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