Investigations and comparative detection ofCryptosporidiumspecies by microscopy, nested PCR and LAMP in water supplies of Ordu, Middle Black Sea, Turkey

ABSTRACTAdenovirus is the most prevalent enteric virus in waters worldwide due to its environmental stability, which leads to public health concerns. Mitigation strategies are therefore required. The aim of this study was to assess the inactivation of human adenovirus type 5 (HAdV-5) by gamma radiation in aqueous environments. Various substrates with different organic loads, including domestic wastewater, were inoculated with HAdV-5 either individually or in a viral pool (with murine norovirus type 1 [MNV-1]) and were irradiated in a Cobalt-60 irradiator at several gamma radiation doses (0.9 to 10.8 kGy). The infectivity of viral particles, before and after irradiation, was tested by plaque assay using A549 cells. D10values (dose required to inactivate 90% of a population or the dose of irradiation needed to produce a 1 log10reduction in the population) were estimated for each substrate based on virus infectivity inactivation exponential kinetics. The capability of two detection methods, nested PCR and enzyme-linked immunosorbent assay (ELISA), to track inactivated viral particles was also assessed. After irradiation at 3.5 kGy, a reduction of the HAdV-5 titer of 4 log PFU/ml on substrates with lower organic loads was obtained, but in highly organic matrixes, the virus titer reduction was only 1 log PFU/ml. The D10values of HAdV-5 in high organic substrates were significantly higher than in water suspensions. The obtained results point out some discrepancies between nested PCR, ELISA, and plaque assay on the assessments of HAdV-5 inactivation. These results suggest that the inactivation of HAdV-5 by gamma radiation, in aqueous environments, is significantly affected by substrate composition. This study highlights the virucidal potential of gamma radiation that may be used as a disinfection treatment for sustainable water supplies.IMPORTANCEHuman adenovirus (HAdV) is the most prevalent of the enteric viruses in environmental waters worldwide. The purposes of this study are to provide new insights on the inactivation of enteric virus by gamma irradiation and to introduce new concepts and reinforce the benefits and utility of radiation technologies as disinfection processes. This may be an effective tool to guarantee the reduction of viral pathogens and to contribute to public health and sustainable water supplies.

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Investigation of a Cryptosporidiosis Outbreak Caused by Cryptosporidium parvum in a Dairy Farm

10.21203/rs.3.rs-55465/v1 ◽

2020 ◽

Author(s):

MUHAMMET KARAKAVUK ◽

Hüseyin Can ◽

MERT DÖŞKAYA ◽

Tuğba Karakavuk ◽

Sedef Erkunt Alak ◽

...

Keyword(s):

Phylogenetic Analysis ◽

Real Time ◽

Cryptosporidium Parvum ◽

Real Time Pcr ◽

Nested Pcr ◽

Environmental Samples ◽

Dairy Farm ◽

Giardia Intestinalis ◽

Water Supplies ◽

Neonatal Calves

Abstract Background Diarrhea caused by parasitic agents is common in neonatal calves and causes significant economic losses in cattle farms worldwide. Cryptosporidium spp. is one of the most frequently detected parasitic agents causing diarrhea in neonatal calves. Also, Giardia intestinalis is shown to cause diarrhea in calves. This study aimed to investigate the presence of Cryptosporidium spp. in calves (n:36), cows (n: 11), drinking water and two different artesian water supplies as well as in environmental swap samples (n:32) obtained from the manger, silage, bottle, and doorknob in a dairy farm which has big diarrhea problems. For this purpose, all fecal samples investigated with using direct microscopy for routine parasitological screening. Then, the presence of Cryptosporidium spp. was examined in feces samples and other environmental samples using Kinyoun acid-fast stained slides and Real-Time PCR targeting Cryptosporidium spp.. In addition, Real-Time PCR positive samples were investigated by nested PCR and subsequently by sequencing, BLAST, and phylogenetic analysis for species identification by MEGA7. Results Giardia intestinalis was detected in 10 feces samples (21,27%; 10/47) during routine microscopic examination. Among them, 9 belonged to calves older than two months without diarrhea, and one belonged to an adult cow. Cryptosporidium spp. was found in 11 calves (30.55%; 11/36) by Real-Time PCR, whereas no cows were found positive. Among the PCR positive samples, only five of them were detected as positive by microscopy. Cryptosporidium spp. positivity value was found higher in younger than two month old calves with diarrhea (9/12; 75%). Also, Cryptosporidium spp. was found in one of two water supplies and five of environmental samples by real-time PCR. Of the 18 real time positive samples, 8 were found positive by nested PCR and all positive samples were detected to be Cryptosporidium parvum by sequencing, BLAST, and phylogenetic analysis. Conclusions Our findings showed the importance of C. parvum infection in diarrhea cases occurred in calves. Besides the correct diagnosis and treatment of Cryptosporidium spp. contaminated water resources, and hygiene measures are very important for preventing the cryptosporidiosis in dairy farms.

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