Short Communication: Prevalence of Listeria monocytogenes in Raw Milk of Healthy Sheep and Goats

Listeria monocytogenes, one of the most important bacterial pathogens transmitted through milk, causes listeriosis in humans and animals. This study aimed to investigate the prevalence of L. monocytogenes in raw milk of healthy sheep and goats in the west of Iran (Lorestan Province) by touchdown PCR (TD-PCR). Listeria spp. were found in milk samples taken from 21 sheep (29.16%) and 3 goats (10.71%) whereas L. monocytogenes was isolated from milk samples taken from 4 sheep (5.55%) and 1 goat (3.75%). The results showed that there was a significant difference between sheep and goats in the prevalence of Listeria spp. in their raw milk (p < 0.05), but no significant difference was observed between them in the prevalence of L. monocytogenes. The study findings suggested that the raw milk of healthy sheep and goats was infected with L. monocytogenes and warned of the risk of human infection with listeriosis following consumption of raw and unpasteurized milk.


Introduction
Listeriosis is one of the most important bacterial infections worldwide. According to the reports published by the Centers for Disease Control and Prevention (CDC), it is estimated that listeriosis is annually the cause of 1,600 cases of disease and 260 deaths [1]. As a severe food-borne illness, listeriosis is often caused by eating foods contaminated with L. monocytogenes. Raw milk is one of the most common environments for the transmission of L. monocytogenes [2].
Sheep milk was formerly known as a possible cause of listeriosis [3]. Since milk and dairy products are of high nutritional value, they are suitable environments for the proliferation and growth of microorganisms including pathogenic bacteria. L. monocytogenes is usually found in dairy products, on farms, and in dairy processing plants [2].
ere are reports on the intermittent, asymptomatic shedding of L. monocytogenes in the milk of goats and sheep [4,5]. It is hence necessary to identify infected animals in order to prevent the possible prevalence of listeriosis [3].
Currently, the standard method for diagnosing L. monocytogenes is culture, but because antibiotics are used in animal feed and this bacterium is intracellular, the sensitivity of the culture is greatly reduced. In addition, isolation and identification of L. monocytogenes using culture media and biochemical methods is a time-consuming process. erefore, using the PCR technique to identify this bacterium in a clinical sample is a valuable method. Listeriolysin O (LLO) is a secretory protein encoded by the hlyA gene and one of the main factors responsible for the virulence of L. monocytogenes so that its isolation from food products can indicate the presence of L. monocytogenes [6,7]. Most studies have investigated the prevalence of L. monocytogenes in cow's milk and a few have dealt with the prevalence of this bacterium in sheep and goat raw milk. Lorestan with a livestock population of about 3750,000 heads, is one of the top 5 provinces in Iran in the field of animal husbandry. is study hence aimed to employ the TD-PCR method for the direct detection of L. monocytogenes to determine its prevalence in sheep and goat raw milk in Lorestan Province, Iran.

Milk Samples.
From January to February 2020 (winter season), 100 milk samples were randomly collected from 72 sheep and 28 goats in livestock farms in four regions, namely, Aleshtar and Azna (cold regions), and Khorramabad and Kuhdasht (tropical regions) of Lorestan province (Iran). 50 milliliters of each sample was gathered in tubes under aseptic conditions and transferred to the laboratory on an icebox. e samples were stored at −20°C for molecular experiments until processing.

DNA Extraction.
Frozen milk was thawed at room temperature, and 10 mL of each sample was added to a 10 mL tube. e tubes were centrifuged at 13, 000 × g for 15 min. en, with a sterile swab, the fatty top layer was removed and the liquid around was poured. Next, 200 μL of precipitate were transferred to a separate tube each to extract DNA using a Blood Genomic DNA Extraction Mini Kit (Favorgen, Taiwan) according to the manufacturer's instructions with a few modifications. e quality and amount of extracted DNA was measured by using a Nanodrop spectrophotometer ( ermo Scientific, Waltham, USA). Finally, the extracted DNA was stored at −20°C for the later use in PCR.
Touchdown (TD)-PCR amplification was performed by using a PCR master kit (Ampliqon Taq DNA Polymerase Master Mix RED 1.25 mL, Ampliqon Denmark) with 25 μL mixtures containing 12.5 μL of 2X master mix, 0.5 μL of each primer, and 4 μL of the extracted DNA. In this study, DNA obtained from L. monocytogenes (laboratory control strain: (PTCC 1294) Iran Scientific and Industrial Research Center) and sterile water were used as the positive and negative controls, respectively. Furthermore, the amplification was conducted by using a Bio-Rad thermocycler (Model T-100, USA) under the following conditions: A for Listeria spp. and B for L. monocytogenes.
(A) e initial step of 94°C for 5 min, followed by 5 cycles of 94°C for 1 min, annealing temperatures starting at 64°C for 45 seconds (decreasing 1°C/ cycle), and finally, at 72°C for 30 seconds for the extension. is step was followed by 30 cycles of 94°C for 30 seconds, 60°C for 45 seconds, 72°C for 45 seconds, and finally, 72°C for 10 min.
(B) e initial step of 95°C for 4 min, followed by 5 cycles of 95°C for 1 min, annealing temperatures starting at 55°C for 45 seconds (decreasing 1°C/ cycle), and finally, at 72°C for 1 min for the extension. is step was followed by 30 cycles of 95°C for 1 min, 52°C for 45 seconds, 72°C for 2 min, and finally, 72°C for 8 min.
e PCR products were separated in a 1.2% (w/v) agarose gel (Merck, Germany) containing 2.5 μg/mL gel stain (Smobio, Taiwan). Electrophoresis was performed in 0.5x Tris/Borate/EDTA (TBE) buffer for one hour at 100 V. e resulting PCR products were visualized under a UV transilluminator (E-Box, Iran) and the 100 bp DNA ladder (Smobio, Taiwan) plus was used as the molecular size marker.

Statistical Analysis.
e obtained data were analyzed by IBM SPSS 20 for Windows (SPSS Inc., Chicago, IL, USA). p value < 0.05 was considered significant.

Results
In this study, 100 samples of raw milk were taken from 72 sheep and 28 goats to detect L. monocytogenes. Listeria spp. identified using 16s rRNA gene sequencing was found in milk samples taken from 21 sheep (29.16%) and 3 goats (10.71%), and L. monocytogenes identified by detecting the hlyA gene was isolated in milk samples taken from 4 sheep (5.55%) and 1 goat (3.75%) (Figure 1). Among the regions, Kuhdasht (tropical region) livestock were more infected with Listeria spp. than other cities and Azna (cold region) livestock were more infected with L. monocytogenes (Table 1). ere was a significant difference observed between milk samples taken from sheep and goat in the prevalence of Listeria spp. (p < 0.05).

Discussion
Listeriosis is an infectious disease in humans and animals in which 99% of cases are caused by consumption of foods contaminated with L. monocytogenes and is rarely transmitted from the environment [10]. Raw milk is one of the most common substrates for the transmission of L. monocytogenes as studies have shown that Listeria spp. isolates can be usually found in raw milk specimens [10][11][12]. Lorestan province has cold and tropical regions due to its geographical location. Also, in this province, more than 80% of the livestock population is sheep in order to preserve pastures and oak trees. erefore, in the present study, milk samples were collected from both tropical and cold regions and were more than sheep. Various studies indicated that the TD-PCR method increases both the specificity and sensitivity of the PCR protocol, since the content of unwanted in the DNA extracted from the milk is high. erefore, using TD-PCR is considered the suitable method for increasing the template copy number leading to an increase in the yield of amplicons [13][14][15][16]. e results of the present study using TD-PCR showed that the prevalence of the Listeria spp.

2
Veterinary Medicine International isolates was equal to 29.16% and 10.71% in samples isolated from sheep and goat milk, respectively. In addition, 5 isolates (20.83%) of Listeria spp. belonged to L. monocytogenes. e prevalence of this bacterium in raw milk samples of sheep and goat was 5.55% and 3.57%, respectively. Many studies have been conducted around the world on the prevalence of L. monocytogenes in cow's milk. eir findings indicated that the prevalence of this bacterium was 4% in the Netherlands [17], 4% in the USA [18], 2% in Turkey [19], 1.1% in Brazil [20], 1.7% in Italy [21], 3.6% in Ethiopia [22], 11% in Iraq [23], 1.7% in Iran [24], and 1.1% in Iran [12]. Nevertheless, few studies have investigated the prevalence of L. monocytogenes in sheep and goat raw milk. Studies conducted by Durmaz et al. [19] in Turkey showed that the prevalence rates of L. monocytogenes in sheep and goat raw milk were 2.7% and 0%, respectively. In another study conducted by Abbas and Jaber [23] in Iraq, the prevalence of L. monocytogenes in sheep milk was 8%. e study conducted by Rahimi et al. [12] in Isfahan Province, Iran, demonstrated that the prevalence rates of Listeria spp. in sheep and goat raw milk were 22.6% and 6.7%, respectively, and the prevalence rates of L. monocytogenes were 6.5% and 1.7%, respectively. In another study conducted by Jamali et al. [25] in Alborz Province, Iran, the prevalence rates of Listeria spp. in sheep and goat raw milk were 16.4% and 4.9%, respectively, and the prevalence of L. monocytogenes was 4% in sheep and goat raw milk. In another study by Osman et al. [26] in Egypt, the prevalence rates of Listeria spp. in sheep and goat raw milk were 3.9% and 5.6%, respectively, and   [12], and Jamali et al. [25], this study showed that the prevalence of Listeria spp. was higher in sheep milk than in goat milk and there was a significant difference between milk specimens taken from sheep and goats in this regard. By contrast, Osman et al. [26] in Egypt reported that the prevalence of Listeria spp. and L. monocytogenes was higher in goat milk than in sheep milk. is study, however, consistent with the results of Rahimi et al. [12] and Jamali et al. [25], demonstrated that the prevalence of Listeria spp. and L. monocytogenes was higher in sheep milk than in goat milk in Iran.
is can be attributed to the higher health status of the udder in goats than in sheep as well as the higher tendency of sheep to graze and rest in compact groups.
Lorestan province with a high livestock population has tropical and cold regions throughout the year. As, the two regions of Aleshtar and Azna are among the cold regions, while Khorramabad and Kuhdasht are two tropical regions of the province. e present study showed that the samples isolated from Kuhdasht as a tropical region are more contaminated with Listeria spp. than other regions of the province. However, samples isolated from Azna as a cold region are more contaminated with L. monocytogenes than in other regions. Considering that all milk samples were collected in winter, but there is no significant difference in the prevalence of Listeria spp. and L. monocytogenes between tropical and cold regions of the province.
ere are various reports that the feeding of livestock on poor-quality silage and poor milking hygiene are two important sources of milk contamination with Listeria spp. especially L. monocytogenes [27,28]. e selected livestock in this study had no history of feeding on silage, and also milking cartridges were disinfected with 70% alcohol. erefore, the isolates identified in this study were not caused by poor hygiene, and the animals were already asymptomatic carriers of listeriosis.
e study findings are also consistent with those of Durmaz et al. [19], Rahimi et al. [12], Mahmoodi [24], and Jamali et al. [25]. Although most studies have focused on the prevalence of Listeria spp. in cow's milk, the prevalence of this bacterium is higher in sheep and goat raw milk. is indicates the importance of pasteurizing sheep and goat milk. Various studies have indicated that this bacterium can survive at pasteurization and even freezing temperatures for a long time depending on its strain type and concentration in milk [18,29]. e presence of bacteria is a serious risk of infection from eating contaminated food even at low prevalence and concentration of the pathogen, especially when unpasteurized dairy products are consumed [26].
Since there are reports of asymptomatic carriage of L. monocytogenes [7,26] and sheep and goat milk is used for preparing local cheese in most rural areas of Iran, the implementation of systematic monitoring in dairy herds is relevant to identify infected animals and prevent and control the presence of L. monocytogenes. Other studies should also be conducted to determine circulating variants and identify resistant strains, and even the inclusion of innovative alternatives for the treatment of milk from positive animals such as the use of lactose oxidase which was recently reported by Flynnet et al. [30] should be performed. All of the above prevent outbreaks among the population of consumers of milk or its by-products, when they do not go through the pasteurization process.

Data Availability
No additional data were used to support this study.

Conflicts of Interest
e authors declare no conflicts of interest.