washing with growing medium and the infected monolayers were further maintained in fresh growing medium for 48 h. Genomic DNA was extracted from each sample and qPCR performed targeting a specific sequence of the 70 kDa heat shock protein gene in order to quantify development. Treatment of oocysts with cresolic disinfectants demonstrated dose dependent reduction of viability of oocysts. More than 98% inactivations were recorded with at least 2% concentration of cresolic disinfectants after 2 h of treatment. Bleach (sodium hypochlorite) at 6% solution induced 92.7% inactivation of C. parvum oocysts after 2 h. Thermally treated oocysts (56 and 70 °C for 20 min) demonstrated complete inactivation, whereas at 38 °C no inactivation was observed. Application of Neopredisan^® 135-1 and Aldecoc^® TGE (4% for 2 h) as recommended according to the current guidelines stipulated by DVG (German Veterinary Society) consistently inactivated more than 99.5% of oocysts. The suggested cc–qPCR method appeared to be suited for standardized testing of inactivation measures, particularly for evaluation of chemical disinfectants and thus cc–qPCR is proposed as an alternative to the established chicken infectivity model for Eimeria tenella for testing anticoccidial disinfectants. A minimum inactivation of 99.5% in cc–qPCR model is claimed as a suitable threshold for certification of chemical products for disinfection of coccidia oocysts.     

球虫消毒剂的筛选及氨水对艾美耳球虫卵囊抑杀条件的优化

为了筛选有效的球虫消毒剂,本研究将柔嫩艾美耳球虫卵囊分别与16种常用消毒剂和1种高效抗球虫药物溶液（250 mg/L妥曲珠利溶液）混合作用2 h然后在2.5%重铬酸钾溶液中孢子化培养,或混合作用4 h后接种14日龄雏鸡,根据卵囊孢子化率、孢子化抑制率、每克粪便卵囊数量（oocysts per gram of feces,OPG）、病变计分和血便情况评价消毒剂和抗球虫药物抑杀球虫卵囊效果。结果显示,8%氨水对未孢子化卵囊和孢子化卵囊均表现为100%的抑杀效果;250 mg/L妥曲珠利、3%甲醛、4%苯酚、0.5%过氧乙酸、5% NaOH+10% NaCl和5% NaOH对球虫卵囊活性具有一定的抑制作用,孢子化抑制率超过10%,但是对孢子化卵囊抑杀作用不明显;而剩余10种常用消毒剂对球虫卵囊没有明显的抑杀作用。氨水抑杀球虫卵囊条件优化的试验结果显示,1%~8%氨水与未孢子化卵囊作用1 h以上,均可100%抑杀球虫卵囊活性。粪便干扰试验结果显示,粪便不影响2%和4%氨水对球虫卵囊的抑杀效果。本研究结果说明常用消毒剂和抗球虫药物不能有效杀灭球虫卵囊的活性,而氨水是一种高效球虫消毒剂。本研究结果可为开发新型高效球虫消毒剂和球虫病控制提供参考。     

Attenuation of Eimeria species: further characterisation of two lines of Eimeria mitis

The immunogenicity of a 'precocious' and attenuated line (HP10) of Eimeria mitis was studied and the stability of attenuation of two precocious lines was compared with that of an embryo-adapted line. Chicks housed in wire-floored cages and given 1 X 10(5) oocysts of the HP10 line were protected against challenge with the parent Houghton strain and two field strains, but remained partially susceptible to the Durham and one other field strain. However, when chicks were kept in litter pens so that reinfection could occur freely, inoculation with as few as 1 X 10(3) oocysts of the precocious line resulted in complete immunity to both the Houghton and Durham strains for at least five weeks afterwards. The stability of attenuation of the precocious and embyro-adapted lines was examined by relaxing the selection pressures used to produce attenuation and then testing the virulence of the resulting lines. The precocious lines remained attenuated after several consecutive relaxed passages, in contrast to the embyro-adapted line which showed a marked reversion to virulence after six passages in chickens.     

Substitution of vaccinia virus Elstree by modified vaccinia virus Ankara to test the virucidal efficacy of chemical disinfectants

After the eradication of variola in 1980, the smallpox vaccination was considered to be no longer required and was subsequently abandoned mainly because of possible adverse effects of vaccinia virus especially in first‐time vaccinees. Despite a growing number of humans without immunity against vaccinia virus, vaccinia virus Lister Elstree (VACV) is still prescribed for testing virucidal efficacy of chemical disinfectants in the guidelines of the German Veterinary Medical Society [Deutsche Veterinärmedizinische Gesellschaft (DVG)], the German Association for the Control of Virus Diseases [Deutsche Vereinigung zur Bekämpfung der Viruskrankheiten (DVV)] and the Robert Koch Institute (RKI). To evaluate a possible substitution of VACV, with the attenuated modified vaccinia virus Ankara (MVA) the virucidal efficacy of four different DVG‐listed commercially available chemical disinfectants representing different groups of chemicals was tested against these two viruses. Quantitative suspension tests and qualitative carrier tests with poplar wood and gauze were performed. Distinction of VACV and MVA was confirmed by cytopathogenic effects, such as differences in plaque morphology. No significant difference in disinfection efficacy between VACV and MVA was observed for any of the disinfectants tested. Implying that vaccinia virus poses a risk after inadvertent inoculation, our results show that MVA, which does not replicate in humans, should replace VACV in the chemical disinfectant testing guidelines.     

Eimeria tenella in chickens: Studies on resistance to the anticoccidial drugs monensin and lasalocid

The response of the Houghton strain of E. tenella to monensin and lasalocid is described. Neither drug was able to completely control this strain at 125 ppm. At this concentration oocyst production was not completely suppressed by monensin in birds given ten oocysts. At higher concentrations both drugs were able to control E. tenella (H) in birds given 1 × 10⁵ oocysts but monensin was toxic. 375 ppm monensin did not prevent oocyst production in birds given 4 × 10⁶ oocysts. Resistance could not be developd to either of these compounds after 12 serial passages in the presence of drug. Serial passage of E. tenella (H) in the presence of monensin however resulted in an increase in pathogenecity. This greater pathogenecity was not due to a better ability to multiply in the host.     

Efficacy of amine-based disinfectant KENO™COX on the infectivity of Cryptosporidium parvum oocysts

Cryptosporidium parvum is a zoonotic protozoan parasite that may cause severe neonatal diarrhoea or even mortality in newborn ruminants: its oocysts are extremely resistant to normal environmental conditions and to most common disinfectants. KENO?COX, a patent pending amine-based formula, was tested for its ability to inactivate C. parvum oocysts. The Daugschies assay (2002), a standardized assay for chemical disinfection initially described for Eimeria spp., was adapted for C. parvum oocysts. KENO?COX diluted in water at 2% and 3% concentration and incubated with oocyst suspensions for 2h, allowed a significant reduction in viability, lysing 89% and 91% of oocysts respectively. Infectivity of the remaining C. parvum oocysts was assessed by inoculation to C57 Bl/6 neonatal mice. Each mouse received 2.5 μl of a suspension initially containing 500,000 oocysts before contact with KENO?COX. Six days post inoculation, the intestinal parasite load was significantly reduced by 97.5% with KENO?COX 2% compared to that of the mice inoculated with untreated parasites. KENO?COX 3% completely eliminated infectivity of oocysts. The number of oocysts remaining infectious in the inoculum treated with KENO?COX 2% was calculated from an inoculated dose-response curve: it was estimated at about 48.6 oocysts among the 500,000 oocysts initially treated corresponding to 99.99% of inhibition. These results demonstrate the high efficacy of KENO?COX against C. parvum oocysts. Combined with an appropriate method of cleaning, the application of KENO?COX may be a useful tool to reduce cryptosporidial infectious load on farm level.     

The anti-microbial activity of electrolysed oxidizing water against microorganisms relevant in veterinary medicine

Standards of the German Association of Veterinary Medicine (DVG) for the evaluation of chemical disinfectants were used to assess the anti-microbial efficacy of electrolysed oxidizing water (EOW). Enterococcus faecium, Mycobacterium avium subspecies avium, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans were exposed to anode EOW (pH, 3.0+/-0.1; oxidation-reduction potential (ORP), +1100+/-50 mV; free chlorine, 400+/-20 mg/l Cl2) and combined EOW (7:3 anode:cathode, v/v; pH, 8.3+/-0.1; ORP, 930-950 mV; free chlorine, 271+/-20 mg/l Cl2). In water of standardized hardness (WSH), all bacterial strains were completely inactivated by a 30 min exposure to maximum 10.0% anode EOW (approximately 40.0 mg/l Cl2) or 50.0% combined EOW (approximately 135.5 mg/l Cl2). The sensitivity ranking order for anode EOW to the bacterial test strains was P. mirabilis>S. aureus>M. avium ssp. avium>E. faecium>P. aeruginosa. P. mirabilis and S. aureus decreased to undetectable levels after 5 min of exposure to 7.5% anode EOW (approximately 30.0 mg/l Cl2). Candida albicans was completely inactivated by a 5-min exposure to 5.0% anode EOW. Both, anode and combined EOW exhibited no anti-microbial activities in standardized nutrient broth or after addition of 20.0% bovine serum to the WSH. Further research is necessary to evaluate the efficacy of EOW as a disinfectant under operating conditions in animal production facilities.     

Drinking water treatment processes for removal of Cryptosporidium and Giardia

Major waterborne cryptosporidiosis and giardiasis outbreaks associated with contaminated drinking water have been linked to evidence of suboptimal treatment. Cryptosporidium parvum oocysts are particularly more resistant than Giardia lamblia cysts to removal and inactivation by conventional water treatment (coagulation, sedimentation, filtration and chlorine disinfection); therefore, extensive research has been focused on the optimization of treatment processes and application of new technologies to reduce concentrations of viable/infectious oocysts to a level that prevents disease. The majority of the data on the performance of treatment processes to remove cysts and oocysts from drinking water have been obtained from pilot-tests, with a few studies performed in full-scale conventional water treatment plants. These studies have demonstrated that protozoan cyst removal throughout all stages of the conventional treatment is largely influenced by the effectiveness of coagulation pretreatment, which along with clarification constitutes the first treatment barrier against protozoan breakthrough. Physical removal of waterborne Crytosporidium oocysts and Giardia cysts is ultimately achieved by properly functioning conventional filters, providing that effective pretreatment of the water is applied. Disinfection by chemical or physical methods is finally required to inactivate/remove the infectious life stages of these organisms. The effectiveness of conventional (chlorination) and alternative (chlorine dioxide, ozonation and ultra violet [UV] irradiation) disinfection procedures for inactivation of Cryptosporidium has been the focus of much research due to the recalcitrant nature of waterborne oocysts to disinfectants. This paper provides technical information on conventional and alternative drinking water treatment technologies for removal and inactivation of the protozoan parasites Cryptosporidium and Giardia.     

Evaluation of two commercial disinfectants on the viability and infectivity of Cryptosporidium parvum oocysts

Cryptosporidiosis is mainly a problem in neonatal ruminants. Not only do Cryptosporidium spp. spread ubiquitously in our environment, but the protozoa are highly resistant to harsh environmental conditions and disinfectants, and a control measure is urgently required. This study investigated the potential biocidal activity on Cryptosporidium parvum oocysts of two commercial disinfectants developed originally to be used in farms and food-processing industries. The products, containing formaldehyde and hydrogen peroxide respectively, both had some anticryptosporidial effects. The viability and infectivity of purified C. parvum oocysts exposed to both disinfectants at different concentrations and exposure times were evaluated by inclusion or exclusion of vital dye (propidium iodide), use of an excystation technique and infection of suckling mice. Viability assays showed a decrease in oocyst viability associated with an increase in exposure time for each of the concentrations used. The intensity of infection in neonatal mice was significantly lower (P<0.05) than in the control litters.     

Susceptibility of Salmonella isolated from fish feed factories to disinfectants and air-drying at surfaces

The objective of this work was to determine whether persistence of certain Salmonella isolates in fish feed factories involved enhanced resistance to disinfectants or air-drying. Salmonella isolates known to be persistent in fish feed factories and Salmonella isolates from other origins were tested for their sensitivity to nine disinfectants by using a suspension test. More than 5log(10)reduction in viable count for all isolates was only achieved by two of the disinfectants at 80% of the lowest recommended user concentration. However, Salmonella isolates from fish feed factories were not more resistant to disinfectants compared to Salmonella from other origins. For four of the disinfectants, presence of fish feed had a more adverse effect on disinfection efficacy compared to the protein bovine serum albumin (BSA). In general, the Salmonella isolates were more resistant to disinfection than Escherichia coli DSM 682, a strain recommended in testing of disinfectants. The Salmonella isolates were also tested for their ability to survive air-drying at stainless steel surfaces, but there were no differences in survival of isolates from fish feed factories compared to isolates of other origin. In conclusion the Salmonella isolates from fish feed factories were not particularly resistant to disinfection or air-drying at surfaces. The data show that disinfectants to be used against Salmonella should be thoroughly tested and selected, since not all disinfectants appear to be effective against Salmonella.     

Protective immunity to toxoplasmosis in pigs vaccinated with a nonpersistent strain of Toxoplasma gondii

The RH strain of Toxoplasma gondii is highly virulent; 1 infective organism is uniformly lethal for mice. Three pigs inoculated SC with 10(3) tachyzoites of the RH strain developed fever, but otherwise remained normal, and T gondii was not demonstrated in their tissues by bioassay into mice. To determine whether vaccination with the RH strain could induce protective immunity to oral challenge with T gondii oocysts, 12 pigs were divided into 3 groups (A, B, C) of 4 pigs each. Pigs in groups A and B were inoculated IM with 10(6) tachyzoites of the RH strain and 4 pigs in group C served as uninoculated controls. Except for fever, the pigs remained clinically normal after inoculation with the RH strain and T gondii was not found by bioassay in mice of tissues from 4 pigs euthanatized 64 days after inoculation. Pigs in groups B and C were challenge-inoculated orally with 10(4) (4 pigs) or 10(5) (4 pigs) T gondii oocysts 72 days after vaccination with the RH strain. The previously uninoculated pigs developed fever, anorexia, and diarrhea from 3 to 8 days after the oocyst challenge. One of the 2 pigs given 10(5) oocysts became moribund because of toxoplasmosis and was euthanatized 9 days after inoculation. Pigs vaccinated with the RH strain remained free of clinical signs after challenge with oocysts. Results of the bioassays indicated that fewer tissue cysts developed in the RH strain-vaccinated pigs than in the previously uninoculated control pigs.     

Therapy and prevention of cryptosporidiosis in animals

Cryptosporidiosis is a common gastro-intestinal illness in animals and man worldwide. The disease is devastating in immune-suppressed individuals but self-limiting in competent hosts. The infectious stages of the organism (oocysts) are shed in the faeces of affected individuals, survive in adverse environmental conditions and spread by direct contact or through contaminants (food, water). Due to the robustness of the oocysts, their tenacity, tiny size, and resistance to common disinfectants, the parasite is difficult to eradicate from contaminated environments. To obtain sufficient control both treatment of infected hosts and inactivation of oocysts are necessary. Several drugs are commonly used to treat cryptosporidiosis in man and very few in animals but none of them are completely effective in terms of both clinical and parasitological response. Only a few chemical agents are able to inactivate oocysts in the environment including water treatment plants but their application has certain limitations. Therefore, control of cryptosporidiosis remains a global challenge in both veterinary and human medicine. Extensive research has been performed on suitable drugs and disinfectants. Thousands of agents have been tested both in vivo and in vitro. Some are excitingly active in vitro but exhibit poor or no response in clinical trials. Currently, no single or combined drug therapy has proven to be completely effective against this disease. This article will focus on therapy and prevention of cryptosporidiosis in animals including perspectives for new drugs.     

Disinfectant tests at 20 and 10 degrees C to determine the virucidal activity against circoviruses

To evaluate virucidal activity against porcine circovirus type 2 (PCV2), four disinfectants were tested under laboratory conditions. As basis to perform the testing the "Guidelines for testing chemical disinfectants" of the German Veterinary Association (DVG-guidelines) were applied. For simulation of field conditions, the tests were carried out in virus carrier tests, at 20 and 10 degrees C, and under protein load (40% foetal calf serum (FCS) in virus suspension). For disinfection of PCV2 at 20 degrees C an exposure time of 120 min in 2% Disinfectant 1 (20% glutaraldehyde, 12% 2-propenal, polymer with formaldehyde) or Disinfectant 2 (55% formic acid, 7% glyoxylic acid) was necessary. 1% of Disinfectant 3 (Component 1: Potassium peroxomonosulphate. Component 2: Active detergents) disinfected PCV2 on carriers within 180 min. After a reaction time of 120 min with 1% and 60 min with 2% Disinfectant 4 (21% glutaraldehyde, 17% formaldehyde) there could not be detected any virus. Reduction on effectivity through temperature reduction to 10 degrees C were more significant for aldehyde containing preparations Disinfectant 1 and Disinfectant 4 than for Disinfectant 2 and Disinfectant 3. These losses on effectivity could be corrected through extension of exposure time or increase of concentration.     

Eimeria tenella: experimental studies on the development of resistance to halofuginone

Resistance to halofuginone has been developed by serially passaging the Houghton strain of Eimeria tenella in chickens medicated with progressively greater concentrations of drug. Attempts to develop resistance to 3 ppm (as recommended for use by the manufacturer) or higher concentrations, by the method of Weppelman et al., were unsuccessful.     

Activity of bleach, ethanol and two commercial disinfectants against spores of Encephalitozoon cuniculi

Encephalitozoon cuniculi is a small protist parasite in the phylum Microspora. Hosts are infected by ingestion or inhalation of spores passed in the urine or feces. Infection with E. cuniculi is usually asymptomatic, except in young or immunocompromised hosts. This study examined the effects of various disinfectants on in vitro infectivity of E. cuniculi spores. Spores of E. cuniculi were exposed to several dilutions of commercial bleach, 70% ethanol and dilutions of commercial disinfectants HiTor and Roccal for 10 min and then loaded onto human fibroblast cells (Hs68 cells). Ten minutes of exposure to these disinfectants was lethal to E. cuniculi spores. Additional exposure time studies were done using dilutions of bleach at 0.1, 1 and 10%, and 70% ethanol. Exposure of E. cuniculi spores to 1 or 10% bleach for 30s rendered them non-infectious for Hs68 cells. Growth of E. cuniculi was observed in Hs68 cells inoculated with spores treated with 0.1% bleach for 30s or 1, 3 and 5 min, but not with spores treated for 7 min or longer. Exposure of E. cuniculi spores to 70% ethanol for 30s rendered them non-infectious for Hs68 cells. Spores of E. cuniculi are more sensitive to disinfectants than are coccidial oocysts and other parasite cysts. The relatively short contact time needed to kill spores indicates that disinfection of animal housing may be a viable means to reduce exposure of animals to E. cuniculi spores.     

Disinfection of caliciviruses at 20 and 10 degrees C

Five disinfectants, Venno FF super, Venno Vet 1 super, Venno Oxygen, M&Enno-Veterin?r B neu und Neopredisan 135-1, were tested to evaluate their efficacy against caliciviruses at 20 and 10 degrees C. As model test virus served feline calicivirus type F9 (FCV F9). All disinfectants were tested according to Guidelines of the German Veterinary Association (DVG). The investigations were performed in suspension tests and germ carrier tests. The suspension tests were carried out without and with protein load. As protein was used foetal calf serum at the concentration of 40%. Venno FF super showed less protein dependence, however a considerable temperature dependence. This matter can be corrected by increase of concentration on 2%. Venno Vet 1 super was without protein especially effective. The losses on the effectiveness through low temperature and protein load can be annulled also here by increase of concentration. Venno Oxygen was more effective in the comparison to that here named both preparations. The effects of temperature can be corrected by extension of reaction time. The most effective preparation was M&Enno Veterin?r B neu. The disinfection occurred at 20 degrees C with 0.5% solution within 120 min and at 10 degrees C with 1.0% solution within 60 min. The fifth disinfectant Neopredisan was in suspension tests without protein load and carrier tests with gauze at 20 and 10 degrees C relative convincing but in germ carrier tests with poplar wood, no complete disinfection could be achieved within tested concentrations and reaction times.     

Disinfection for dermatomycoses in veterinary practice

Sporulating cultures of two strains of Trichophyton equinum, two strains of Trich. mentagrophytes and one strain of Microsporum canis were tested by a simple dipping technique for their sensitivity to ten currently used disinfectants. Antifungal effects on all cultures of dermatophytes were observed already after one-minute treatment with 0.5%-4% peracetic acid, 4% formaldehyde solution and concentrated solution of Lastanox super. The above strains were inactivated after five-minute treatment with 1% and 2% formaldehyde solution, 20% Iodonal B solution and Ajatin solution with 10% active substance. Fungistatic effects of various intensity were observed in the other disinfectants.     

Immunogenicity of Mycoplasma gallisepticum

The serological response and protective immunity elicited in the chicken by the pathogenic Ap3AS strain and the moderately pathogenic 80083 strain of Mycoplasma gallisepticum and variants of strain 80083 attenuated by repeated passage in mycoplasma broth were investigated. Strain 80083 elicited a substantial serum antibody response after administration either in drinking water or by conjunctival sac instillation to 7-week-old SPF chickens. No vaccinated chickens developed air sac lesions when challenged by intra-abdominal (IA) injection with the virulent Ap3AS strain. Chickens vaccinated with strain 80083M (50 broth passages) showed only a weak serological response but were substantially protected when challenged 4 weeks after vaccination. Chickens vaccinated with 80083H (100 broth passages) were serologically negative 4 weeks after vaccination and developed severe air sac lesions after challenge. Thirty-seven-week-old hens vaccinated 6 months previously with strain 80083 had high serum antibody levels and were completely protected against IA challenge with the homologous strain. However, 4/6 showed mild air sac lesions when challenged intra-abdominally with strain Ap3AS. Another group showed high M. gallisepticum serum antibody levels 6 months after vaccination with strain Ap3AS but 4/6 and 2/6 showed mild lesions after IA challenge with strains Ap3AS or 80083, respectively. Strains 80083 or 80083M were administered by conjunctival sac instillation to susceptible 11-week-old commercial pullets at the time of fowl pox vaccination. The concurrent use of both vaccines had no apparent adverse effect on the health of the chickens. Similar protection against IA challenge with strain Ap3AS was produced with the M. gallisepticum vaccines whether used alone or in combination with fowl pox.     

Sensitivity of the standardized pseudorabies virus neutralization test varies with the test strain used.

The effect of altering the strain of the test virus used in the standardized pseudorabies virus neutralization (VN) test on the sensitivity of the assay was evaluated. Comparative VN tests were performed using 4 different strains: the avirulent Bartha parental, the avirulent recombinant Bartha gIIIKa, the moderately virulent Shope (currently used for the VN test at the National Veterinary Services Laboratory, Ames, IA), and the highly virulent P2208 (Funkhauser). A radioimmunoassay and a Western immunoblotting technique were employed to verify the presence of anti-pseudorabies virus (PrV) antibodies in sera. Statistical analysis indicated that replacement of the Shope strain by the Bartha gIIIKa or the P2208 strain resulted in VN titers that were 4.23- and 2.00-fold higher, respectively. Despite these differences, specificity with regard to PrV diagnosis was unaltered. This apparent enhancement of the sensitivity of the PrV VN test would be beneficial for the serologic identification of PrV-infected animals during an eradication effort.     

Isolation of Cryptosporidium andersoni Kawatabi type in a slaughterhouse in the northern island of Japan

Fecal samples were collected from 325 adult cattle and 108 pigs in a slaughterhouse in Hokkaido, the northern island of Japan. Five adult cattle were found to be positive for oocysts of Cryptopsoridium (1.5%). The oocysts were morphologically similar to those of Cryptosporidium andersoni. The partial sequence of the 18S rRNA gene of the isolate was 100% identical with that of the C. andersoni Kawatabi strain. SCID mice were infected after oral administration. Based on the morphology of the oocysts, the sequence of the 18S rRNA gene and the infectivity to SCID mice, the isolate was concluded to be of the same type as the C. andersoni Kawatabi strain that has been isolated in Honshu, the main island of Japan.