Giardia duodenalis Styles, 1902 Prevalence in Cattle (Bos taurus Linnaeus, 1758) in Europe: A Systematic Review

Giardia duodenalis Styles, 1902 is an infectious agent which can cause enteritic disease in cattle (Bos taurus Linnaeus, 1758) worldwide. As a zoonotic protozoan, it is important to acknowledge Giardia prevalence and assemblages found in cattle and risk factors associated with the Giardia infection in herds. This systematic review aims to estimate the prevalence of G. duodenalis and its assemblages in cattle and to identify the risk factors associated with Giardia infection in cattle in Europe. A systematic review was performed according to Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines to collect information from published studies in Europe. A total of 1414 studies were identified and 17 relevant studies were included in this review. Mean Giardia prevalence in cattle in Europe was 35.1%, with the highest prevalence found in neonatal animals (39.6%), but mean heard prevalence was 67.0%. Mixed infections of Giardia assemblages A and E were found most frequently (55.6%), while assemblages A and B were found more frequently in animals under 24 months old. Risk factors, such as deep litter with run-out, prolonged calf contact with the dam, and seasonality, such as winter and spring, were found to be potential risk factors for Giardia presence in the herds.

Giardiasis is G. duodenalis-caused infection in cattle with enteric manifestations accompanied with diarrhoea. The severity of clinical signs are age-dependent, with symptoms being more prominent in calves younger than 30 days old. Clinically, giardiasis may be observed in cattle until they are six months of age [3][4][5][6]. After exposure of calves to G. duodenalis infection, gastric acid disrupts the cyst wall with initiation of excystation after a short excyzoite stage. Later, Giardia turns into vegetative stage trophozoite, which colonizes the epithelium of the small intestine through attachment with an adhesive disc and multiplies by longitudinal binary fission every 6-12 h [7]. Giardiasis may develop as malabsorption induced by reduced jejunum brush border surface area and decreased absorption of fluids, electrolytes, and nutrients. These conditions may lead to increased intestinal motility, reduced production, and activity of intestinal enzymes, including disaccharidase, maltase, and lactase, hence decreased weight gain [6,8,9].
Giardiasis causes immense economic losses, especially in meat-and milk-oriented cattle farms, attributed to high treatment costs, calf mortality, and decreased weight gain in animals [10].
Giardia has been found in beef and dairy cattle worldwide [11]. Prevalence can vary between farms with different management systems, such as differences in animal husbandry and production strategies. Geographic areas and climate conditions have also been associated with differences in Giardia prevalence in studies [4]. Study design may also affect the reported prevalence, and longitudinal epidemiological studies have consistently demonstrated prevalence of up to 100% in beef and dairy cattle herds [8,9]. Transmission of Giardia occurs between infected and non-infected calves, but chronically infected, asymptomatic adults may also be involved. The increased cyst excretion in dams has been demonstrated during the periparturient period that involves the period between two weeks before calving and four weeks post-calving; thereby, dams can also infect calves [12]. Infected calves can start to excrete the cysts as early as four days after the animal has been infected. Typically, the maximum number of colonizing cysts is reached within five weeks after infection, and infected animals can shed the cysts for several months [4,6,13]. Giardia transmission occurs between calves, despite the use of extensive disinfection and antiparasitic treatment [8]. Broad-spectrum anthelmintic drugs-benzimidazoles (albendazole, fenbendazole) group and paromomycin-are used for the treatment of giardiasis, and antiparasitic therapy has been shown to reduce cyst excretion and length of diarrhoea [8,14]. Although there are no licensed drug treatments for giardiasis for ruminants in Europe, benzimidazoles are relatively safe to use, if used appropriately and in combination with proper control to prevent reinfection, such as heat, disinfection with 10% ammonia, and desiccation. Giardia cysts are resistant to chlorine and may survive in that environment [14].
Calves should be considered a major source of environmental contamination since the infected animal may excrete as much as 10 6 cysts per gram of faeces [14]. The infective dose of G. duodenalis can be as low as ten cysts [15]. Cysts are infectious immediately after excretion and can survive for a long period in the environment: in water, soil, and faeces at +4 • C up to nine weeks; and in cattle slurry for three months [16][17][18][19]. Although the prevalence of G. duodenalis in dairy and beef cattle farms in Europe is extensively studied, there is a necessity to recognize the risk factors associated with the carriage of G. duodenalis on the farm. The systematic review aims to estimate the prevalence of G. duodenalis and its assemblages in cattle to identify the most important risk factors for cattle giardiasis in Europe.

Search Strategy and Criteria Selection
A systematic review was performed to collect information on the prevalence of G. duodenalis in cattle; the presence of G. duodenalis assemblages in cattle; and the most important risk factors related to the prevalence of giardia in cattle in Europe. International online databases PubMed, Web of Science, ScienceDirect (all fields), and Scopus (title, abstract, and keywords) were searched for all published data on the topic. The "PRISMA" "Preferred reporting items for systematic reviews and meta-analyses (PRISMA)" guidelines were followed (Supplementary file S1) [20]. The following search phrases were used: Giardia (AND) Cattle (AND) Prevalence (AND) Risk factors. The databases were searched for all published studies in English, from 1960 to 1 November 2022.

Eligibility Criteria, Study Selection, and Data Extraction
The search results from four databases were combined and duplicates were excluded. Eligible studies were initially selected based on the title and abstract using the inclusion and exclusion criteria. Inclusion criteria were: study was done on cattle and contained information about the prevalence of G. duodenalis; study was done in European countries (European Union, European Economic Area, and the United Kingdom); and study was published from 1960 to 1 November 2022. Exclusion criteria were: studies of Giardia prevalence in animal species other than cattle, humans, or environment; studies done outside Europe; letters, editorials, notes, books, comments, case reports, reviews, and conference abstracts. Primary screening of the titles and abstracts of each study was conducted to judge the eligibility for the present systemic review. Additionally, the reason(s) for exclusion during the primary screening was recorded. If two reviewers could not reach an agreement on inclusion or exclusion after the primary screening, the titles and abstracts were screened for eligibility by a third independent reviewer till a consensus was reached. Studies were further screened for eligibility based on the full text by two independent experts. Exclusion criteria for the second screening were: studies with no full text available; conference abstracts; and no prevalence data included. The data representing the eligible studies for the full-text screening were collected and processed in Microsoft Excel 365 spreadsheets. The following variables were extracted from each study: country, study year, number of farms visited, collected samples, positive samples, prevalence, cattle age, sample preparation technique, diagnostic tools, diagnostic kit for cyst detection, DNA isolation technique, PCR type, targeted gene, assemblage, and on-farm risk factors (Supplementary file S2).

Statistical Analysis
The total prevalence and the pooled prevalence were calculated using the open-source software for epidemiological statistics OpenEpi (OpenEpi: Open Source Epidemiologic Statistics for Public Health, Version. www.OpenEpi.com, accessed on 2 November 2022) [21] by calculating 95% confidence interval using the Z-test for estimation of differences between the population. To determine significances of all statistical analyses, p-value of 0.05 was used. Fisher's exact test (p < 0.05) was used to calculate the differences in Giardia prevalence between cattle of different age groups.

Search Results and Eligible Studies
In the literature search, a total of 1414 studies were identified ( Figure 1). Among the screened papers, 33 studies were duplicates and 1364 studies did not meet the inclusion criteria during either the title and abstract, or the full-text screening. Altogether, 17 studies were found relevant for the present review from following countries-Belgium, Denmark, France, Germany, Italy, Norway, Scotland, Spain, The Netherlands, and the United Kingdom (one from 1996, nine from 2000 to 2010, and seven from 2011 to 2020)-after thorough screening for the inclusion and exclusion criteria (Table 1).

Study Design
In all studies, there was no unified age distribution for cattle, therefore for pooled prevalence calculations, age groups were divided as follows: neonatal (≤1 month old), calves (1-6 months old), heifers (7-24 months old), and adults (≥24 months old). If the included study aimed to detect the prevalence of giardia within the farm independently of animal age, the prevalence was shown as the total prevalence (Table 2). Table 2. Summary of estimated pooled Giardia duodenalis prevalence in cattle by age groups (neonatal (≤ 1 month old), calves (1-6 months old), heifers (7-24 months old), adults ≥24 months old) and total prevalence of all cattle.

Country
No Sample collection per rectum was mentioned in thirteen studies, but the sampling technique was not mentioned in four studies.

Cyst Detection
Microscopic methods were used for cyst detection in 13 studies. Five different protocols were used for faecal sample preparation for microscopy: a purification technique with saturated saline and glucose flotation fluid was used in two studies, a flotation technique with sucrose fluid, a formalin-ethyl acetate sedimentation technique, a Merthiolate-iodineformaldehyde concentration, and a sodium acetate-acetic acid-formaldehyde technique were used in one study each, and no sample preparation was used in seven studies.

Prevalence of G. duodenalis in Europe
Giardia prevalence in cattle in Europe was 35 Table 2). In neonatal animals, a significantly higher prevalence of Giardia than in other age groups (p < 0.05) was found.
Assemblages were successfully differentiated in nine studies. Mixed infections of A and E assemblages were found in five studies (55.6%, 95% CI: 26.6-81.2), only assemblage E was found in three studies (33.3%, 95% CI: 11.7-64.9), and mixed infection with A, B, and E assemblages was found in one study (11.1%, 95% CI: 0.0-45.7). In one study, no data about assemblages were obtained because of failure in DNA isolation. Assemblages A and B were found in cattle younger than 24 months.

Farm-Related Factors
Farm-related factors were analysed in six studies. Factors-herd size, organic or conventional farm, cattle breed, cattle kept outdoors or indoors, and floor type (slatted or solid)-did not show an impact on G. duodenalis prevalence and cyst excretion intensity [23,25,30,38]. Higher G. duodenalis prevalence in dairy cattle compared to beef cattle was observed in one study [31]. Significantly higher prevalence of Giardia was observed in German Angus calves kept on deep litter with run-out compared to deep litter housing without run-out, winter run-out yarding, or slatted floor management systems [26]. The regular cleaning of maternity pens (4x/year), instead of cleaning them only after calving, reduced the Giardia infection risk in calves [25].

Calf Husbandry-Related Factors
In three studies, factors related to calf rearing practice were discussed. Individual or group housing of calves did not reveal significant impact on the presence of Giardia, while a higher risk was identified in calves after contact with the dam [25]. The disinfection of housing units significantly reduced giardiasis in calves [25]. Maddox-Hyttel et al. [23] found that excretion of Giardia cysts was significantly lower in farms which had implemented high-pressure cleaning and maintained the empty period prior to the introduction of new calves into a pen. A significantly lower prevalence was observed in individually fed calves older than three weeks as compared to group-fed calves (p = 0.0030) [30].

Seasonality
In three studies, the differences in the prevalence of G. duodenalis between seasons were observed. In The Netherlands and Norway, the prevalence of Giardia in cattle was higher in winter than in summer or autumn, respectively [30,37]. In contrast, in Spain, prevalence was significantly higher in spring (p < 0.05) than in summer and winter [36].

Age
In five studies, animal age was identified as a risk factor of for Giardia infection. Higher risk was observed in calves younger than eight weeks of age [23], calves under one month of age [23,35], pre-weaned [31], suckling, and weaning calves [33].

Presence of Diarrhoea
In five studies, the correlation between diarrhoea and Giardia infection was calculated. However, links between G. duodenalis infection, diarrhoea, and changes in faecal consistency were not established [23,25,26,33,38]. No other clinical signs were studied.

Discussion
The limited number of published studies shows that Giardia is common but neglected in cattle in Europe, despite its relevance as a food and waterborne parasite [15,39]. The overall pooled prevalence in Europe reached 35.1%. Different sample preparation techniques were used for the microscopic analysis of animal faeces. Staining with iodine is a fast, cheap, and easy to perform the diagnosis of Giardia; however, lower sensitivity and specificity compared to IMF has been reported [40]. Indirect detection methods possess higher sensitivity than iodine staining or light microscopy as opposed to IMF, but may lead to incorrect results, if a low number of cysts is present. Indirect detection methods were reported to be more expensive than IMF, which might affect their availability and practical application [41]. Despite the IMF being "the gold standard" for the detection of Giardia cysts in faeces, expensive equipment and trained staff are required for testing. In contrast, ELISA is an easy to perform, cost-effective, and commercially widely available protocol for the routine diagnosis of giardia. Additionally, only minor differences between the sensitivity and specificity of direct immunofluorescence and ELISA methods were found, which demonstrates the practical importance of both laboratory investigations [42]. The classical detection methods include the microscopic detection of Giardia cysts by flotation and iodine staining; however, false negative results which arise due to the application of those methods have been reported [43].
Direct DNA isolation from faeces was done in most of the studies. Better results from the application of DNA isolation were attributable to the additional cleaning of debris, aiming to remove large amounts of bacteria and other DNA present in the faeces, which is also less time consuming than purifying [44]. The β-giardin gene is unique to Giardia, and is therefore frequently targeted for the detection of the pathogen. Additional advantages include the absence of cross-reaction with other pathogens, such as fungi or bacteria as compared to 18S rRNA gene which lacks specificity, and the 18S rRNA gene-amplified specimens were negative for other genes (bg, TPI, and GHD) [38,44,45]. Even though a single gene can be targeted for the detection of Giardia, simultaneous identification of all three Giardia genes is required for genotyping and subtyping [18]. Conventional PCR was mostly used, even though semi-nested and nested PCR can retrieve better results if low numbers of cysts are present in the sample [46]. Conventional PCR is the gold standard for the routine diagnosis of Giardia because of the lower costs and wider availability, even though there is a need for experienced technical staff [47,48].
Results on the prevalence of Giardia between age groups differed, but the highest prevalence was found in neonatal animals. Overall, the highest prevalence was recorded in calves younger than one month [23,31,37] or in calves between one and six months of age [33,39]. Observed differences in the prevalence of Giardia between the cattle age groups could be related to prolonged periods of cyst excystation, which may extend to 112 days after initial infection [6,8]. Assemblage E has been found in all cattle age groups; however, an association between the presence of this assemblage and the clinical diarrhoea was not identified [48][49][50]. The prevalence of assemblages A and B in calves is of importance since both share zoonotic potential and expressed pathogenicity in humans [50][51][52]. Similar findings were reported from South Korea, the USA, and Canada, where the highest prevalence was observed in calves 7-8 and ≥12 weeks (15.4%) old, calves 2-11 months (52.0%) old, and calves under 6 months old (53.0%), with zoonotic assemblage A (3.8%; 12.2%; 13.0%, respectively) presence [53][54][55].
In several studies, the risk analysis has been conducted to identify the factors affecting the distribution of Giardia between farms. It has been observed that beef cattle herds had higher prevalence compared with dairy cattle, which could be due to different management systems [22,31,54]. Even though multiple studies recorded from which dairy or beef cattle breed samples were collected [22,25,56], information about potential prevalence differences between specific breeds is lacking.
Deep litter housing without run-out, use of straw bedding, and slatted floor types were associated with a potential risk of excretion and accumulation of cysts in the environment [23,26]. Incorrect use of deep litter housing and the use of straw bedding can retain moisture that creates appropriate microclimatic conditions for parasite survival [23,57]. Prolonged dam-calf contact or contact with other calves increased the odds of infection, because of the potential rise in cyst excretion in the dams' peri-parturient period and the large accumulation of cysts in the environment over time from other infected calves were observed in France, Italy, Germany, and the United Kingdom, as well as the USA [25,58]. Regular cleaning of maternity pens reduced the shedding of Giardia, and routine highpressure cleaning of pens with disinfection and an empty period before the housing of calves reduced cyst excretion in calves [23,25]. Similar results were observed in the USA [59]. These farm management-related risk factors show that proper prevention and improvement in animal farming practice could significantly reduce the prevalence of acute or chronic giardiasis in cattle farms, thereby reducing the risk of economic losses. Reduced environmental contamination that may arise from the minimized emission of Giardia from cattle farms should be considered a potential benefit of improved animal farming strategy.
Higher prevalence of Giardia was observed in winter and spring, rather than summer or autumn, months. A higher prevalence of giardia in cattle in winter months could be linked to lower immunity, higher density of animals indoors, and prolonged survival of cysts in colder temperatures. Additionally, widespread occurrence of respiratory infections in colder months may provoke the development of giardiasis as a secondary infection [30,37,60].
Age as a risk factor was discussed in several studies, with calves under eight weeks of age being more affected. Newborn calves are protected with the dam's colostrum, that contains immune cells and may contain anti-giardia antibodies, providing passive immunity against giardia with the maximum protective effect lasting in calves up to two weeks of age [8,13].
Diarrhoea was observed in cattle, but associations between diarrhoea and infection were not found, apart from one study done by Xiao et al. [61]. Diarrhoea in infected calves could be induced by other enteric pathogens, such as Cryptosporidium or rotavirus. Giardia may cause chronic, intermittent diarrhoea that makes it difficult to establish a connection between the clinical signs and the presence of the pathogen [23,25,26,33,37,62].
Even though sex as a risk factor was not included in these studies, there are studies done in Turkey and South Korea that show no differences in prevalence between males and females [53,63]; however, other studies have shown that prevalence was higher in male cattle in Nepal and Bangladesh, which could be due to different housing conditions for male calves [64][65][66].

Conclusions
This review provides a limited, but comprehensive overview of the presence of Giardia in cattle in Europe. The prevalence of giardia was significantly different between the animal groups, with neonatal cattle being more clinically affected, while older cattle could be potential asymptomatic carriers. Assemblages A and B were identified in cattle of all ages that could indicate a potential zoonotic threat for humans. Application of DNA-based methods may improve the sensitivity and specificity of the detection of Giardia infection in animals. The estimation of potential risk factors may be helpful in establishing pathogen on-farm control programmes for the reduction of giardia in cattle.

Conflicts of Interest:
The authors declare no conflict of interest.