Zoonotic risks of pathogens from dairy cattle and their milk-borne transmission

Abstract Dairy products are major sources of high-quality protein and bioavailable nutrients and dairy production contributes to local, regional and national-level economies. Consumption of raw milk and raw milk products does, however, carry a zoonotic risk, as does direct contact with cattle by farm husbandry staff and other employees. This review will mainly focus on the latter, and deal with it from the standpoint of a well-developed dairy industry, using the example of the Netherlands. With regard to dairy cattle, the main bacterial pathogens are Salmonella spp., Listeria monocytogenes and Leptospira hardjo as well as Brucella abortus and Chlamydia abortus. The main viral pathogens associated with dairy are Rift Valley fever virus, rabies virus, cowpox virus and vaccinia virus. The main parasitological infections are Echinococcus granulosis, Cryptosporidium parvum and Giardia duodenalis, however, the last mentioned have mainly swimming pools as sources of human infection. Finally ectoparasites such as lice and mites and Trichophyton verrucosum may affect employees. Some pathogens may cause health problems due to contamination. Bacterial pathogens of importance that may contaminate milk are Campylolobacter jejuni, Escherichia coli, Mycobacterium avium subsp. paratuberculosis, Leptospira hardjo and Salmonella typhimurium. Excretion of zoonotic viruses in milk is negligible in the Netherlands, and the endoparasite, Toxocara vitulorum is mainly found in suckling and fattening calves, whilst the risk in dairy cattle is limited. Excretion of transmissible spongiform encephalopathies (TSEs) or mycoses in milk are not expected and are, therefore, not of importance here. Being aware of the risks and working according to hygiene standards can substantially limit zoonotic risks for employees. Additionally, diseased employees are advised to limit their contact with cattle and to indicate that they work with cattle when consulting a physician. To prevent zoonotic risks through excretion of pathogens in milk, standard hygiene measures are necessary. Further, using only pasteurised milk for consumption and/or processing of milk can considerably limit the risks. If these measures are not possible, well-constructed monitoring can be followed. Monitoring programmes already exist for pathogens such as for Salmonella spp., Leptospira hardjo and Mycobacterium avium subsp. paratuberculosis. For others, like Campylobacter jejuni and E. coli, programmes are not available yet as far as we know.


Introduction
Dairy production and consumption have mainly positive effects on society and individual consumers, but can have negative effects on human health also (Hawkes and Ruel, 2006).Dairy products are major sources of high-quality protein and bioavailable nutrients (eg calcium; Todd et al., 2006).Dairy production can also contribute to local, regional and national-level economies and provide opportunities for employment and income generation (Hawkes, 2006), which are critical determinants of health (Marmot et al., 2008).However, a number of potential health risks associated with dairy production and consumption have also been identified, such as diet-related chronic diseases like milk allergy, environmental change, foodborne and occupational hazards and zoonotic diseases (Horrigan et al., 2002;Hawkes, 2006;Kimman et al., 2013).Globally, there is strong demand for milk and dairy products (IDF, 2016;USDA, 2021).This is largely due to global population growth (IDF, 2016), although increases per capita in dairy intake have also driven global demand (OECD and FAO, 2016).As demand for food increases, agricultural sectors have sought to increase production to meet that demand, and the dairy sector is no exception.In 2020, more than 906 million tons of milk were produced by the global dairy sector (FAO, 2021), and global production is projected to increase by 23% in 2025, compared to years 2013-2015(OECD and FAO, 2016).
Direct or indirect contact with contaminants such as bacteria, viruses and other pathogens is a potential risk when working with animals (WOAH, 2022).Exposure to contaminants can occur by respiration or by contact with excreta such as urine, faeces, milk and abortive fluids.Individuals may also have direct contact with the animal's coat and skin.Contact with the pathogenic agents can cause an infection or even disease and the same risk of transmission of pathogens from animals to humans applies to the consumption of raw milk products (Maunsell and Donovan, 2008).Fortunately, not all infectious agents and infections lead to health problems for cows and humans, though some pathogens unmistakably contain a markedly increased zoonotic risk.The objective of this paper is to provide an overview of the micro-organisms that may affect dairy cows under Dutch circumstances, the risk that these micro-organisms are presented to herd mates and to the employees working with dairy cattle and their effect on safety of the milk.Based on this overview, the pathogens with the highest zoonotic risk are identified and listed in the database.A distinction is made between pathogens that pose a risk to employees that work with dairy cattle and those that threaten the safety of milk and milk products.Reviewing the health impacts associated with dairy production and consumption will enhance understanding of the potential consequences associated with intensification of the dairy sector.To the authors' knowledge, no other comprehensive reviews of the potential health impacts of bacterial, viral, parasitological and mycotic infections associated with dairy production and consumption have been published.
With these objectives into mind, a broad review was undertaken in an effort to provide a comprehensive overview of the linkages between the dairy sector and public health.Specifically, the review aimed to identify the potential public health risks associated with dairy production.The content of this review can be used to support improved decision making for the future development of the dairy sector, from a public health perspective.Such decisions include: • Prioritisation of potential health hazards associated with the dairy sector that require specific risk communication and management actions; • Resource allocation for the management of specific hazards associated with dairy production and consumption and • Identification of knowledge gaps that require further research to improve understanding and management of the public health impacts associated with dairy production and consumption.
There are several methods that can be used to support these decision-making processes by providing systematic assessments of the public health impacts of dairy production and consumption at a wide range of spatial and temporal scales and with varying levels of detail.This inventory covered the clinical symptoms in cattle, the impact on animal welfare, the route and estimated risk of transmission to herd mates and to humans, the excretion in milk, the production of endotoxins, possible biosecurity measurements, vaccinations, diagnostic tools and the prevalence in our country (The Netherlands) as an example of a developed dairy sector.

Materials and methods
The infectious contaminants that may be found on Dutch dairy farms for now and in the near future were identified and listed in a database.Next, relevant background information for these micro-organisms was added, including their prevalence, the risk of excretion in milk, available diagnostic tests and preventive measures that can be taken to minimise the risk of infection.The relevant information was obtained from the literature, specialists of and the diagnostic results from the Royal GD laboratory in the last decade.
The pathogens with the highest zoonotic risk were identified based on the following criteria: causes of diseases of infectious origin in dairy cattle, characteristics of the agents, zoonotic aspects of the agents, route and risk of transmission to herd mates and humans, prevalence or the risk that the disease will be introduced in the Netherlands, laboratory diagnosis and excretion in or contamination of milk.
Scope of the literature review to support the risk analysis First, lists were composed of micro-organisms including bacteria, viruses, parasites, TSEs, mycoses and emerging diseases that may be found on Dutch dairy.The following information was included in the list for each micro-organism: • Species • General information including size, RNA-or DNA, presence of an envelope (viruses), Gram status and production of endotoxins (bacteria), route of transmission (all pathogens).This information was obtained from the literature, experts and laboratory staff employees of Royal GD Animal Health.The Dutch information was combined with papers about zoonotic infectious diseases from other countries with modern dairy production systems.Attention was given to TSEs and viral, bacterial, parasitological and mycotic infections.Specific attention was paid to list A diseases (ie diseases regulated by the EU Animal Health Law: https://eur-lex.europa.eu/EN/legal-content/summary/the-euanimal-health-law).Literature regarding production systems other than the dairy system (suckler cows, for example) was not included in this study.A comprehensive literature search was conducted in July-August 2021 to identify all relevant publications addressing infectious diseases in dairy cows, excretion of agents in milk, faeces and urine, as well as zoonotic risks.Literature search terms included specific phrases such as 'salmonella', 'trichophyton' and 'dairy cows' in the title, abstract, or as a keyword.The search terms were entered into the following three search databases: • Web of Science (http://apps.webofknowledge.com).
To complete this systematic review in a reasonable period of time, we included literature published in the last five years as far as possible, thus, publications between January 2015 and August 2021.An exception was made in the case of high-quality reviews published before 2015 or where there were no publications in period mentioned.The database search of scientific articles resulted in papers published predominantly in Western countries.All of the information was presented in an Excel file data base that distinguished between bacterial, mycotic, parasitological and viral infections as well as TSEs.

Risk analysis
The pathogens with the highest zoonotic risk were identified based on the following criteria: • Causing diseases of infectious origin in dairy cattle • Characteristics of agents • Zoonotic aspects of agents • Route and risk of transmission to herd mates and humans • Prevalence in the Netherlands or the risk of being introduced The prioritisation of the pathogens in this report is based on knowledge and discussion with the scientific staff of the Bovine Health Department and the Laboratory of Royal GD, which manages diseases in cattle with zoonotic consequences on a daily basis.Among the 223 publications identified in the literature, 119 were not considered useful because better or more recent examples or reviews of a given pathogen were consistently identified.In total, 104 papers provided usable information, but the pathogens discussed were either not all present or not emerging in the Netherlands.Ultimately, about 60 papers were selected to support the conclusions presented in this paper.

Results
An alphabetic overview of the pathogens of importance in the Netherlands or from regions important for the Netherlands was compiled.Viral, bacterial and parasitological infections are presented in Tables 1-3, respectively.Mycoses are presented in Table 4 transmission of pathogens from cattle to humans is especially possible through direct or indirect contact with the skin or excreta, and through faecal contaminated milk produced by clinically healthy animals.Transmission by excretion of pathogens in the milk is considered to be very limited (with exception of Salmonellae), especially if milk from sick cows is treated with care.

Viral infections
There are said to be a total of 42 viruses, including Toro or Breda virus, that are causing serious infections in cattle and are of potential zoonotic risk in The Netherlands (Hoet and Saif, 2004).Many viruses are species-specific, in which case the risk for transmission to humans is considered to be minimal.Other viruses (such as Enterovirus) have a low zoonotic potential but currently (January 2022) are limited present in the Netherlands.They are also found in other European regions and in the US (Gomez and Weese, 2017) and may become a concern in the near future in our region.
Rabies and Rift Valley fever (RVF) were identified as viral infections with the highest risk to employees working with cattle.Rabies can occur in all warm-blooded animals and is principally transmitted via direct contact with the saliva of an infected animal.Infection with rabies can be fatal without rapid intervention, which is the main reason for it being classified as a high-risk pathogen.Rabies is found in wildlife in Eastern Europe, in Africa, Asia, Indonesia, Bolivia, Mexico and Cuba (WHO, 2019).RVF, genus Phlebovirus, order Bunyavirales, is most commonly seen in domestic animals in sub-Sahara Africa and considered a serious risk to animals by the World Organisation for Animal Health (WOAH, 2022), with high economic impact.The virus can be transmitted to humans by contact with the body fluids of infected animals or through bites from infected mosquitoes (Culicoides).Most infected humans do not show signs of clinical illness or have only mild symptoms.However, a small percentage develop severe symptoms such as eye disease, haemorrhage and encephalitis (Wright and Kortekaas, 2019).The risk of future RVF introduction in Europe is relatively high given intercontinental traffic and storms.
Cattle warts, caused by bovine papillomavirus, are highly prevalent in Dutch cattle but appear to be species-specific and transmission to humans is unproven (Lawson et al., 2018).In contrast, cowpox (mainly observed in cats) and related vaccinia virus may infect humans (Lapa et al., 2019).These viruses usually cause skin lesions, although the ocular form may lead to serious complications.Both viruses are not present in cattle in the Netherlands, but may become a threat in the near future through worldwide travelling and trade.At present, viruses with high zoonotic risk that are excreted in the milk of dairy cows have not been identified in the Netherlands.For an overview of potential zoonotic viruses, the laboratory test to detect them, their clinical symptoms in cattle, their presence in milk, control measures and their total occurrence and importance in our region, see Table 1.

Bacterial infections
There are a total of 37 bacteria species and their various subspecies, causing infections in cattle and of which roughly 17 species present a potential zoonotic risk.Some species such as Salmonella typhimurium, Bacillus cereus and Brucella abortus have a high  As said before, mastitis pathogens themselves are normally not a problem in food-borne diseases because these products are not used for human consumption.In the milk of dairy cows with mastitis, endotoxins (lipopolysaccharides) may be present at the moment of bacterial death (ie after treatment with antimicrobials that kill mastitis pathogens).Experts at our company estimated that endotoxins remain in milk for roughly seven days after removal of the bacterial infection (vd Merwe, Royal GD, personal communication).These endotoxins can cause fever and local inflammatory reactions in the gastro-intestinal tract of humans if the milk of cows cured of mastitis is consumed (Wang and Quinn, 2010).However, this risk is limited because cows that contract clinical mastitis will be treated with antibiotics and the milk of treated cows is not allowed for consumption during the withdrawal period.Special attention must be paid to mastitis caused by potentially methicillin resistant S. aureus (MRSA;Vanderhaeghe et al., 2010).However, these MRSA are mostly linked to the intensive beef industry (van Loo et al., 2007).For an overview of potential zoonotic bacteria, the laboratory test to detect them, their clinical symptoms in cattle, their presence in milk, control measures and their total occurrence and importance in our region, see Table 2.

Parasitological and mycotic infections
Parasitological infections can be distinguished as being caused by endo-or ectoparasites.Endoparasites affecting host tissues and organs of live cattle include: Some of these parasites, such as Cryptosporidium parvum (Thomson et al., 2017), Toxocara ventilorum (Borgsteede et al., 2012), Echinococus granulosis (Eckert and Deplazes, 2004) and Giardia duodenalis (G.duodenalis; Geurden et al., 2004;Olson et al., 2004), are of zoonotic importance.C. parvum infections in humans are frequently related to contact with surface water (e.g. in swimming pools).The T. ventilorum parasite is known to be excreted in milk, but is mainly found in the colostrum of suckling cattle from southern Europe.The prevalence of E. granulosis in the Netherlands is also low and the main risk is consumption of imported raw meat of cattle from Eastern Europe (Berends et al., 2009).G. duodenalis is predominantly found in young calves (Geurden et al., 2004).Therefore, the overall zoonotic risk of endoparasites from dairy cattle in the Netherlands is estimated as low.
Ectoparasites such as lice and mites may cause problems of the coat and skin.They can be a risk for employees working with cattle, and may be principally responsible in humans for zoonotic dermatitis symptomsred spots and itch, in the case of infection with mites (Pérez de León et al., 2020).Consuming milk from cattle infected with ectoparasites does not carry a zoonotic risk.
By far the most important mycotic infection with a serious zoonotic risk is Trichophyton verrucosum (Lund et al., 2014) which results in proliferative dermatitis with crust.The spores of this infection are highly resistant and are mostly seen in animal crusts but can also be present throughout the barn.Therefore, eliminating this infection from the herd is very challenging.Trichophyton verrucosum (commonly known as ringworm) can be transmitted to humans by direct contact and causes circular skin lesions (Lund et al., 2014).The agent is not excreted in milk.For an overview of potential zoonotic parasitological and mycotic infections of importance in Western Europe, the laboratory test to detect them, their clinical symptoms in cattle, their presence in milk, control measures and their total occurrence and importance in our region, see Tables 3 (parasitological infections) and 4 (fungal infections).

Transmissible spongiform encephalitis
The most important TSE in the last several decades has been bovine spongiform encephalitis (BSE), which has been responsible for a considerable number of outbreaks with most economic damage in the UK (Alarcon et al., 2022).The Netherlands has observed 31 clinical cases and a total of 89 confirmed cases (58 after slaughterhouse control, www.wur.nl).The last confirmed case was in 2023.Humans have been diagnosed with variant Creutzfeldt-Jacob disease, but a relationship with BSE has not been proven.Evidence does not support transmission of TSE by milk consumption.Therefore, the zoonotic risk of BSE is estimated as very low.

Conclusions and recommendations
Dairy cattle can be a source of various types of zoonotic infections.Therefore, working with cattle includes a risk that farmers or employees become infected with a pathogen.Some infections may cause serious symptoms such as fever, diarrhoea, respiratory problems or worse in humans.The risk of transmission of infectious agents from dairy cattle to humans is mainly through air, by direct or indirect contact with manure, urine or abortive material (where indirect contact is largely through contaminated milk) and by direct contact with the coat.Risks can be limited by taking good preventive hygiene measures.We advise that all employees working with cattle or milk be aware of the risks and take preventive measures, for example using coveralls, gloves and protective glasses and washing hands frequently with disinfectant soap after contact with cows or their milk.Additionally, sick dairy farm employees are advised to limit their contact with cattle and to indicate they work with cattle when consultation with a physician is required.Excluding milk from infected dairy cattle also limits the risk of pathogen transmission.Other measures include the use of cattle that are free of specific pathogens such as L. hardjo, S. typhimurium and M. paratuberculosis.
Bacterial infections caused by pathogens excreted in milk (Salmonella spp.and paratuberculosis) or faecal contamination of milk (eg Campylobacter jejuni and E. coli) and mycosal infections (eg Trichophyton verrucosum) are particular risks for farmers and employees working with cattle.They should be aware of possible risks, avoid the consumption of raw milk and take protective measures such as those just described.More extreme measures, like having lunch in dedicated rooms, must be considered as well.Dairy farms are advised to follow certification programmes for L. hardjo, Salmonella spp.and M. paratuberculosis.All these measures should result in an acceptably low risk of becoming affected by a zoonotic disease.

Table 1 .
Potential zoonotic viruses of importance in Western Europe, DNA or RNA, the laboratory test to detect them, their clinical symptoms in cattle, their presence in milk, control measures and their importance in The Netherlands

Table 2 .
Potential zoonotic bacteria of importance in Western Europe, the laboratory test to detect them, their clinical symptoms in cattle, their presence in milk, control measures and their importance in The Netherlands potential pathogenic character.Bacillus cereus can cause clinical mastitis in cows.In case of clinical mastitis, milk delivery for consumption is forbidden, so the zoonotic risk for direct transmission by milk is considered low, if mastitis milk is removed and if hygiene measures are followed by employees.Brucella abortus can cause substantial health problems in

Table 3 .
Potential zoonotic parasitological infections, their clinical symptoms in cattle, their detection methods, their presence in milk, control measures and their importance in The Netherlands

Table 4 .
Potential zoonotic fungal infections, their clinical symptoms in cattle, their route of transmission, their presence in milk, control measures and their risk of transmission Journal of Dairy Research 329 humans, but the Netherlands has been declared by the European Union to be officially free of bovine brucellosis for over 20 years.In the Netherlands, salmonellosis, campylobacteriosis and possibly paratuberculosis were identified as bacterial infections with serious zoonotic risk.Other bacterial pathogens with nonnegligible zoonotic risk are Leptospira hardjo, Escherichia coli and Listeria monocytogenes.Most of these bacterial zoonotic infections are a consequence of direct excretion in or contamination of the milk or contact with manure (eg Salmonella spp., Mycobacterium avium subsp.paratuberculosis, E. coli [STEC O157], Campylobacter spp.; Christidis et al. 2016; Whittington et al. 2019; Ameer et al., 2021; Stevens and Kingsley, 2021).Other vectors are excreta associated with abortion (e.g.Brucella abortus, Listeria monocytogenes and Chlamydia abortus; Walker et al., 2015; Chlebicz and Śliżewska, 2018; Whatmore and Foster, 2021), with urine (e.g.Leptospira hardjo; Ellis, 2015) or with cadavers (e.g.Clostridium botulinum; Holzhauer et al., 2009).