Enterocytozoon bieneusi in raw milk of cattle, sheep and water buffalo in Turkey: Genotype distributions and zoonotic concerns

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Highlights

  • The overall prevalence of Enterocytozoon bieneusi in raw milk of dairy animals was determined as 10.2%.

  • A Significant relation was determined between the presence of E. bieneusi and mastitis.

  • Two known (ERUSS1, BEB6) and six novel genotypes (TREb1 to TREb6) were identified.

  • This study contributes to the current knowledge on the molecular epidemiology and transmission dynamics of E. bieneusi.

Abstract

Raw milk is a continued threat to public health due to possible contamination with zoonotic pathogens. Enterocytozoon bieneusi is one of the most prevalent pathogenic fungi in a wide range of vertebrate hosts, causing diarrheal disease. Although there has been some evidence, the role and potential risk of raw milk of dairy animals in the transmission dynamics of E. bieneusi is not clear. Therefore, we aimed to determine the occurrence and genotypes of E. bieneusi in raw milk of dairy animals in several farms of the Central Anatolia Region. We also investigated if there is a relation between the presence of E. bieneusi and mastitis. Genomic DNAs from a total of 450 raw milk including 200, 200 and 50 samples from cattle, sheep and water buffalo respectively were analyzed using nested PCR, targeting the internal transcribed spacer of E. bieneusi. Totally milk samples of 9 (4.5%) dairy cattle, 36 (18.0%) sheep, and 1 (2.0%) water buffalo were PCR-positive. A significant relationship was determined between mastitis and the presence of E. bieneusi. Sequence analysis revealed the presence of eight genotypes: two known (ERUSS1, BEB6) and six novel genotypes (named as TREb1 to TREb6). The genotype ERUSS1 and BEB6 were the most common genotypes, found in all cattle and sheep farms. Phylogenetic analysis clustered all the identified genotypes in Group 2. This study provides novel findings that contribute to the transmission dynamics and molecular epidemiology of E. bieneusi. Our study also highlighted the potential risk of raw milk for public health with respect to microsporidia infections.

Introduction

Raw milk and milk products have increasing demand among consumers who believe that raw milk and products provide better nutritional quality and beneficial microbiota with lower risk of lactose intolerance, allergy and asthma although limited scientific evidence is available for this claim (Lucey, 2015). However, raw milk and milk products might pose a risk for human health with regard to various zoonotic agents like Campylobacter, Salmonella, Yersinia, Listeria monocytogenes, Escherichia coli O157 and Staphylococcus aureus. In a study conducted in the United States; during 2007–2012, a total of 81 outbreaks were reported associated with unpasteurized milk (Mungai et al., 2015). Unpasteurized milk can pose serious health risks especially for the high-risk population groups such as children, elderly, pregnant or immunocompromised individuals.

The intestinal biota and the udder of healthy dairy animals consist of many pathogens that can easily contaminate milk during the production process (Gopal et al., 2015). Microsporidia, a group of various mandatory intracellular parasitic protists related to fungi, is frequently seen in humans and various other animals and causes gastroenteritis (Abe and Kimata, 2010; Udonsom et al., 2019; Wang et al., 2018). Enterocytozoon bieneusi is the major microsporidiosis agent of healthy and immunocompromised individuals and is responsible for more than 90% of human microsporidiosis cases (Matos et al., 2012). The spores of E. bieneusi are ubiquitous in nature especially in water sources, and contaminated food or water and the fecal-oral route are the transmission ways for E. bieneusi. All animal species can be infected with E. bieneusi due to its wide range of host diversity (Santín and Fayer, 2011). In addition, several animals might act as potential reservoirs, environmental pollutants, and continuous transmission sources for E. bieneusi (Galván-Díaz et al., 2014). Although the potential risk of raw milk and milk-derived products for transmission of several microbial pathogens is well known, limited knowledge is available concerning the presence of microsporidia in milk and related risk factors for public health so far. Lee (2008) reported that 8.33% of the milk specimens from 180 dairy cows in Korea were found positive for E. bieneusi using molecular assays. Kváč et al. (2016) detected Encephalitozoon cuniculi in raw milk of one cow among totally examined 50 milking cows in the Czech Republic and also reported continued infectivity of the spores after pasteurization, using mice model in in-vivo experiments. Both studies provided evidence on the risk of raw milk as a potential source of zoonotic microsporidia infections even pasteurization process was applied.

Sequencing of the internal transcribed spacer (ITS) region of the rRNA gene is widely used to identify the genotypes of E. bieneusi from various animal and human hosts (Santín and Fayer, 2009). To date, over 500 E. bieneusi genotypes have been identified with this ITS genotyping. Most of the zoonotic genotypes were commonly clustered in Group 1 and Group 2 that pose major risk for public health and contamination of the environment with E. bieneusi (Gong et al., 2019; Li et al., 2019; Liu et al., 2017; Prasertbun et al., 2019; Wang et al., 2018; Zhang et al., 2018). Cattle and sheep are known as the common hosts of E. bieneusi. Both zoonotic and potentially host-adapted genotypes were identified from these animals and a majority of genotypes belonged to the so-called bovine or ovine-adapted Group 2 (Chang et al., 2020; Chen et al., 2018; Jiang et al., 2015; Wang et al., 2019, Wang et al., 2013; Zhang et al., 2018). On the other hand, there have been few reports on E. bieneusi in water buffaloes (Al-Herrawy and Gad, 2016; Ma et al., 2015) and the genotype CHN11 from potentially zoonotic Group 1 was the only identified genotype (Ma et al., 2015). The occurrence of zoonotic genotypes in cattle and sheep, and sporadic infections with ruminant-adapted genotypes (I, J, BEB4, and BEB6) in humans (Jiang et al., 2015; Sak et al., 2011; Wang et al., 2013; Zhang et al., 2011) draw attention to the importance of these livestock animals in zoonotic transmission of E. bieneusi.

There is limited data on the presence of zoonotic microsporidia in raw milk of livestock and their genetic characterization. To our knowledge, the distribution and genotypes of E. bieneusi in both animal and human hosts in Turkey are not fully understood to date. Therefore, to fill this knowledge gap, we aimed to determine the occurrence of E. bieneusi in the raw milk from dairy cattle, sheep and water buffaloes using molecular tools and sequence analysis to reveal the genotypes of E. bieneusi. We also aimed to assess the potential role of raw milk of livestock in the zoonotic transmission dynamics of E. bieneusi. The association between the presence of mastitis based on somatic cell count (SCC) and E. bieneusi infection was also evaluated in the study.

Section snippets

Study area and collection of milk samples

A total of 450 raw samples including bovine (n = 200), ovine (n = 200), and water buffalo (n = 50) milk were randomly collected during April to September of 2018 from several farms of the Central Anatolia Region in Turkey. Cattle included to survey were from intensive dairy farms, while sheep and water buffaloes were from free-range farms. Prior to collection of milk samples from each animal, teats were cleaned with sterile saline and initial milk was discarded. A total of 40 ml milk from teats

Distribution of mastitis

Among the examined bovine, ovine and water buffalo milk samples, totally 103 (51.5%), 112 (56.0) and 15 (7.5%) samples were evaluated as mastitis positive based on the SSC, respectively. The mean SSC with minimum and maximum values with respect to mastitis for each animal species is presented in Table 1.

Prevalence of E. bieneusi and association with mastitis

The DNA of E. bieneusi was detected in 46 out of 450 gDNA isolates of raw milk samples by ITS-nested PCR analysis resulting in the overall prevalence of 10.2% (Table 2). The overall prevalence

Discussion

Milk and milk products can harbor a variety of microorganisms and could be important sources of various pathogens such as bacteria, protozoa, and viruses originating from blood, mastitis, or contamination of milk with feces, skin, or environment. The presence of zoonotic pathogens in raw milk could arise from direct contaminations from the environment of dairy farms and infected dairy animals. Although milk is commonly pasteurized in the dairy industry, it is seen that concerns remain about the

Conclusions

Our study provides unique data on the occurrence and potential risk factors of E. bieneusi in raw milk of dairy animals including cattle, sheep and water buffalo raised in Turkey. Our findings indicate wide occurrence of two known (ERUSS1 and BEB6), and the presence of further six novel (TREb1-TREb6) genotypes in Group 2 of E. bieneusi. Raw milk contaminated with Group 2 genotypes should not be ignored in the zoonotic transmission dynamics of E. bieneusi due to host range expansion. Perishable

Declaration of competing interest

The authors have no conflict of interest concerning the work performed in this paper.

Acknowledgement

This study was supported by “The Scientific and Technological Research Council of Turkey” with project number 117O702.

References (64)

  • M. Qi et al.

    Dominance of Enterocytozoon bieneusi genotype J in dairy calves in Xinjiang, Northwest China

    Parasitol. Int.

    (2017)
  • M. Qi et al.

    Zoonotic Cryptosporidium spp. and Enterocytozoon bieneusi in pet chinchillas (Chinchilla lanigera) in China

    Parasitol. Int.

    (2015)
  • J. Reetz et al.

    First detection of the microsporidium Enterocytozoon bieneusi in non-mammalian hosts (chickens)

    Int. J. Parasitol.

    (2002)
  • M. Santín et al.

    Microsporidiosis: Enterocytozoon bieneusi in domesticated and wild animals

    Res. Vet. Sci.

    (2011)
  • S.S. Wang et al.

    Novel genotypes and multilocus genotypes of Enterocytozoon bieneusi in pigs in northwestern China: a public health concern

    Infect. Genet. Evol.

    (2018)
  • Y. Wu et al.

    Occurrence and molecular characterization of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi from Tibetan sheep in Gansu, China

    Infect. Genet. Evol.

    (2018)
  • N. Abe et al.

    Molecular survey of Enterocytozoon bieneusi in a Japanese porcine population

    Vector Borne Zoonotic Dis.

    (2010)
  • N. Abu Samra et al.

    Enterocytozoon bieneusi at the wildlife/livestock interface of the Kruger National Park, South Africa

    Vet. Parasitol.

    (2012)
  • A.Z. Al-Herrawy et al.

    Microsporidial spores in fecal samples of some domesticated animals living in Giza, Egypt

    Iran. J. Parasitol.

    (2016)
  • T. Bilgin et al.

    Molecular prevalence and phylogenetic characterization of Enterocytozoon bieneusi in healthy cattle

    Türk. Parazitol. Derg.

    (2020)
  • A. Bradley et al.

    Use and interpretation of somatic cell count data in dairy cows

    In Pract.

    (2005)
  • M.A. Buckholt et al.

    Prevalence of Enterocytozoon bieneusi in swine: an 18-month survey at a slaughterhouse in Massachusetts

    Appl. Environ. Microbiol.

    (2002)
  • Y. Chang et al.

    Molecular characterization of Giardia duodenalis and Enterocytozoon bieneusi isolated from Tibetan sheep and Tibetan goats under natural grazing conditions in Tibet

    J. Eukaryot. Microbiol.

    (2020)
  • E.S. Didier et al.

    The immunology of microsporidiosis in mammals

  • R.C. Edgar

    MUSCLE: a multiple sequence alignment method with reduced time and space complexity

    BMC Bioinf.

    (2004)
  • N. Ercan et al.

    Molecular prevalence and phylogenetic characterization of microsporidian parasites in chicken

  • R. Fayer et al.

    First detection of microsporidia in dairy calves in North America

    Parasitol. Res.

    (2003)
  • R. Fayer et al.

    Enterocytozoon bieneusi in mature dairy cattle on farms in the eastern United States

    Parasitol. Res.

    (2007)
  • A.L. Galván-Díaz et al.

    Microsporidia detection and genotyping study of human pathogenic E. bieneusi in animals from Spain

    PLoS One

    (2014)
  • B. Gong et al.

    First survey of Enterocytozoon bieneusi and dominant genotype Peru6 among ethnic minority groups in southwestern China's Yunnan Province and assessment of risk factors

    PLoS Negl. Trop. Dis.

    (2019)
  • N. Gopal et al.

    The prevalence and control of Bacillus and related spore-forming bacteria in the dairy industry

    Front. Microbiol.

    (2015)
  • S. Guindon et al.

    A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood

    Syst. Biol.

    (2003)
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