Infection of humans and animals with trematodes of the Opisthorchiidae family—Opisthorchis viverrini and O. felineus—as a result of ingestion of raw or undercooked fishes from the Cyprinidae family, negatively affects bile excretion–related functions of the liver. Opisthorchiasis contributes to the development of purulent cholangitis, cholecystitis, biliary strictures, chronic hepatitis, liver abscesses, obstructive jaundice, pancreatitis, and even cholangiocarcinoma (Sripa et al., 2009, 2012).

It has been established that excessive alcohol drinking is the cause of more than 200 severe diseases (e.g., cardiomyopathy, arrhythmia, gastritis, pancreatitis, dementia, and mental and behavioral disorders) (WHO, 2018). Nonetheless, the first consequence of excessive alcohol consumption is alcoholic liver disease, which, according to the ICD-10-CM Coding Guide (codes K700–K709), includes hepatitis, fibrosis, sclerosis and cirrhosis of liver, alcoholic hepatic failure, and other health problems. These effects are primarily due to damage to hepatocytes, which represent 70% of liver weight (Osna et al., 2017).

It is obvious that the combination of chronic trematode infection with long-lasting alcohol consumption can have irreversible consequences for human health. However, a popular misconception among people in some nations is that certain drinks, including alcohol, can kill the parasites that enter the human body via ingestion of uncooked cyprinoid fish in traditional dishes (Sriraj et al., 2013b).

Meanwhile it is known that both helminth infections and excessive alcohol consumption weaken the body's immune defenses. In this context, concentrations of inflammatory markers—proinflammatory cytokines, in particular interleukin 6 (IL-6)—increase in the blood (McClain et al., 2004; Swiatkowska-Stodulska and Bakowska, 2004; Sripa et al., 2009, Sripa et al., 2012; Mandrekar et al., 2014). In both diseases, an active response of the immune system implies an involvement of the spleen, which is the immune system's main peripheral effector (Bronte and Pittet, 2013). Experimentally, we have demonstrated significant splenomegaly when mice and hamsters are infected with O. felineus helminths (Vishnivetskaya et al., 2019; Lvova et al., 2020).

Therefore, our aim was to model a combination of chronic O. felineus infection and long-term ethanol consumption in mice of the inbred C57BL/6 strain, which, unlike many other inbred strains of mice, prefer to consume ethanol (Belknap et al., 1993; Yoneyama et al., 2008) and are susceptible to the infection by O. felineus (Orlovskaya et al., 2018). Even though alcohol catabolism is fivefold faster in rats and mice than in humans (Holmes et al., 1986), C57BL/6 mice are often used in numerous experimental models for studying the mechanisms underlying alcohol-induced organ damage to advance our understanding of alcoholism in humans (D'Souza El-Guindy et al., 2010). The “alcohol in drinking water model” (single bottle – no choice) is regarded as physiological and inexpensive and can be adjusted to mimic human drinking. In this model, mice are gradually accustomed to the consumption of a high dose (up to 25.32%, v/v) of ethanol in water; further consumption of ethanol at this dose by these mice can last up to 8–18 months (Cook et al., 2007; D'Souza El-Guindy et al., 2010). As for O. felineus infection, earlier in our studies, it has been shown that the acute and chronic stages of opisthorchiasis can be successfully modeled in C57BL/6 mice (Avgustinovich et al., 2016; Orlovskaya et al., 2018; Vishnivetskaya et al., 2019). In this model, mature marita of O. felineus helminths are found in the liver of the mice 6 months after they are infected.

Considering all of the above, in this work, we modeled a combination of prolonged consumption of ethanol in drinking water by C57BL/6 mice with subsequent infection by liver flukes to comprehensively assess, first of all, the liver state: including whole-liver visualization and microhistological analysis. In addition, we decided to estimate liver pathology–mediated inflammation in the mice by means of spleen involvement, changes in blood IL-6 levels, absolute and relative blood cell counts, and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities.